structure function analysis of mitochondrial dna

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Doctoral Dissertations Dissertations and Theses

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STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA

POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST

MAINTENANCE MAINTENANCE

Jonathan Miller University of Massachusetts Amherst

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STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST

MAINTENANCE A Dissertation Presented by

JONATHAN C. MILLER

Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

May 2020

Department of Microbiology

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© Copyright by Jonathan C. Miller 2020

All Rights Reserved

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STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE

A Dissertation Presented

By

Jonathan C. Miller

Approved as to style and content by:

Michele M. Klingbeil, Chair

Steven J. Sandler, Member

Yasu S. Morita, Member

Peter Chien, Member

James F. Holden, Department Head Department of Microbiology

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DEDICATION

To my father Michael who gave me the resources and support to find my own path to success,

To my brother Shawn for being the voice of reason and always protecting me,

To my mother Kimlien for her tireless support to provide a better future for her children,

To Cindy who without I wouldn’t be the person I am today.

Thank you.

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ACKNOWLEDGEMENTS

First and foremost I must thank my supervisor and mentor Dr. Michele Klingbeil, who enrolled

me in biochemistry bootcamp many summers ago and taught me how to become an

independent thinker at the chalkboard and a leader in laboratory. I thank my committee

members Drs. Steven Sandler, Yasu Morita, and Peter Chien for their intellectual guidance and

support throughout this process.

Thank you to all the teaching faculty I had the privilege of teaching under and alongside as they

gave me a love and respect for teaching and inspiring students.

To the many members of the Klingbeil lab over the years: Jeny CA, Mike B, Rebecca G, Garvin D,

Stephanie D, Yadi B, Maria CR, Jirka T, David A, Sam K, Dan B, Elsie H, Paris J, Jon K, Parker C,

James H, Thanh B, Loan V, and Matt F. I have learned so much from you and I hope I could

teach you something helpful in your respective journeys as well.

Thank you to my UMass graduate school family spanning over a half dozen departments across

the Amherst campus, especially my dear friends Jen H and Michael U who I wouldn’t have

made it through my first graduate school years without their unconditional support. I would

also be remiss to not mention Emily R, Julia P, Gina C, and Bia D for all our adventures across

the state and abroad! Also, thank you to the house members for adopting me during my

surgery recovery process.

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Thank you to my family and friends for a what was quite a number of years of supporting me.

Thank you to Cindy for completing my graduate experience with much-needed love and care.

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ABSTRACT

STRUCTURE FUNCTION ANALYSIS OF MITOCHONDRIAL DNA POLYMERASE IC REVEALS MULTIPLE ROLES IN KINETOPLAST MAINTENANCE

MAY 2020

JONATHAN C MILLER

B.S., UNIVERSITY OF CONNECTICUT, STORRS

PH.D., UNIVERSITY OF MASSACHUSETTS AMHERST

Directed by: Professor Michele M. Klingbeil

Kinetoplastid organisms including medically relevant species like Trypanosoma brucei,

Trypanosoma cruzi, and Leishmania are distinguished by their single flagellum and unique

mitochondrial DNA called kDNA, among other features. While there is heterogeneity in copy

number and sequence classes among different species, kDNA is most often found as a network

of thousands of DNA molecules catenated together. This unique biological property of disease-

causing organisms has been a subject of study as a potential drug target since it is essential for

parasite survival. Trypanosoma brucei is the causative agent of African Sleeping Sickness in

humans and related diseases in other mammals and is fatal if left untreated. Recent advances in

drug development have produced an oral medication to treat most forms of the disease,

hopefully alleviating the medical burden of the organism and enabling us to appreciate and study

its fascinating mitochondrial biology.

Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and

POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have

roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear.

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In Chapter 2, we show that POLIC localization changes throughout kDNA S phase and in order to

look for potential interactions with kDNA segregation machinery, we utilize structured

illumination microscopy to follow localization dynamics with a TAC marker, TAC102. Additionally,

we show that kDNA replication proteins POLIB and POLID do not affect TAC102 localization

further reinforcing them as strictly kDNA replication proteins. In Chapter 3, we use an RNAi-

complementation system to dissect the functions of the distinct POLIC domains: the conserved

family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While

RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization,

active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB

and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci

formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a

crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This

is the first report of a DNA polymerase that impacts DNA segregation.

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TABLE OF CONTENTS Page

ACKNOWLEDGEMENTS ............................................................................................................... v

ABSTRACT ................................................................................................................................. vii

LIST OF TABLES .......................................................................................................................... xii

LIST OF FIGURES ....................................................................................................................... xiii

CHAPTER

1. LITERATURE REVIEW AND STUDY BACKGROUND .................................................................... 1

1.1 The global health impact of Trypanosoma brucei ................................................... 1

1.2 Infection and immunity of Trypanosoma brucei ..................................................... 3

1.3 Structure, sequence, and function of kDNA ........................................................... 5

1.4 Spatiotemporal and structural aspects of kDNA network replication ..................... 9

1.5 Mechanistic insight into kDNA replication ............................................................ 10

1.6 Segregation of kDNA networks ............................................................................ 15

1.7 Goal of this study ................................................................................................. 17

1.8 Significance and contribution of this study ........................................................... 17

1.9 Bibliography ......................................................................................................... 23

2. INVESTIGATION OF POLIC CELL CYCLE LOCALIZATION IN RELATION TO TRIPARTITE ATTACHMENT MARKER, TAC102 ............................................................................................... 34

2.1 Abstract ............................................................................................................... 34

2.2 Introduction ......................................................................................................... 34

2.2.1 Widefield fluorescence microscopy and its limitations .................................. 34

2.2.2 Principles of super-resolution microscopy ..................................................... 36

2.2.3 Principles of Structured Illumination Microscopy .......................................... 38

2.2.3 Cell cycle dependent localization of kDNA-associated proteins ..................... 39

2.3 Materials and Methods ........................................................................................ 40

2.3.1 DNA constructs. ............................................................................................ 40

2.3.2 Trypanosome cell culture and transfection ................................................... 41

2.3.3 Widefield Immunofluorescence .................................................................... 41

2.3.4 Structured Illumination Microscopy .............................................................. 42

2.3.5 Three-dimensional reconstruction and projection ......................................... 43

2.4 Results ................................................................................................................. 43

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2.4.1 Cell cycle analysis of POLIC and TAC102 by structured illumination microscopy............................................................................................................ 43

2.5 Discussion ............................................................................................................ 44

2.6 Bibliography ......................................................................................................... 51

3. DNA POLYMERASE IC IS CRITICAL FOR REPLICATION AND SEGREGATION OF MITOCHONDRIALDNA IN THE AFRICAN TRYPANOSOME TRYPANOSOMA BRUCEI. ................................................ 53

3.1 Abstract ............................................................................................................... 53

3.2 Introduction ......................................................................................................... 53

3.3 Materials and Methods ........................................................................................ 60

3.3.1 DNA constructs. ............................................................................................ 60

3.3.2 Trypanosome cell culture and transfection. .................................................. 61

3.3.3 RNA isolation and RT-qPCR analysis. ............................................................. 62

3.3.4 SDS-PAGE and Western blot analysis. ............................................................ 62

3.3.5 DNA isolation and Southern blot analysis. ..................................................... 63

3.3.6 Immunoprecipitation and DNA polymerase activity assay. ............................ 63

3.3.7 Immunofluorescence microscopy.................................................................. 64

3.4 Results ................................................................................................................. 65

3.4.1 POLIC 3ʹUTR RNAi and rescue with epitope-tagged wild-type protein. .......... 65

3.4.2 POLIC DNA polymerase activity is essential for kDNA replication and cell cycle localization. ........................................................................................................... 68

3.4.3 The POLA domain partially rescues minicircle replication defects.................. 71

3.4.4 The UCR domain partially rescues IC-UTR RNAi defects. ................................ 72

3.5 Discussion ............................................................................................................ 73

3.6 Bibliography ....................................................................................................... 103

4. LOOKING FORWARD............................................................................................................ 109

4.1 Looking backwards (briefly) ............................................................................... 109

4.2 Aligning our data with the kDNA field ................................................................ 110

4.2.1 Further defining role of POLIC utilizing the cell lines generated ................... 110

4.2.2 Interconnections among kDNA polymerases ............................................... 111

4.2.3 Narrowing in on the antipodal site determinant.......................................... 112

4.2.4 Addressing ancillary kDNA........................................................................... 112

APPENDIX: GENETIC COMPLEMENTATION WITH IC-WT IN A POLIB OR POLID RNAI BACKGROUND......................................................................................................................... 118

A.4 Bibliography ...................................................................................................... 127

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BIBLIOGRAPHY ........................................................................................................................ 128

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LIST OF TABLES Table Page

1.1 Known kDNA replication and maintenance proteins ………………………………………………………….19

1.2 Known TAC proteins. …………………………………………………………………………………………………………20

3.1: Primers used in this study. .......……………………………………………………………………..…………….…80

4.1: Table summary of experiments that induce ancillary kDNA. ……………………………………………116

xiii

LIST OF FIGURES Figure Page

1.1: Illustration of microscopic analysis of haematozoa in the blood of the cow, Bruce 1895.

Republished under the Creative Commons, Public Domain Mark. ….…………………………………….…21

1.2: Localization of kDNA replication and TAC proteins. …….…………………………………………………..22

2.1: Simplified Jablonski Diagram. …….…………………………………….……………………………………………..46

2.2: Diagram of Structured Illumination Apparatus. …….…………………………………………………………47

2.3: Widefield fluorescence of IC-PTP and TAC102 during kDNA S phase. …….………………………..48

2.4: Three-dimensional SIM imaging of POLIC-PTP, TAC102, and basal bodies. ………………………49

2.5: Linear intensity plots of kDNA disks. …….…………………………………….…………………………………..50

3.1: Phenotype of POLIC 3’UTR-targeted depletion. …….………………………………………………………..82

3.2: TAC102 colocalizes with ancillary kDNA. …….…………………………………….………………………….….84

3.3: Rescue with ectopically expressed IC-WT in the IC-UTR RNAi cell line. ……………………….……86

3.4: Overexpression of ectopically expressed IC-WT in the absence of POLIC RNAi. ………....……88

3.5: IC-WT displays nucleotide incorporation activity. …….……………………………………………………..89

3.6: Overexpression of IC-DEAD results in a dominant-negative phenotype while

complementation exacerbates loss of fitness. …….………………………………………………………………….91

3.7: Characterization of IC-POLA and IC-UCR overexpression cell lines. …….…………………………...93

3.8: kDNA replication phenotype analysis of IC-DEAD overexpression and

complementation.…………….…………………………………….……………………………………………………………..95

3.9: IC-POLA complementation is not sufficient to rescue the RNAi phenotype ……………………..97

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3.10: kDNA replication phenotype analysis of IC-POLA and IC-UCR

complementation.…………………………………….…………………………………….…………………………..……….…99

3.11: IC-UCR complementation is not sufficient to rescue the RNAi phenotype. …………….……101

3.12: Summary of POLIC variant phenotypes. ……….…….…………………………………….…………………102

4.1: Illustration of the localization of proteins whose depletion or mutant induction leads to the

detection of ancillary kDNA during kDNA S phase. …….…………………………………………………….…117

A.1: Overexpression of IC-WT in an SLIB RNAi background. …….……………………………………….…..124

A.2: Overexpression of IC-WT in an SLID RNAi background. …….…………………………………….……..125

A.3: Overexpression of IC-WT in an SLDB RNAi background. ………..…….………………………………..126

1

CHAPTER 1

1. LITERATURE REVIEW AND STUDY BACKGROUND 1.1 The global health impact of Trypanosoma brucei In the late 1800’s while cattle and horses died in the thousands from a disease called nagana by

the indigenous people of then-Zululand, the Scottish microbiologist Sir David Bruce of the British

Royal Army was dispatched to discover its etiology. Prior to this, Bruce recently discovered the

cause of Malta fever (Brucellosis) and before that had worked for a year at Dr. Robert Koch’s

institute in Berlin. With infectious bacteria on his mind, Bruce and his wife Mary Elizabeth Steele

(described as a brilliant laboratory scientist) discovered haematozoa in the blood of infected

cows and within a few months published on the causative agent for nagana in his Preliminary

Report on the Tsetse Fly Disease or Nagana, in Zululand (Bruce, 1895) (Figure 1). The haematozoa

responsible for nagana was subsequently named Trypanosoma brucei by Plimmer and Bradford

in 1990 (Plimmer and Bradford, 1900). By 1915, Bruce and colleagues had discovered what

transmitted this flagellated protozoan parasite to human and mammalian hosts (the tsetse fly,

Glossina morsitans), the parasite’s developmental life cycle, and two other distinct patterns of

trypanosomal disease in humans which were later discovered to be the T. b. rhodesiense and T.

b. gambiense subspecies, responsible for the acute and chronic forms of the disease, respectively

(Cook, 1994).

Trypanosomatid organisms like T. brucei are a global family of early-branching protists. Three key

members responsible for causing serious human diseases are T. brucei (human African

trypanosomiasis or HAT), Trypanosoma cruzi (Chagas disease), and leishmania spp.

(leishmaniasis). These three organisms are among 17 others classified as neglected tropical

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diseases (NTDs) by the World Health Organization that prevail in 149 countries in tropical and

sub-tropical climates, affecting more than one billion people worldwide (Addisu et al., 2019).

NTDs historically have garnered little attention for pharmaceutical development of treatments

and include many decades-old chemotherapies, some with poor efficacy, high patient toxicity,

and requires weeks-long administration of intravenous infusions to complete treatment (Barrett

and Croft, 2012). No vaccines are available for HAT. Advances in combination therapy led to the

introduction of nifurtimox plus eflornithine (NECT) in 2009 to combat late stage T. b. gambiense

infection to supplant existing monotherapies of pentamidine for early-stage and melarsoprol for

late-stage (Yun et al., 2010).

The WHO has targeted HAT for elimination as a public health problem by the year 2020 (Chan,

2012). Robust control and surveillance activities over the last decade have kept on target with a

92% decrease in reported cases since 2000 and a 61% reduction in cases/10,000 inhabitants/year

as compared to the 2000-2004 baseline rate (Franco et al., 2018). These two primary indicators

show encouraging progress towards elimination and this is further facilitated by the recent

approval of fexinidazole (nitroimidazole) in 2019 as an oral medication for both the early and late

stage infections of T. b. gambiense subspecies which makes up over 90% of reported cases.

Clinical trials to treat T. b. rhodesiense, the second most-common subspecies responsible for a

rapid onset of the disease, and Chagas disease (T. cruzi) are being prepared (Deeks, 2019). This

nitoreductase was discovered through successful compound-mining efforts in 2005 and was

subsequently tested in clinical trials beginning in 2009 under a partnership between the Drugs

for Neglected Disease initiative (DnDi) and Sanofi (Robinson et al., 2019).

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1.2 Infection and immunity of Trypanosoma brucei Endemic to sub-Saharan Africa, HAT early-stage infection (haemo-lymphatic) begins with a bite

from an infected Glossina. Metacyclic trypomastigote parasites primed for infection in the fly’s

salivary glands are released into and proliferate in the blood, lymphatic systems, and interstitial

organ space of the infected host and recent research suggests that T. brucei can utilize adipose

tissues as a “fat haven” and replicate in the skin (Kennedy, 2013; Trindade et al., 2016). These

parasites differentiate into slender bloodstream form (BSF) parasites and quickly adapt to the

new environment to cause early-stage infection. A subpopulation will utilize quorum sensing to

differentiate into transmissible non-replicative stumpy forms which is partly controlled by an

orphan G protein-coupled receptor, TbGPR89 (Mony et al., 2014; Reuner et al., 1997; Rojas et al.,

2018; Vassella et al., 1997). These stumpy forms further differentiate into procyclic forms (PCF)

to proliferate in the tsetse fly. BSF T. brucei eventually crosses the blood-brain barrier (BBB) into

the central nervous system (CNS) to cause late-stage infection (meningo-encephalitic) and almost

always leads to death if left untreated (von Geldern et al., 2011; Kennedy, 2013). This process

may take several weeks for the acute infection caused by T. b. rhodesiense or several months to

years for a more chronic T. b. gambiense infection (Kennedy and Rodgers, 2019). While early-

stage symptoms are vague (intermittent fever, headache, lassitude, arthralgia), once in the CNS,

neurological symptoms manifest including the characteristic sleep disorder identified by daytime

somnolence and nocturnal insomnia, giving the disease its common name, Sleeping Sickness.

Heterogeneity in both the patient and parasite’s genetics, possible co-incidence of other

infectious disease (notably malaria), and a wide range of possible symptomology can make it

difficult to diagnose (Kennedy and Rodgers, 2019). Thankfully, the development of rapid

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diagnostic tests (RDT) such as the SD BIOLINE HAT are in prototype phases and will hopefully

supercede or work in conjunction with current standards such as the Card Agglutination Test for

Trypanosomiasis (CATT) and PCR-based methods (Bisser et al., 2016; Lumbala et al., 2018).

Unfortunately, identification of late-stage infection is still reliant on cerebrospinal fluid (CSF)

examination by lumbar puncture, though the quantification of certain biomarkers (CSF IgM,

neopterin, combined panels of chemokines and proteins) to circumvent this strategy are under

consideration (Hainard et al., 2009; Lejon et al., 2002; Tiberti et al., 2012).

T. brucei cannot rely on immune evasion by hiding in host cells in the way that the South American

trypanosome T. cruzi does. As an extracellular parasite, it expresses a single variant surface

glycoprotein (VSG) on its surface and once an immune response is mounted, changes expression

to another VSG gene. Out of the ~9,000 predicted genes in the parasite’s genome, 10% code for

VSG molecules that are expressed on the cell surface and are critical for determining immune

specificity (Berriman et al., 2005). Glycosylphosphatidylinositol (GPI) anchors attach the VSG to

the cell’s outer membrane. BSF parasites will differentially express a single VSG molecule during

infection by switching VSG genes in and out of an expression site (ES) through a process called

antigenic variation. This phenomenon is a nearly insurmountable barrier to vaccine development

(Kennedy, 2013).

Some innate defense against trypanosome infection exist in humans and include a defense

mechanism called trypanolytic factors (TLFs). Proteins present in serum (TLF) comprised of

apolipoprotein A1 (APOA1), L1 (APOL1), and haptoglobin related protein (HPR) together form

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two serum protein complexes, trypanosome lytic factor 1 (TLF1) and 2 (TLF2) (Capewell et al.,

2015). These are able to induce lysis of animal trypanosomes (T. b. brucei). To counter this, the

human-infective subspecies T. b. rhodesiense contains the SRA gene to express the serum

resistance associated (SRA) protein, a truncated VSG molecule, that binds to and prevents lysis

by TLF. T. b. gambiense has a more complex resistance mechanism involving reducing binding

affinity to TLF, increase cysteine protease activity, and the expression of a truncated VSG called

T. b. gambiense-specific glyocoprotein (TGSgp) (Capewell et al., 2015). Recently, is has been

shown that G1 and G2 APOL variants affect susceptibility to HAT, where the G1 variant was

associated with asymptomatic infection and failure to detect trypanosomes and the G2 variant

showed a protective effect against T. b. rhodesiense infection but more rapid disease progression

from T. b. gambiense infection (Cooper et al., 2017).

1.3 Structure, sequence, and function of kDNA Much of the impetus in studying trypanosome biology is to develop new treatments due to the

multitude of diseases they cause. One classic example of a biological property essential for T.

brucei survival that have no structural homologues in the mammalian host is the genome of the

parasite’s single mitochondrion called the kineoplast DNA (kDNA). Unlike other eukaryotic

organisms that have varying numbers of mitochondria per cell and mitochondrial nucleoids per

organelle, T. brucei harbors only one mitochondrion containing a single mtDNA nucleoid in the

form of a catenated network of circular DNA molecules (Vickerman, 1976). Prior to understanding

the connection between the flagellum and mtDNA, different parts of the structure have

historically been called a blepharoplast, para-basal body, and kinetonucleus until researchers

finally settled on kinetoplast fifty years after discovering HAT (Trager, 1965). Among the kDNA-

6

containing organisms are the medically important trypanosomatids previously described:

Leishmania (Leishmanisasis), T. cruzi (Chagas disease), and T. brucei (HAT), hence why most of

our knowledge on the kinetoplast comes from this group. Not to be excluded, however, are

Crithidia species, including the mosquito parasite C. fasiculata, whose ease of culture and

amenability to biochemistry led to many early discoveries on kDNA-related proteins beginning in

the 1980’s with the characterization of a type II topoisomerase (TopoIIMT) (Melendy et al., 1988).

A kDNA network exists as thousands of catenated duplex DNA moecules that form a disk-shaped

structure inside the parasite’s single mitochondrion. By light microscopy, the kDNA network

appears as disk-shaped structure situated near the parasite’s single flagellum. The advent of

electron microscopy (EM) led to the resolution of kDNA networks seen in vitro either as intact or

Topoisomerase II-treated networks (to decatenate and resolve individual DNA circles) by

spreading isolated networks on an EM grid, or in vivo to observe the kDNA structure inside the

parasite’s mitochondrion via thin sectioning (Ogbadoyi et al., 2003; Shapiro and Englund, 1995).

Much of our understanding on kDNA structure is by networks purified from the insect parasite C.

fasiculata. Isolated networks form a planar elliptical structure approximately 10 to 15 microns in

size and in vivo is condensed into a disk-shaped structure approximately 1 micron in diameter

and 0.3 microns thick (Pérez-Morga and Englund, 1993). When spread out on an EM grid they are

reminiscent of medieval chainmail which for T. brucei contain dozens of 23 kb maxicircles and

thousands of 1 kb minicircles topologically linked with an average valence of three, while EM thin

sections reveal the network is condensed into a disk-shaped structure in vivo (Chen et al., 1995;

Rauch et al., 1993). Maxicircle catenanes appear to be mutually interlocked and create a

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“network within a network” (Shapiro, 1993). For other kinetoplastid organisms, the number, size,

and topology of kDNA varies and in some, maxicircles are not even present in the genome (Blom

et al., 2000; Lukes et al., 2005; Shlomai, 2004). The kDNA network is a highly ordered structure

and physically linked by a filamentous protein structure called the tripartite attachment complex

(TAC) that traverses the mitochondrial membrane and connects to the flagellar basal body

(Ogbadoyi et al., 2003). To maintain its structure, there are at least four histone-like proteins

called kinetoplast-associated proteins (KAP1-4) that maintain the network’s disk shape

(Avliyakulov et al., 2004; Lukeš et al., 2001; Xu et al., 1996).

The complement of the dozens of 23 kb maxicircles are the genetic equivalent of mitochondrial

DNA (mtDNA) in other eukaryotes and encode ribosomal RNAs and proteins involved in energy

metabolism and respiratory complexes, but lack tRNA genes (Benne et al., 1983; Eperon et al.,

1983; Fairlamb et al., 1978; Feagin et al., 1985; Hensgens et al., 1984; Jasmer et al., 1985; Johnson

et al., 1984; Payne et al., 1985). Each maxicircle contains an 8 kb noncoding variable region with

repeated sequences called the divergent region, a 15 to 17 kb stretch of coding sequences called

the conservative region, and two copies of a sequence associated with the minicircle origin of

replication (Shapiro and Englund, 1995). The variable region is called so due to major differences

in size and sequence within different stocks of the same species (Borst et al., 1980; Maslov et al.,

1984).

Unlike other circular DNA found in nature, kDNA minicircles exist as topologically relaxed

covalently closed circles though supercoiling can be induced by the addition of DNA intercalating

8

agents (Rauch et al., 1993). It is possible that the absence of supercoiling was a property

evolutionarily abandoned to allow for kDNA catenation, although minicircles are relaxed

molecules even in kinetoplastids with non-catenated DNA (Lukes et al., 2002). In a non-dividing

network each minicircle is catenated with a single interlock (Hopf link) to three other neighbors

(Chen et al., 1995a; Rauch et al., 1993). The 1,000 base pair sequence contains replication origins

identified by conserved sequence analysis called conserved sequence blocks (CSB1-3) (Ray,

1989). Specifically, there is an invariant 12 base pair universal minicircle sequence (UMS) (CSB-

3), a 10 base pair sequence (CSB-1), and an eight base pair sequence (CSB-2) highly conserved in

all minicircles. Interestingly, most minicircles also contain a sequence of adenine tracts which

structurally produce a bent helix region that is susceptible to endonuclease digestion (Linial and

Shlomai, 1987; Marini et al., 1982).

While maxicircles encode genes for mitochondrial function, their cryptic immature transcripts

require additional processing by the sequence-specific insertion or deletion of uridylate residues.

This process, called RNA editing, is regulated by minicircle-encoded guide RNAs (gRNAs) that act

as templates for insertion or deletion. The approximately 5,000 minicircles in T. brucei contain

over 250 sequence classes with each minicircle encoding between two to five gRNAs (Hong and

Simpson, 2003). gRNAs contain several key features: a 5’ triphosphate which marks them as a

primary transcript, an initiation sequence with a 5’-RYAYA consensus sequence, an anchor

sequence for cognate cryptogene recognition, an information region that is complementary to

the stretch of edited mRNA, and a 3’ oligo(U) tail that likely stabilizes the gRNA-mRNA interaction

(Koslowsky et al., 2004; Pollard et al., 1990). Nine of the cryptogenes (pre-mRNA) require

9

extensive pan-editing with the insertion of hundreds and deletion of dozens of uridylate residues

per transcript. The enzymatic activities of the RNA editing pathway are retained in the RNA

editing core complex (RECC) which dynamically associates with other multi-subunit complexes to

form a holo-editosome (Aphasizheva and Aphasizhev, 2016; Read et al., 2016). The most recent

sequencing data revealed that there are over 900 canonical gRNAs and ~370 putative non-

canonical gRNAs present in the kinetoplast genome (Cooper et al., 2019). Retaining the kDNA

including the maxicircle genes and gRNAs from minicircles is essential for the parasite to

complete its lifecycle and therefore its long-term viability (Dewar et al., 2018; Schnaufer et al.,

2002). Currently, the only known way to bypass a dyskinetoplastic-induced transmission block is

a mutation in the gamma subunit of the ATP synthase which allows the parasite to maintain a

mitochondrial membrane potential (Dean et al., 2013).

1.4 Spatiotemporal and structural aspects of kDNA network replication kDNA replication occurs once per cell cycle and is coordinated with the duplication and

segregation of the flagellar basal bodies (Lacomble et al., 2010). Network replication is initiated

just prior to nuclear S phase and the first cytological event is the duplication of the basal body

(Woodward and Gull, 1990). From here, there are five distinct stages of kDNA S phase based on

the size and structure of the kDNA disk and the number and position of the basal body/pro-basal

body and flagellar pocket from 3-dimensional reconstructions of electron micrographs and

fluorescent images (Gluenz et al., 2011). In stage I, the kDNA is a unit-sized disk and only one

basal body/probasal body pair. In stage II, the kDNA has prominent antipodal sites and is

associated with two basal bodies. Antipodal sites in kDNA replication refer to sites flanking the

kDNA disk 180o apart during S phase where replicated minicircles accumulate (Ferguson et al.,

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1992). This stage is further divided into stage IIa and stage IIb, where the newly forming flagellum

is positioned anterior to the old flagellum and still contained in the flagellar pocket, and the kDNA

is dome-shaped, respectively. In stage III, the kDNA forms a bilobed shaped made of two joined

disks and antipodal sites are found on one or both of the disks. Here, each disk is associated with

a basal body/probasal body pair and two flagellar pockets are present. In stage IV, the two disks

are distinct but are still connected by a filament of basic proteins connecting daughter networks

exist even as far as 1.3 um apart, a structure called the nabelshnur (Gluenz et al., 2007).

Unlinking of maxicircle DNA in the nabelshnur structure marks the final stage in kinetoplast

segregation and in stage V the two disks are fully separated with the same morphology in stage I

(Gluenz et al., 2011). Additionally, there are differences between kDNA disk rotational dynamics

between T. brucei and Crithidia relative to the antipodal sites (Pérez-Morga and Englund, 1993).

In C. fasiculata, progeny minicircles accumulate around the periphery of the kDNA disk according

to networks pulse-labeled with [3H] thymidine while newly replicated minicircles in T. brucei are

detected antipodal 180o apart from each other. Additional experiments using fluorescent

methods have culminated into a model that while the C. fasiculata kDNA network rotates relative

to the antipodal sites during replication, the T. brucei network oscillates (Liu and Englund, 2007).

1.5 Mechanistic insight into kDNA replication Mechanistic and spatial considerations for replication differ between maxicircles and minicircles,

and much more is known about the latter scheme. Maxicircles are replicated while still catenated

to both minicircles and other maxicircles and were originally thought to undergo a rolling circle

replication scheme (Hajduk et al., 1984). Follow-up studies introducing interstrand DNA

11

crosslinks with trixosalen (4,5’,8-trimethylpsoralen) in vivo with decatenation by TopoIIMT or

restriction enzyme digestion and visualized via electron microscopy showed that in both Crithidia

and T. brucei maxicircles undergo unidirectional theta structure replication (Carpenter and

Englund, 1995). While eight bacterial PIF-like helicase enzymes were discovered (six being

mitochondrial), one of these, PIF2, was shown to be integral in regulating maxicircles where

induced RNAi of PIF2 resulted in complete loss of maxicircles and little effect on minicircles after

one day and PIF2 overexpression resulted in a three-fold to six-fold increase in the total number

of maxicircles after one day (Liu et al., 2009). Furthermore, PIF2 overexpression impacted kDNA

segregation where cells had a heterogenous mix of small or large kDNA and induction of a

catalytically inactive PIF2 reduced maxicircle numbers. DNA replication initiation requires a

primase to synthesize a short RNA primer and two kinestoplast-specific primers, PRI1 and PRI2,

have been discovered (Hines and Ray, 2010; Hines and Ray, 2011). PRI1 RNAi predominantly

depleted maxicircle content and its transcript expression was found to be cyclically regulated

with its peak corresponding to kDNA S phase, suggesting that this is the maxicircle primase. A

maxicircle-specific replicase has not yet been discovered. Post-replication, maxicircles

concentrate in the center of 2n kDNA just prior to network division (Carpenter and Englund, 1995;

Hoeijmakers and Weijers, 1980). As the two daughter networks begin to segregate, a thread of

maxicircles identified by fluorescence in situ hybridization (FISH) and a concentration of basic

proteins creates a structure called the nabelschnur (Gluenz et al., 2007; Gluenz et al., 2011).

TopoII is likely responsible for cleaving the maxicircle threads and unlinking the daughter

networks and a leucine aminopeptidase called LAP1 may act on proteins located at the

nabelschnur to complete segregation (Peña-Diaz et al., 2017).

12

Minicircle replication is initiated by release from the network as covalently closed (CC) monomers

mediated by a topoisomerase II (Drew and Englund, 2001; Englund, 1979). These free minicircles

are localized by unknown mechanisms to a region between the kDNA network and the

mitochondrial membrane closest to the flagellar basal body, called the kinetoflagellar zone (KFZ)

where minicircles undergo unidirectional replication by theta structure intermediates resolved

buy topoisomerase IA (TOPIA) (Scocca and Shapiro, 2008). Both progeny minicircles contain gaps,

where the leading strand molecule contains a single gap and the lagging strand molecule contains

multiple gaps between Okazaki fragments, collectively called nicked/gapped molecules or

species (N/G) (Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987; Kitchin et al., 1984; Kitchin

et al., 1985) N/G molecules migrate to two antipodal sites (AS) flanking the kDNA disk where

they are thought to undergo primer removal and the multiply-gapped species undergo some

repair by structure-specific endonuclease 1 (SSE1) (Engel and Ray, 1999) Here, distribution of

replicated minicircles in the growing network differs between T. brucei and Crithidia and other

related kinetoplastid organisms. Replicated minicircles in T. brucei distribute in a polar manner,

accumulating at the AS while in Crithidia and other organisms, replicated minicircles are

uniformly distributed around the network periphery (Pérez-Morga and Englund, 1993a). Once

minicircle replication is completed with each still containing a single gap (perhaps as a counting

mechanism), the gaps are subsequently repaired and the network divides (Pérez-Morga and

Englund, 1993a). How this final step is done is not well understood but is likely carried out by a

topoisomerase II specific unlinking of the minicircles along the scission line of network division

(Morris et al., 2001).

13

How minicircle release, replication, and reattachment is carried out mechanistically is relatively

well-studied compared to knowledge on maxicircle replication. Covalently closed minicircles are

released from the network into the KFZ by a topoisomerase. TopoIIMT from T. brucei was

discovered and cloned in 1990 and has 66% identity with C. fasiculata mtTopoII which localized

to the AP sites flanking the kDNA disk (Melendy and Ray, 1989; Melendy et al., 1988; Strauss and

Wang, 1990). Further studies on T. brucei TopoIIMT resulted in parasite inhibition and shrinking

to eventual loss of the kDNA network (dyskinetoplasty) due to inefficient attachment of

minicircles to the network (Wang and Englund, 2001; Wang et al., 2000). Replication is initiated

in the KFZ by the universal minicircle sequence binding protein (UMSBP) and p38, a high

abundance protein discovered in a proteomics assay. UMSBP binds to both the universal

minicircle sequence (which is part of the origin of replication) and the complementary strand

hexamer (Abu-Elneel et al., 1999). It is not known if binding to the hexamer is required for

initiation or provides some other function. Crithidia contain a single UMSBP protein while T.

brucei have two where knockdown of either inhibits minicircle replication initiation, daughter

network segregation, and blocks nuclear division (Milman et al., 2007). RNAi knockdown of p38

results in loss of kDNA and the accumulation of fraction S, a species composed of highly

underwound DNA containing regions of Z-DNA that compensate for extreme unwinding. p38 also

binds to the UMS and complementary hexamer (Liu et al., 2006).

Following initiation, replication is conducted by a number of DNA helicases (6), primases (2), and

polymerases (6). Unlike most other eukaryotic organisms which have one or two enzymes for a

14

specific function, trypanosomes have multiple proteins with similarity in conserved sequence and

activities but different functions. Of the six known kDNA helicase, three have been shown to be

essential (PIF1, PIF2, PIF8). PIF1 RNAi results in the accumulation of a smear of minicircle dimers

known as fraction U which is also formed after RNAi of POLIB and topoIIMT (Liu et al., 2010). PIF2

regulates maxicircle replication and PIF8 is so divergent it is not predicted to have helicase activity

but may have an indirect role in kDNA synthesis (Liu et al., 2009; Wang et al., 2012). Maxicircles

are likely primed by PRI1 due to RNA knockdown displaying rapid loss of maxicircles and slow loss

of minicircle DNA, whereas PRI2 knockdown results in an increase in covalently closed minicircles

and a decrease in replicated N/G molecules, providing strong evidence that it is the minicircle

primase (Hines and Ray, 2010; Hines and Ray, 2011). Additionally, a protein identified as p93

localized to AS and RNAi resulted in loss of N/G replication intermediates, suggesting a role in

minicircle replication (Li et al., 2007).

Of the six identified kDNA polymerases, four are related to bacterial DNA Pol I (repair and Okazaki

fragment processing) and two are related to eukaryotic DNA Pol β (base excision repair and other

maintenance). An additional DNA pol in T. cruzi is related to Pol κ (translesion synthesis).

Distinctly lacking from this list is a DNA polymerase related to the canonical replicative DNA

polymerase found in most other eukaryotic mitochondria, Pol γ. RNAi studies in T. brucei suggest

that three of the four Pol I-like proteins, POLIB, POLIC, and POLID, are essential for kDNA

replication and POLIB and POLID may be replicative kDNA polymerases (POLIA showed no loss of

fitness upon RNAi induction and is likely involved in kDNA repair) (Bruhn et al., 2010; Bruhn et

al., 2011; Chandler et al., 2008; Klingbeil et al., 2002). The two Pol β-like proteins are likely

15

involved in gap-filling replicated minicircles. Pol β localizes to AP sites and repairs gaps prior to

reattachment to the network with ligase kβ (Downey et al., 2005; Saxowsky et al., 2003). Pol β-

PAK, named for its N-terminal extension rich in prolines, alanines, and lysines, and works with

ligase kα to repair final gaps at the end of kDNA replication.

1.6 Segregation of kDNA networks Once kDNA replication is complete the TAC provides the physical linkage of daughter kDNA

networks to their respective basal body/pro-basal body pairs and drives genome segregation.

One additional function of the TAC is the proper positioning of the kDNA disks to the posterior

region of the mitochondrial organelle (Jakob et al., 2016). A hallmark study utilizing electron

microscopy detailed the morphology of the TAC by defining three TAC subdomains (Ogbadoyi et

al., 2003). Exclusion-zone filaments (EZFs) extend from the proximal end of the basal body to the

mitochondrial membrane and are named so due to the appearance of an electron-dense and

ribosome-free region in the cytoplasm. These filaments connect to the mitochondrial membrane

in an area that lacks cristae and is resistant to detergent extraction, called the differentiated

membrane (DM). Finally, a packed mass of filaments called unilateral filaments (ULF) reach out

from the DM and directly connect to one side of the kDNA disk. Further studies show that the

ULFs closest to the kDNA disk are made of DNA and basic proteins, while the ULFs closest to the

DM are free of DNA and are more acidic (Gluenz et al., 2007). A table of currently known TAC

components can be found in Table 1.2.

The cytological event in kDNA S phase that distinguishes the end of replication and beginning of

segregation are when two full-size disks are detected but still connected by the nabelschnur.

16

What proteins or signals that contribute to this transition stage are not well understood but

partial overlap between the KFZ and ULF regions is a likely area where proteins involved in this

transition may be located. Two proteins that may be involved are MiRF172 (mitochondrial

replication factor) which localizes between the kDNA disk and mitochondrial membrane and

could be involved in the reattachment of minicircles, and LAP1 (leucine aminopeptidase) which

localizes to the nabelschnur and is required for proper segregation (Amodeo et al., 2018; Peña-

Diaz et al., 2017). TAC102 is a ULF protein and is the most kDNA-proximal TAC protein discovered

which makes it the best marker currently to identify potential interactions between kDNA

replication (driven by the replication machinery) and segregation (driven by the TAC machinery)

(Trikin et al., 2016).

How kDNA replication and segregation are coordinated is not a well understood process. Proper

segregation requires the correct distribution of two fully-replicated kDNA networks to be

correctly positioned and distributed in the cell prior to cytokinesis. A distribution defect would

result in a cell displaying unequal network content incorrectly positioned and could be due to the

replicated kDNA network improperly positioned for the scission event into the two daughter

networks. This would lead to asymmetric division of two kDNA networks of different sizes in the

same cell. When one of the kDNA polymerases, POLIC is experimentally depleted, it displays a

pleiotropic phenotype as both a replication and distribution defect. This phenotype is unusual

because it is not a classical replication defect showing complete loss of kDNA or a classical

segregation defect that typically results in one cell containing a double-sized network and one

that lacks kDNA. It is possible that this enzyme may be multifunctional.

17

1.7 Goal of this study Genetic studies indicate that two kDNA polymerases, POLIB and POLID, have distinct functions in

kDNA replication. The resulting RNAi phenotype and localization data of POLIC suggest an

additional role involved in kDNA distribution. We aimed to identify these two potential functions

of POLIC by utilizing a rationally designed structure-function approach based on conserved

sequence data. Here, we address three major questions:

1. What is the cell cycle dependent localization pattern of POLIC in relation to TAC marker,

TAC102?

2. Are the conserved POLA residues essential for kDNA replication or distribution?

3. Does the conserved POLA domain or the large uncharacterized region of POLIC have essential

functions in kDNA replication or segregation?

1.8 Significance and contribution of this study The single mitochondrial nucleoid of Trypanosoma brucei offer a unique opportunity to study

mitochondrial biology in ways that are not amenable in more classical systems. This, in

conjunction with the public health concerns of this disease-causing organism make it an

attractive organism to study its unique biological properties. Replication and segregation of kDNA

are two processes that have always been studied in their own silos, yet some coordination must

exist to bridge these two processes and provide appropriate distribution of replicated kDNA

networks in the cell. This study demonstrates a rigorous interrogation of kDNA polymerase IC

and finds a unique role for a DNA polymerase that has not been seen before: notably a role in

genome distribution and provides additional mechanistic insight into the linking of kDNA

replication and segregation functions.

18

Protein Suggested Function

Localization Species References

p38 Minicircle replication initiation

AS PCF T. brucei (Liu et al., 2006)

p93 Minicircle replication

AS PCF T. brucei (Li et al., 2007)

TOPOIImt Minicircle dimer resolution, reattachment at AP sites, mending holes

AS C. fasiculata, PCF T. brucei

(Lindsay et al., 2008; Melendy and Ray, 1989; Melendy et al., 1988)

TOPIAmt Late theta structure resolution

AS (cell cycle-dependent)

PCF T. brucei (Scocca and Shapiro, 2008)

PRI1 Maxicircle primase

AS PCF T. brucei (Hines and Ray, 2010)

PRI2 Minicircle primase

AS PCF T. brucei (Hines and Ray, 2011)

Pol β Okazaki fragment repair

AS C. fasiculata, PCF T. brucei

(Saxowsky et al., 2002; Saxowsky et al., 2003)

UMSBP Minicircle replication initiation

KFZ (two foci) C. fasiculata, PCF T. brucei

(Abu-Elneel et al., 1999; Abu-Elneel et al., 2001; Milman et al., 2007)

KAP2, p18 Histone H1-like, condense kDNA

kDNA disk C. fasiculata (Xu, Hines, Engel, Russell, Ray, 1996) KAP3, p17

KAP4, p16 Edges of disk SSE1 RNA primer

removal AS C. fasiculata (Engel and Ray, 1999)

PIF1 Minicircle helicase

AS PCF T. brucei (Liu et al., 2009; Liu et al., 2010)

PIF2 Maxicircle helicase

Matrix (overexpressed)

PCF T. brucei (Liu et al., 2009)

PIF4 nd kDNA disk PCF T. brucei PIF5 nd AS PCF T. brucei PIF7 nd kDNA disk PCF T. brucei PIF8 kDNA

segregation, structure

Distal face of kDNA disk

PCF T. brucei (Liu et al., 2009; Wang et al., 2012)

POLIA Repair and maintenance

Matrix PCF T. brucei (Klingbeil et al., 2002)

POLIB Replicative DNA polymerase

KFZ (two foci) PCF and BSF T. brucei

(Bruhn et al., 2010; Bruhn et al., 2011; Klingbeil et al., 2002)

POLIC nd KFZ (cell cycle-dependent)

PCF and BSF T. brucei

(Bruhn et al., 2011; Concepción-Acevedo et al., 2018; Klingbeil et al., 2002)

POLID Replicative DNA polymerase

AS (cell cycle-dependent)

PCF and BSF T. brucei

(Bruhn et al., 2011; Chandler et al., 2008;

19

Concepción-Acevedo et al., 2012; Klingbeil et al., 2002)

POL β-PAK Repair of N/G kDNA disk (Saxowsky et al., 2003)

Pol κ Homologous recombination, translesion synthesis

AS T. cruzi (Rajão et al., 2009)

ligase kα Repair of N/G kDNA disk (two foci)

PCF T. brucei (Downey et al., 2005)

ligase kβ Base excision repair

AS PCF T. brucei

Table 1.1 Known kDNA replication and maintenance proteins. Nd = not determined.

20

Protein Suggested Function Localization Species References

Mab22 antigen Unknown EZF BSF T. brucei (Bonhivers et al., 2008)

AEP-1 kDNA maintenance ULF (IM) BSF T. brucei (Ochsenreiter et al., 2008)

p166 Unknown IMM PCF T. brucei (Zhao et al., 2008)

p197 kDNA segregation EZF PCF T. brucei (Gheiratmand et al., 2013)

α-KDE2 kDNA segregation (dual function)

ULF matrix BSF T. brucei (Sykes and Hajduk, 2013)

TBCCD1 Structural subunit EZF PCF T. brucei (Andre et al., 2013)

TAC65 Complex with pATOM36

EZF PCF and BSF T. brucei

(Käser et al., 2016)

TAC102 Connects to DM ULF PCF and BSF T. brucei

(Trikin et al., 2016)

TAC40 Complex with TAC42, TAC60

OMM PCF and BSF T. brucei



OMM PCF and BSF T. brucei



DM (OM) PCF and BSF T. brucei


BBA4 antigen Lines flagellar basal body

EZF PCF T. brucei (Woods et al., 1989)

pATOM36 Mitochondrial import DM (OM) PCF and BSF T. brucei

(Käser et al., 2016; Vitali et al., 2018)

Table 1.2 Known TAC proteins.

21

Figure 1.1: Illustration of microscopic analysis of haematozoa in the blood of the cow, Bruce 1895. Republished under the Creative Commons, Public Domain Mark.

22

Figure 1.2: Localization of kDNA replication and TAC proteins.

23

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CHAPTER 2

2. INVESTIGATION OF POLIC CELL CYCLE LOCALIZATION IN RELATION TO TRIPARTITE ATTACHMENT MARKER, TAC102

2.1 Abstract The unique catenated mitochondrial DNA of Trypanosoma brucei is replicated once per cell cycle

in near synchrony with nuclear S phase. This process requires coordination of over 30 identified

kDNA replication proteins which include kDNA polymerases: POLIB, POLIC, and POLID. For most

of these proteins, how they dynamically localize throughout the cell cycle and how their functions

are coordinated during replication is not well understood. POLID has been previously reported

to undergo cell cycle dependent localization coupled to kDNA replication events and its role is

well established. Contrary to this, the function of POLIC is not clear and its cell cycle dependent

localization is not known. Here, we show that POLIC localization changes throughout kDNA S

phase and in order to look for potential interactions with kDNA segregation machinery we utilize

structured illumination microscopy to follow localization dynamics with a TAC marker, TAC102.

Additionally, we show that kDNA replication proteins POLIB and POLID do not affect TAC102

localization further reinforcing them as strictly kDNA replication proteins. Taken together, POLIC

has cell cycle dependent localization and may be involved in kDNA transactions beyond a purely

replicative role, unlike POLIB or POLID.

2.2 Introduction 2.2.1 Widefield fluorescence microscopy and its limitations Fluorescence microscopy allows biologists to interrogate microorganism and subcellular

structures through fluorophore detection in a non-destructive way. The power of this tool is

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perhaps best exemplified by the discovery of one fluorophore, GFP, which led to the Nobel prize

in Chemistry in 2008 awarded to Osamu Shimomura and two American scientists, Martin Chalfie

and Robert Y. Tsien for its development (Sanderson, 2008). A large range of fluorescent dyes are

now available ranging from near infrared to ultraviolet and proteins can be labeled with dyes that

provide high sensitivity, specificity, and contrast (Fernández-Suárez and Ting, 2008). A sample

labeled with a fluorophore will absorb and emit light at a longer wavelength if illuminated with

the proper excitation wavelength. This basic principle of fluorescence is summarized in the

simplified Jablonski diagram (Fig 2.1). Repeated transitions from the ground state to the excited

state cause photon-induced chemical damage to the fluorophore through a process called

photobleaching, a potential limiting factor to the imaging capabilities of the experiment

(Ghauharali and Brakenhoff, 2000). A typical fluorescence microscope will utilize homogenous

light at a specific emission wavelength to illuminate the sample and excited light is filtered

through an emission filter so that a camera captures solely the fluorescence intensity. How a

digital camera converts the detection of incident light on its sensor to an electronic signal relies

on a linear relationship between the amount of light detected and the signal output. In

conjunction with this linearity, fluorescence recorded by the camera can be modeled as the

convolution of the fluorescence distribution with a point spread function (PSF) corresponding to

the image of a single fluorophore.

Conventional widefield microscopy uniformly illuminates a sample. This allows out of focus light

to pass to the detector which contributes to image degradation in the form of blur, as the

constructed image contains the fluorescence intensity of out of focus light from points originating

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above or below the focal plane in addition to the in-focus light. The predominant source of blur

is light diffraction and image resolution is considered diffraction-limited when this is thought to

be the only source. Blur is the nonrandom spreading of light as it passes through the optical path

of the imaging system (primarily through the objective lens) and because it is nonrandom, it can

be mathematically modelled and employed through image deconvolution to reduce or remove

its effects. A single point source of light cannot be perfectly imaged in three dimensions but

instead appears as a three-dimensional diffraction pattern, creating the point spread function

(PSF) which in widefield microscopy appears as an oblong vertical ellipse with flares of concentric

rings. To address this issue, one can mitigate out of focus light by employing a physical solution

via confocal microscopy utilizing a pinhole, or by mathematics via deconvolution. Deconvolution

reconstructs an image to remove the blurring of an image originating from the convolution of the

point objects with the known or estimated PSF and there are multiple mathematical methods to

achieve this (Sarder and Nehorai, 2006).

2.2.2 Principles of super-resolution microscopy Despite the ability to utilize image deconvolution by defining the point spread function of the

instrument (either through mathematical modeling or empirically using fluorescent beads to

capture a three-dimensional image), standard widefield fluorescent microscopes are at best

capable of achieving spatial resolution of 200 nm lateral (x-y) and 500 nm axial (z) (Yamanaka et

al., 2014). To overcome the resolution limitations of widefield fluorescent microscopy, a number

of superresolution techniques have been developed to observe otherwise unresolvable biological

structures. Confocal microscopy is one of the earliest approaches to reduce out-of-focus blur by

using a pinhole to filter out light coming from the background away from the focal plane.

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Information is collected by a single laser rasterizing through the volume of the sample and

improved technology has built further on this principle with multiple beam scanning (spinning

disk) and more advanced detection systems using synthetic pinholes that can improve axial

resolution down to 400 nm.

Other superresolution techniques that break the diffraction limit include stimulated emission and

depletion (STED), single molecule localization superresolution such as photoactivation

localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), and

structured illumination microscopy (SIM) (Betzig et al., 2006; Ghauharali and Brakenhoff, 2000;

Gustafsson, 2000; Hell and Kroug, 1995; Hess et al., 2006; Rust et al., 2006). STED uses a

combination of two lasers: an excitation beam to excite fluorophores and a donut-shaped

depletion beam to decrease the effective diameter of the emission area, ultimately providing

increased resolution without any image processing. While the spatial resolution is dependent on

the intensity of the depletion beam, photobleaching is a heightened issue. STORM and PALM

widefield techniques utilize sophisticated single-molecule emitters that change their emission

properties based on light irradiation with a low energy laser. Hundreds of thousands of images

are recorded using photoswitchable or blinking fluorophores, and a final image is produced from

the precise calculation of each single molecule emission using intensive data processing. Unlike

confocal or STED, SIM is a widefield technique that requires few numbers of acquisitions and can

utilize conventional probes unlike PALM/STORM. The drawback is that SIM resolution is limited

to approximately 100 nm while PALM/STORM can reach 20 nm resolution. Each technique has

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advantages and drawbacks related to experimental setup, acquisition speed, photobleaching,

complex data processing and spatial resolution.

2.2.3 Principles of Structured Illumination Microscopy SIM improves the capabilities of widefield fluorescence imaging by illuminating the sample with

multiple acquisitions from non-uniform patterns that are combined to improve axial and lateral

resolution. Despite the diffraction limit of widefield fluorescence optics, principles of frequency

information in images can be utilized to extract additional information from a sample. Structural

information is commonly visualized as a matrix with each value having an intensity value (0, 1 for

black and white; 0-255 for grayscale; 0-255 in a color spectrum, i.e. RBG). However, spatial

information can also be described as a combination of sine waves modulating in amplitude

through a so-called Fourier Transform (FT), and the PSF of the microscope in real space is

converted to the optical transfer function (OTF) in Fourier space. Visible structures in a sample

are in a specific frequency range while very low or high frequency information is not visible. In

particular, very small structures beyond the diffraction limit (less than 200 nm laterally) are

represented by very high frequencies. In order to extract this high frequency information, a SIM

microscope will capture the same image with a grid pattern of illumination at varying angles and

deconvolve (perform a reverse FT) these patterns of light to reconstruct a final image with

additional high spatial-frequency (beyond diffraction-limited) information, effectively doubling

the resolution (Gustafsson, 2000).

39

2.2.3 Cell cycle dependent localization of kDNA-associated proteins Utilizing traditional widefield microscopy, cell cycle localization of kDNA replication protein POLID

revealed that it forms foci flanking the kDNA during kDNA S phase but is present throughout the

mitochondrial matrix at other cell cycle stages (Concepción-Acevedo et al., 2012). Foci formation

was striking enough with these methods where fluorescence intensity increased four-fold over

what was seen in the matrix. For proteins that localize at/around the kDNA or in the KFZ region,

it can be impossible to resolve additional structural information.

Superresolution has previously been used to analyze protein localization at the kDNA at more

detail. Localization of MiRF172 at the kDNA disk during the cell cycle at widefield fluorescence

resolution was probed in greater detail with STED. 3D reconstructions of 1N1K disks revealed

curved structures along the disk perimeter facing the KFZ which helped further support its role

in minicircle reattachment (Amodeo et al., 2018). α-KDE2 is a multifunctional protein that

supports kDNA replication, and 3D reconstructions resolved two discrete points around the kDNA

from detergent-treated cells (Sykes and Hajduk, 2013). Lastly, TAC102 is a ULF component that

was determined to localize to the mitochondrion using STED (Trikin et al., 2016).

Previous work on POLIC demonstrated a less severe kDNA replication defect compared to the

striking defects of POLIB or POLID depletion (Bruhn et al., 2010; Chandler et al., 2008; Klingbeil

et al., 2002). Additionally, non-replication defects that were detected during depletion suggest

that it may have a role additional to replication. Therefore, we utilized a single expresser cell line

where one POLIC allele was knocked out and the other was endogenously tagged to observe

kDNA S phase localization and compare it to a known TAC marker most proximal to the kDNA:

40

TAC102. Here, we demonstrate that increased resolution utilizing SIM shows a more refined

kDNA S phase localization pattern and the potential for transient interactions with TAC102. These

data taken together suggest POLIC as an additional role distinct from kDNA replication.

2.3 Materials and Methods

2.3.1 DNA constructs.

2.3.1.1 POLIC Knockout

For generation of pKOPOLICPuro, a 430–base pair TbPOLIC 5ʹ UTR fragment was PCR amplified

and ligated into XhoI and HindIII sites in the upstream polylinker of the pKOPuro vector (Lamb et

al., 2001). Subsequently, a 448–base pair TbPOLIC 3ʹ UTR fragment was PCR amplified and ligated

into SpeI and XbaI sites in the downstream polylinker portion of pKOPuro to generate

pKOPOLICPuro. The puromycin resistance cassette from pKOPOLICPuro was replaced with a

blasticidin cassette (AscI/PacI fragment) from pKOBSR to generate pKOPOLICBSR.

2.3.1.2 Allelic tagging

pPOLIC-PTP-PURO was generated as previously described (Bruhn et al., 2010). For generation of

pPOLIC-PTPNEO, the TbPOLIC C-terminal coding sequence (2226 base pairs) was PCR amplified

from the T. brucei genomic DNA and ligated into the ApaI and NotI restriction sites of the pC-PTP-

NEO vector (Schimanski et al., 2005). pPOLID-PTP-NEO was generated as previously described

(Concepción-Acevedo et al., 2012)

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2.3.2 Trypanosome cell culture and transfection

For POLIC-PTP–tagged cells, 427 wild-type cells were transfected with XhoI/XbaI-digested

pKOPOLICPuro (15 μg). Stable transfectants were selected with 1 μg/ml puromycin (Puro)

followed by limiting dilution as described previously (Klingbeil et al., 2002). Southern blot

analyses confirmed single-allele deletion in clonal cells. Clonal cell line P1A8 expressing a single

TbPOLIC allele was then transfected with AatI-linearized pPOLIC-PTP-NEO. POLICKOPuro/IC-PTP

cells were selected in media containing 1 μg/ml Puro and 50 μg/ml G418. After limiting dilution

of POLICKOPuro/IC-PTP cells, POLIC-PTP expression and localization, cell growth, and kDNA mor-

phology were monitored in three individual clones (P2C2, P2A1, and P2C1). The average doubling

times were 12 h, proper chromosomal integrations and no defects in kDNA morphology were

detected (unpublished data). Data presented in this study correspond to clonal cell line

POLICKOPuro/IC-PTP P2C1, which we named TbIC-PTP. For RNAi transfections, 10 µg of linearized

pSLDB was introduced by electroporation, stable transfectants were subsequently selected with

2.5 µg/ml phleomycin (Invivogen), and clonal cell lines obtained by limiting dilution described

previously (Chandler et al., 2008). The data presented in this study correspond to clonal cell line

SLDB 1A10, which we named SLDB10. To induce for RNAi, growth medium was supplemented

with tetracycline (1 µg/mL).

2.3.3 Widefield Immunofluorescence Cells were harvested, washed, resuspended in phosphate-buffered saline (PBS), and adhered to

poly-l-lysine (1:10)-coated slides for 5 min. Cells were then fixed in 4% paraformaldehyde (5 min)

and washed three times (5 min) in PBS containing 0.1 M glycine (pH 7.4). Cells were permeabilized

42

(0.1% Triton X-100 in PBS, 5 min) and washed in PBS three times (5 min). PTP-tagged proteins

were detected with anti–protein A serum (1 h, Sigma, 1:3000) followed by Alexa Fluor 594 goat

anti-rabbit (1 h, 1:250). POLIC-HA was detected with anti-HA 3F10 (1 h, Roche, 1:100) followed

by Alexa Fluor 594 goat anti-rat (1 h, 1:100). Detection of bbs (YL1/2) and DNA (DAPI) was

described previously (Concepción-Acevedo et al., 2012). It should be noted that YL1/2 recognizes

RP2, a protein that localizes to transitional fibers and is therefore a marker only for mature bbs

(Andre et al., 2014; Harmer et al., 2017). Slides were then washed three times in PBS before being

mounted in Vectashield (Vector Laboratories).

2.3.4 Structured Illumination Microscopy

Cells were harvested, washed, resuspended in phosphate-buffered saline (PBS), and adhered to

poly-l-lysine (1:10)-coated slides for 5 min. Cells were then fixed in 4% paraformaldehyde (5 min)

and washed three times (5 min) in PBS containing 0.1 M glycine (pH 7.4). Cells were permeabilized

(0.1% Triton X-100 in PBS, 5 min) and washed in PBS three times (5 min). Antibody concentrations

were as follows: anti–protein A (1:3000), YL1/2 (1:3000), TAC102 (1:10,000) and Alexa Fluor 647

goat α-mouse 1:1000, Alexa Fluor 488 goat α-rabbit 1:250, and Alexa Fluor 546 goat α-rat 1:400

secondary. Slides were then washed three times in PBS before being mounted in Vectashield

(Vector Laboratories). Three-dimensional SIM (3D-SIM) was performed using a Nikon N-SIM E

superresolution microscope equipped with an ORCA-Flash 4.0 sCMOS camera (Hamamatsu

Photonics K.K.) and a CFI SR Apochromat TIRF 100× oil-objective (NA 1.49) lens. Z-stacks (6 μm,

240 nm thickness each) were acquired using the NIS-Elements Ar software.

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2.3.5 Three-dimensional reconstruction and projection

Image slices were reconstructed default software parameters and deconvolved using the

Automatic method in NSIM modality. The 3D SIM videos display a top-down x,y coordinate view,

then rotate along the y-axis to show three-dimensionality on the x-axis and z-axis, and finally

along various transverse rotations for a complete 360° view. Videos were created using the Movie

Maker option in the NIS-Elements Ar software at 50% volume zoom, high-resolution setting and

were projected using alpha blending mode.

2.4 Results

2.4.1 Cell cycle analysis of POLIC and TAC102 by structured illumination microscopy

TAC102 is a core protein of the TAC that is essential for proper kDNA segregation. The protein is

detected throughout the cell cycle in the ULF zone and is assembled de novo into the TAC shortly

after bb duplication (Trikin et al., 2016). Electron microscopy (EM) cytochemistry defined inner

and outer ULF domains (Gluenz et al., 2007). It is possible that subdomains exist within the ULF

zone for protein localization; one that occupies replication processes and others that occupy

structural or other related processes. Although the precise role of POLIC in kDNA maintenance is

not known, the presence of ancillary DNA (mislocalized kinetoplast-derived DNA; Miyahira and

Dvorak, 1994) during POLIC RNA interference (RNAi) suggests a role in kDNA segregation.

Traditional widefield microscopy displayed possible co-localization between POLIC and TAC102,

especially during later stages of kDNA S phase (Figure 2.2). However, the limited resolution made

this difficult to determine.

44

To investigate whether POLIC-PTP colocalizes with a known ULF marker TAC102, we used high-

resolution structured illumination microscopy (SIM). TAC102 signal was always detected in the

ULF zone, closely resembling the previously published pattern (Trikin et al., 2016), and POLIC-PTP

signal was detected only during kDNA synthesis phases, as described earlier. In three-dimensional

(3D) reconstructions of cells at stage IIa or IIb of the kDNA duplication cycle, POLIC-PTP and

TAC102 signals rarely overlapped with representative cells shown in Figure 2.4 and linear

intensity profiles (Figure 2.4). POLIC-PTP pattern varied the most at stage IIa, appearing as a single

focus or more diffuse signal patterns that showed varying overlap with the TAC102 signal (Figure

2.3A). Three clearly defined localization patterns were detected for stage IIb cells that correlated

with the transition through kDNA synthesis as measured by inter-bb distance (Figure 2.3A):

POLIC-PTP foci localized only to the antipodal sites with no TAC102 overlap; POLIC-PTP signal

appeared mainly at the antipodal sites with signal detected between the foci in the ULF that

overlapped with TAC102; and POLIC-PTP signal started to become less focused, taking on a

crescent shape that localized adjacent to TAC102 in the ULF zone. These data demonstrate that

the great majority of POLIC-PTP signal does not overlap with TAC102 signal even when both

proteins localize to the KFZ/ULF zone (Figure 2.3B).

2.5 Discussion

The increased resolution of SIM provided a more refined localization pattern for POLIC with

respect to a known ULF zone marker, TAC102 (Figure 2.3). During early kDNA replication (stage

IIa), POLIC may transiently interact with TAC102 or other TAC proteins to facilitate POLIC activity.

As kDNA synthesis proceeds (stage IIb), the POLIC association with newly synthesized DNA at the

45

antipodal sites appears to shift back to the KFZ/ULF zone, but not with TAC102 association,

suggesting that sub-complexes of proteins likely exist in the KFZ/ULF zone. Previously, RNAi of

POLIC resulted in smaller kDNA networks, perturbation of minicircle replication, and ancillary

DNA in ∼12% of the cells (Klingbeil et al., 2002). Accumulation of ancillary DNA (small extra kDNA

mislocalized to the anterior of the cell) is associated with kDNA segregation defects for TAC102

and PNT1, a cysteine peptidase, which supports the hypothesis that POLIC may have multiple

roles (Grewal et al., 2016; Trikin et al., 2016).

46

Figure 2.1: Simplified Jablonski Diagram. Energy levels of a fluorescent molecule are shown as horizontal black lines with energy increasing upwards in the diagram. Colored arrows represent energy transfer between molecular states. The blue arrow indicates the promotion of ground state to excited state by photon absorption. Excited molecules undergo internal conversion to a lower electronic state (downward-pointing black arrows). Lastly, the molecule undergoes a radiative transition to ground state and fluoresces, where the according to the Stokes-Shift where the fluorescence occurs at a longer wavelength than the absorption (green arrows).

47

Figure 2.2: Diagram of Structured Illumination Apparatus. Laser light is collimated onto a linear phase grating, producing diffraction orders -1, 0, and +1. These are refocused onto the back of the focal plane of the objective lens and intersect at the focal plane in the sample. This produces interference and generates an intensity pattern with lateral and axial structure. Emission light from the sample is observed by a charge-coupled device camera via diachromic mirror. Adapted from Gustafsson et al., 2008.

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Figure 2.3: Widefield fluorescence of IC-PTP and TAC102 during kDNA S phase. TbIC-PTP cells were labeled with anti–protein A (green), anti-TAC102 (red), YL1/2 (red), and DAPI (blue). Scale bar: 5 μm.

49

Figure 2.4: Three-dimensional SIM imaging of POLIC-PTP, TAC102, and basal bodies. TbIC-PTP cells were labeled with anti–protein A (green), anti-TAC102 (red), YL1/2 (white), and DAPI (blue). More than 20 cells were analyzed by 3D-SIM at each stage. Scale bar: 5 μm. (A) Representative images of POLIC-PTP, TAC102, and bb localization at stage IIa and stage IIb of kDNA replication. (B) Schematic model of localization of POLIC-PTP (green), TAC102 (red), and YL1/2 (gray) during stages IIa and IIb of kDNA replication.

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Figure 2.5: Linear intensity plots of kDNA disks. (A) Linear intensity plot for the Stage IIa representative sample. Plotted lines are TAC102 (red), YL ½ (black), IC-PTP (green) and DAPI (blue) passing through the kDNA disk either across the disk edge/KFZ interface (left plot) or transversing the disk face through the center of a single or pair of basal bodies (right plot). Arrow represents the line and direction of the corresponding intensity plot. Y-axis, arbitrary units for Intensity; X-axis, distance in microns. (B-D) Linear intensity plots for the Stage IIb representative images.

51

2.6 Bibliography

Amodeo, S., Jakob, M. and Ochsenreiter, T. (2018). Characterization of the novel mitochondrial genome replication factor MiRF172 in Trypanosoma brucei. J Cell Sci 131, jcs.211730. Andre, J., Kerry, L., Qi, X., Hawkins, E., Drižytė, K., Ginger, M. L. and McKean, P. G. (2014). An Alternative Model for the Role of RP2 Protein in Flagellum Assembly in the African Trypanosome. J Biol Chem 289, 464–475. Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, W. O., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. F. (2006). Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 313, 1642–1645. Bruhn, D. F., Mozeleski, B., Falkin, L. and Klingbeil, M. M. (2010). Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes. Mol Microbiol 75, 1414–1425. Chandler, J., Vandoros, A. V., Mozeleski, B. and Klingbeil, M. M. (2008). Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes▿. Eukaryot Cell 7, 2141–2146. Concepción-Acevedo, J., Luo, J. and Klingbeil, M. M. (2012). Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID. Eukaryot Cell 11, 844–855. Fernández-Suárez, M. and Ting, A. Y. (2008). Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Bio 9, 929–943. Ghauharali and Brakenhoff (2000). Fluorescence photobleaching-based image standardization for fluorescence microscopy. J Microsc-oxford 198, 88–100. Gluenz, E., Shaw, M. K. and Gull, K. (2007). Structural asymmetry and discrete nucleic acid subdomains in the Trypanosoma brucei kinetoplast. Mol Microbiol 64, 1529–1539. Grewal, J. S., McLuskey, K., Das, D., Myburgh, E., Wilkes, J., Brown, E., Lemgruber, L., Gould, M. K., Burchmore, R. J., Coombs, G. H., et al. (2016). PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast. J Biol Chem 291, 9492–9500. Gustafsson, M. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc-oxford 198, 82–87. Harmer, J., Qi, X., Toniolo, G., Patel, A., Shaw, H., Benson, F. E., Ginger, M. L. and McKean, P. G. (2017). Variation in Basal Body Localisation and Targeting of Trypanosome RP2 and FOR20 Proteins. Protist 168, 452–466.

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Hell, S. and Kroug, M. (1995). Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit. Appl Phys B 60, 495–497. Hess, S. T., Girirajan, T. and Mason, M. D. (2006). Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys J 91, 4258–4272. Klingbeil, M. M., Motyka, S. A. and Englund, P. T. (2002). Multiple Mitochondrial DNA Polymerases in Trypanosoma brucei. Mol Cell 10, 175–186. Lamb, J. R., Fu, V., Wirtz, E. and Bangs, J. D. (2001). Functional Analysis of the Trypanosomal AAA ProteinTbVCP with trans-Dominant ATP Hydrolysis Mutants. J Biol Chem 276, 21512–21520. Miyahira, Y. and Dvorak, J. A. (1994). Kinetoplastidae display naturally occurring ancillary DNA-containing structures. Mol Biochem Parasit 65, 339–349. Rust, M. J., Bates, M. and Zhuang, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793–796. Sanderson, K. (2008). Great glowing jellyfish! It’s the Nobel Prize in Chemistry. Nature. Schimanski, B., Nguyen, T. N. and Günzl, A. (2005). Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. Eukaryot Cell 4, 1942 1950. Sykes, S. E. and Hajduk, S. L. (2013). Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei. Eukaryot Cell 12, 78–90. Trikin, R., Doiron, N., Hoffmann, A., Haenni, B., Jakob, M., Schnaufer, A., Schimanski, B., Zuber, B. and Ochsenreiter, T. (2016). TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes. Plos Pathog 12, e1005586. Yamanaka, M., Smith, N. I. and Fujita, K. (2014). Introduction to super-resolution microscopy. Microsc Oxf Engl 63, 177–92.

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CHAPTER 3

3. DNA POLYMERASE IC IS CRITICAL FOR REPLICATION AND SEGREGATION OF MITOCHONDRIAL DNA IN THE AFRICAN

TRYPANOSOME TRYPANOSOMA BRUCEI. 3.1 Abstract The mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network

containing thousands of minicircles and tens of maxicircles called kinetoplast DNA (kDNA).

Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and

POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have

roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear.

Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC

domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-

terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA

content and cell cycle localization, active site point mutations in the POLA domain impaired

minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA

domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype.

Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization that

facilitates proper distribution of progeny networks. This is the first report of a DNA polymerase

that impacts mitochondrial nucleoid distribution.networks. This is the first report of a DNA

polymerase that impacts DNA segregation.

3.2 Introduction Maintenance of mitochondrial DNA (mtDNA) is vital for most eukaryotic cells. Accumulation of

mutations or failure to maintain copy number can result in loss of mtDNA- encoded proteins

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essential for energy generation and mitochondrial dysfunction that is associated with aging,

metabolic disease, neurodegenerative disorders, and cancer (Viscomi and Zeviani, 2017). As a

result, mtDNA maintenance is a focal point of biomedical research. Despite decades of

investigation, the mechanisms that regulate replication, copy number maintenance and

segregation of mammalian mtDNA still remain unclear.

Although many studies on mitochondrial biology have focused on a few eukaryotic lineages

(yeast and mammals), great diversity exists in mitochondrial genomes and proteomes across

eukaryotes (Chen and Butow, 2005; Gray, 2015; Zíková et al., 2016). Eukaryotic microbes

constitute much of this evolutionary diversity, and the parasitic protists of medical importance

are by far the most well-studied. One of the most intriguing and structurally complex

mitochondrial genomes is found in trypanosomatid protists such as Trypanosoma brucei, the

parasite responsible for African sleeping sickness. Trypanosomatids harbor a single

mitochondrion (often branched) and the mtDNA consists of two genetic elements, the minicircles

and maxicircles that are catenated into a network called kinetoplast DNA (kDNA) (Jensen and

Englund, 2012; Verner et al., 2015). In vivo, kDNA is condensed into a single disk-shaped nucleoid

always present near the flagellar basal body and is replicated once every cell cycle in near

synchrony with other single unit organelles, including the nucleus and flagellum (Woodward and

Gull, 1990). The topological complexity of kDNA provides a striking reporter system in which

lesions in the network and disruption of replication intermediates help to define mechanistic

defects and therefore represents a good model to study mtDNA dynamics.

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Variations on kDNA structure and the sizes of minicircles and maxicircles exist (for review see

Lukes et al., 2002). In T. brucei, the 23 kb maxicircles (approximately 25 copies) are considered

homologs to mammalian mtDNA as they contain similar protein-coding genes for respiratory

complex subunits and rRNAs. However, a majority of the maxicircle genes are cryptic and

undergo an extensive post-transcriptional RNA editing process of uridine insertion and deletion

to generate translatable mRNAs. This process is mediated by minicircle-encoded guide RNAs

(gRNAs) (for reviews see Read et al., 2016; Stuart et al., 2005). Therefore, both genetic elements

are essential for fitness and completion of the parasite’s digenetic life cycle (Dewar et al., 2018).

Moreover, the ~5000 minicircles (1 kb) constitute the majority of the network with each

minicircle Hopf-linked to three others (Chen et al., 1995). The maxicircles are linked to the

minicircles and are also likely interlocked with other maxicircles forming a network within a

network (Shapiro, 1993). How this complex structure is replicated and segregated are areas of

focused study (for reviews see Jensen and Englund, 2012; Povelones, 2014; Schneider and

Ochsenreiter, 2018).

One key feature is the topoisomerase II-mediated release of minicircle monomers to replicate

free of the network. Briefly, unreplicated covalently closed (CC) minicircles are released into a

region between the kDNA disk and mitochondrial membrane nearest the flagellar basal body, the

kinetoflagellar zone (KFZ), where initiation and synthesis occur via unidirectional theta

replication (Abu-Elneel et al., 2001; Melendy et al., 1988). Later stages such as Okazaki fragment

processing and topoisomerase-mediated attachment of minicircle progeny occur in two protein

assemblies located at opposite poles of the network periphery called antipodal sites (AS) (Hines

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et al., 2001; Melendy et al., 1988). Minicircle progeny are attached to the network while still

containing at least one nick or gap (N/G); a presumed counting mechanism that distinguishes

between replicated versus non-replicated minicircles (Ntambi et al., 1986). The process results in

a spatial and temporal separation of replication events. Maxicircles replicate via theta replication

within the network and progeny also contain N/G (Carpenter and Englund, 1995; Liu et al., 2009).

During the later stages of network replication the remaining nicks and gaps are repaired and the

network undergoes topological remodeling prior to network scission, distribution of equal

progeny networks and finally segregation into daughter cells.

A second key feature is the tripartite attachment complex (TAC) that physically links the kDNA

network to the basal body of the flagellum and mediates segregation of the progeny kDNA

networks (Ogbadoyi et al., 2003). The TAC is composed of three distinct regions: the exclusion

zone filaments (EZF) that extend from the basal body to the mitochondrial outer membrane, the

differentiated mitochondrial membrane (DM) located between the basal body and the kDNA

disk, and the unilateral filaments (ULF) that extend from the mitochondrial inner membrane to

the kDNA network (in the KFZ). Elegant electron microscopy (EM) cytochemistry studies defined

inner and outer ULF domains (Gluenz et al., 2007) that could represent subdomains; one that

occupies replication processes and others that occupy structural or segregation processes. Lastly,

several components of each region have been identified and genetic studies indicate a

hierarchical organization for TAC biogenesis starting with components that are most proximal to

the basal body (Hoffmann et al., 2018; Schneider and Ochsenreiter, 2018). One core TAC

component, TAC102, localizes to the ULF and is essential for proper kDNA segregation, although

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it does not directly interact with the kDNA network it is the most proximal ULF component (Jakob

et al., 2016; Trikin et al., 2016).

A third feature is the multiplicity of proteins with similar activities but non-redundant roles in

kDNA maintenance. Among the many additional factors not present in mammalian nucleoids are

2 topoisomerases, 2 origin-binding proteins, 2 primases, 6 helicases, 6 DNA polymerases, 2 ligases

and several other factors (Abu-Elneel et al., 2001; Downey et al., 2005; Lindsay et al., 2008; Liu

et al., 2006; Saxowsky et al., 2003; Scocca and Shapiro, 2008). For example, TbPIF2 helicase and

TbPRI1 primase have defined roles in maxicircle replication (Hines and Ray, 2010; Liu et al., 2009),

while TbPIF1 and TbPRI2 have roles in minicircle replication (Hines and Ray, 2011; Liu et al., 2010).

Three Family A DNA polymerase paralogs are required for T. brucei growth and kDNA

maintenance in both life cycle stages, the procyclic and bloodstream forms (PCF and BSF): POLIB,

POLIC, and POLID (Bruhn et al., 2010; Bruhn et al., 2011; Chandler et al., 2008; Klingbeil et al.,

2002). While POLIB and POLID have demonstrated roles in minicircle replication, the precise role

for POLIC in kDNA maintenance has not been determined.

Approximately 30 kDNA replication proteins have been characterized at the single protein level

and localize to specific sites surrounding the kDNA disk including the KFZ, the AS flanking the disk,

or within the disk. Given the unique spatiotemporal replication mechanism of the network,

several studies have recently uncovered a complex choreography of kDNA replication proteins

that likely coordinate subsets of proteins to specific sites during cell cycle progression (Abu-Elneel

et al., 2001; Concepción-Acevedo et al., 2012; 2018; Johnson and Englund, 1998; Peña-Diaz et al.,

58

2017). POLID is the most dramatic example of cell cycle localization in which the protein

colocalized with replicating minicircles at the AS only during kDNA S phase (1N1K*), and localized

in the mitochondrial matrix during all other cell cycle stages (Concepción-Acevedo et al., 2012).

Cell cycle stages are identified based on the single unit structures the nucleus (N) and kDNA (K),

thus 1N1K are cells in G1, 1N1K* are cells undergoing kDNA S phase, 1N2K cells have segregated

two progeny networks and are completing nuclear S phase and mitosis, and 2N2K cells have not

yet undergone cytokinesis (2N2K). Another kDNA polymerase, POLIC, is initially detected in the

KFZ/ULF, then accumulates at the AS only during kDNA S phase colocalizing with a fraction of

POLID, thus supporting the idea of highly transient kDNA interactions (Concepción-Acevedo et

al., 2018). The majority of the POLIC signal did not overlap with TAC102 signal. Additionally, when

both proteins localized to the KFZ/ULF region, POLIC was more proximal to the kDNA than

TAC102 (Concepción-Acevedo et al., 2018). What regulates the timing and recruitment

associated with the protein choreography remains unknown.

The minicircle kDNA polymerases POLIB and POLID contain both a predicted C-terminal Family A

DNA polymerase domain (POLA) and a 3’-5’ exonuclease domain. In contrast, POLIC is a 1649 aa

protein (180 kDa) with only a conserved C-terminal POLA domain (315 aa). The remainder of this

large protein is an uncharacterized N-terminal region we termed the UCR. Gene silencing of POLIC

resulted in loss of fitness (LOF), a decrease in both minicircle and maxicircle copy number, and

progressive loss of the kDNA network; hallmarks of a kDNA replication defect (Bruhn et al., 2010;

Chandler et al., 2008; Liu et al., 2010). However, its silencing also resulted in cells displaying a

predominant “small” kDNA phenotype rather than complete loss, an increase in the free

59

minicircle population, and the appearance of ancillary kDNA, extra-kDNA material (determined

via FISH) abnormally positioned anterior to the nucleus in addition to the normal positioned

kDNA network (Miyahira and Dvorak, 1994). Ancillary kDNA has been detected when other

mitochondrial proteins were silenced and presumed to be a secondary defect (Archer et al., 2009;

Clayton et al., 2011; Grewal et al., 2016; Trikin et al., 2016; Týč et al., 2015). The POLIC phenotype

is unusual by not displaying as a classical replication defect or as a classical segregation defect

that typically results in one cell containing a double-sized network and one that lacks kDNA. In

addition to decreased kDNA content, POLIC depletion appears to exhibit a distribution defect

where the kDNA network is not positioned properly for the scission event leading to asymmetric

division of the incompletely-replicated networks.

Therefore, the aim of this study was to apply a genetic complementation approach to dissect the

pleiotropic RNAi phenotype and further define the function of POLIC in kDNA maintenance. We

present data that POLIC has two functional domains that contribute to kDNA maintenance; one

for nucleotidyl incorporation and another that facilitates cell cycle localization and distribution

of the kDNA network. An inactive POLA domain causes a dominant negative effect and

exacerbates the kDNA replication defect of POLIC RNAi. Interestingly the UCR domain alone can

restore localization to the AS during kDNA S phase. Both domains appear to contribute to proper

segregation of kDNA. To our knowledge this is the first time a DNA polymerase is implicated in

the proper distribution of progeny mitochondrial nucleoids and provides the strongest evidence

thus far for interactions between the kDNA replication and segregation machineries.

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3.3 Materials and Methods 3.3.1 DNA constructs. 3.3.1.1 RNAi.

A pStL (stem-loop) vector for inducible gene silencing of POLIC was constructed as previously

described (Wang et al., 2000). Briefly, 517 bp of POLIC 3¢ UTR sequence was PCR amplified from

TREU 927 gDNA using primers listed in Table 3.1 to generate the two fragments for subsequent

cloning steps, The resulting vector, pStLIC3¢UTR was EcoRV-linearized for genome integration.

3.3.1.2 Site-directed mutagenesis.

Full-length wild-type POLIC (Tb927.7.3990) was PCR amplified from TREU 927 gDNA using

Phusion DNA polymerase and subcloned into the pCR®-Blunt II-TOPO vector (Invitrogen).

Mutation of 1380Asp and 1592Asp to Ala was performed by site-directed mutagenesis using the

QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent) generating pTOPO-IC-DEAD.

3.3.1.3 Inducible expression of POLIC variants. To create pLew100-PTPPuro for inducible expression of PTP tagged POLIC variants, the Myc tag of

pLew100-MycPuro (gift from Dr. Laurie Read, SUNY Buffalo) was replaced with the PTP tag. Briefly,

the PTP tag was PCR amplified from pC-PTP-NEO (Schimanski et al., 2005) and cloned into pCR®-

Blunt II-TOPO vector. Site-directed mutagenesis was used to introduce a silent mutation and

eliminate an internal BamHI site generating pTOPO-PTP-ΔBamHI. The Myc tag was replaced with

PTP-ΔBamHI to generate pLew100-PTPPuro. The full-length open reading frames of wild-type

POLIC and with the point mutations were cloned into pLew100-PTPPuro (pLewIC-WT-PTPPuro).

Gibson assembly was used to generate truncated versions of POLIC that retained only the C-

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terminal POLA domain or the N-terminal uncharacterized region (UCR). Briefly, PCR fragments

containing the predicted mitochondrial targeting sequence (MTS) (nucleotides 1-174), the POLA

fragment (nucleotides 3637-4947), and the UCR fragment (nucleotides 1-3816) were generated.

Gibson Assembly (NEB) was then performed using HindIII/XbaI digested pLew100-PTPPURO with

the MTS and POLA PCR fragments or with the UCR fragment. All DNA constructs and point

mutations were verified by direct sequencing. Due to the lack of sequence conservation beyond

the POLA domain, PSIpred prediction software was used to locate an region upstream of the

POLA domain that would be least likely to affect protein folding based on the secondary structure

prediction (Jones, 1999).

3.3.2 Trypanosome cell culture and transfection. Procyclic form T. brucei strain 29-13 (Wirtz, Leal, Ochatt, Cross, 1999) was cultured at 27˚C in

SDM-79 medium supplemented with FBS (15%), G418 (15 µg/mL) and hygromycin (50 µg/mL).

To generate the parental RNAi cell line IC-UTR, 15 µg of pStLIC3¢UTR was transfected by

electroporation into 29-13 cells, selected with 2.5 µg/mL phleomycin and cloned by limiting

dilution as previously described (Chandler et al 2008). Four individual clones were analyzed and

clone P6E2 was chosen based on growth rate, POLIC knockdown efficiency, and kDNA defects. All

POLIC variant expression constructs (15 µg) were transfected by nucleofection using the Amaxa

Nucleofection Parasite Kit (Lonza) into 29-13 cells to generate inducible overexpression POLIC-

PTP variant cell lines (OE) and into IC-UTR RNAi cell line to generate complementation cell lines

(RNAi+OE). Following selection with 1 µg/mL puromycin, cell lines were cloned by limiting

dilution. A single clone of each cell line was selected based on variant expression for OE and

confirmed POLIC knockdown and variant expression for RNAi+OE cell lines. All cell lines were

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maintained between 1x105 – 1x107, and were not continuously cultured for more than three

weeks. Cell density was determined using a Beckman Coulter Z2 particle counter. RNAi and/or

overexpression were induced by the addition of tetracycline (1 µg/mL). Cultures were

supplemented with 0.5 µg/mL Tet on non-dilution days to maintain POLIC variant expression

and/or RNAi (Rusconi, Durand-Dubief, Bastin, 2005).

3.3.3 RNA isolation and RT-qPCR analysis. Total RNA was isolated from 5x107 cells with Trizol (Thermo Fisher) using standard procedures.

RNA concentration was determined by Nanodrop and 100 ng of total RNA was reverse

transcribed to cDNA with random primers and RNase inhibitor (Applied Biosystems). qPCR was

performed using QuantiNova SYBR Green PCR (Qiagen) with 1 µg of cDNA and 0.2 µM of primer

per reaction (qPCR primers can be found in Table 3.1) with a Stratagene MxPro 3000x

thermocycler. All reactions were performed in triplicate and qPCR results were determined from

three biological replicates. Gene knockdown was normalized using TERT (Brenndörfer and

Boshart, 2010).

3.3.4 SDS-PAGE and Western blot analysis. Cells were harvested and washed once with PBS supplemented with protease inhibitor cocktail.

Samples were fractionated by SDS-PAGE and transferred overnight onto a PVDF membrane in 1X

transfer buffer containing 1% methanol. Membranes were blocked in Tris-buffered saline (TBS)

+ 5% non-fat dry milk for at least one hour. Peroxidase-Anti-Peroxidase soluble complex (PAP)

reagent (1:2000, 30 min) (Sigma, P1291) was used for PTP tag detection. For subsequent

detections where stripping was required because proteins of interest were similarly sized to the

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loading control, membranes were stripped with 0.1 M glycine (pH 2.5, 15 min, 37 °C), washed in

TBST (0.1 % Tween-20), blocked and re-probed with specific Crithidia fasciculata anti-Hsp70

(1:5000, 1 h) (Johnson and Englund, 1998) and secondary chicken anti-rabbit-HRP (1:2000, 1 h)

(Santa Cruz, sc-516087). Signal was detected with Clarity™ ECL Blotting Substrate (NEB) and on a

GE Imagequant LAS 4000 mini. Quantification of Western blot intensities was performed using

ImageJ software (http://imagej.nih.gov/ij/).

3.3.5 DNA isolation and Southern blot analysis. Total DNA was isolated from 1x108 cells using Puregene Core Kit A (Qiagen). Free minicircles and

total kDNA content were analyzed as previously described (Bruhn, Mozeleski, Falkin, Klingbeil,

2010). Quantification was performed using a Typhoon 9210 Molecular dynamics Phosphoimager

(GE Healthcare) with background subtraction and normalized against the tubulin signal using

ImageQuant 5.2 software.

3.3.6 Immunoprecipitation and DNA polymerase activity assay. Immunoprecipitation of PTP-tagged POLIC variants was conducted according to (Brandenburg et

al., 2007) with modifications. Briefly, 3 x 108 mid-log phase cells were harvested (9 x 108 IC-UCR

cells were used due to low expression), washed with cold PBS and lysed in 970 μL of 150 mM KCl,

20mM Tris-HCl (pH 8), 3 mM MgCl2, 0.5% NP-40, 0.5 mM DTT supplemented with [1X] cOmplete™

EDTA-free protease inhibitor (Roche). Cell lysate was bound to IgG-Sepharose beads (Roche) and

washed by centrifugation with 100 mM NaCl, 20 mM Tris-HCl (pH 8), 3 mM MgCl2, 0.1% Tween

20 buffer five times at 1,000 x g for 5 minutes each. All steps were performed at 4˚C. Samples

bound to beads were assayed for DNA polymerase activity. Reactions contained 50 mM Tris-HCl

http://imagej.nih.gov/ij/

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(pH 8), 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, 20 μg/mL activated calf thymus DNA as template,

10 μM dTTP, dGTP, and dCTP, 5 μL [α-32P] dATP (Perkin Elmer) mixed with 10 μM cold dATP,

either the specific units of Klenow fragment (NEB) or the specified volumes of

immunoprecipitated POLIC variants. Reactions were incubated at 37oC for 30 minutes, stopped

by adding 5 μL of 500 mM EDTA, and spotted onto Whatman DE81 disks, dried, and then washed

four times with 0.5 M NaPO4 (15 min each). Disks were then washed with 80% EtOH for 15 min

and dried before analyzing with an LS2 Beckman Coulter scintillation counter.

3.3.7 Immunofluorescence microscopy. Procyclic cells were pelleted at 975 x g, washed and resuspended at approximately 5x106 cells/mL

in PBS, then adhered to poly-L-lysine-coated slides (5 min). Cells were then fixed in 4%

paraformaldehyde (5 min) and washed three times (5 min) in PBS/0.1 M glycine (pH 7.4). Cells

were permeabilized with 0.1% Triton X-100 (5 min) and washed in PBS 3 times (5 min). PTP-

tagged proteins were detected by incubating with anti-protein A serum (1:3000, 1 h) (Sigma,

P3775) followed by Alexa Fluor® 594 goat anti-rabbit (1:250, 1 h) (Invitrogen, A-11012). Detection

of basal bodies (YL1/2), TAC102 and DNA (DAPI) was previously described (Concepción-Acevedo

et al., 2018). Slides were then washed 3 times (5 min) in PBS prior to mounting in Vectashield

(Vector Laboratories). Images were captured at the same exposure time for each channel within

the same experiment using a Nikon Eclipse E600 microscope equipped with a cooled CCD Spot-

RT digital camera (Diagnostic Instruments) and a 100X Plan Fluor 1.30 (oil) objective.

Quantification for kDNA morphology was performed as described previously (Bruhn et al., 2010;

Chandler et al., 2008). Asymmetric networks were classified as one cell with two detectable kDNA

networks and “other” was classified as a cell with a karyotype that did not match either a normal

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karyotype (1N1K, 1N2K, 2N2K) or aberrant kDNA morphology (small, none, ancillary, or

asymmetric).

3.4 Results 3.4.1 POLIC 3ʹUTR RNAi and rescue with epitope-tagged wild-type protein.

Previously, POLIC was silenced using an intermolecular dsRNA trigger targeting the open reading

frame that led to the distinct pleiotropic small, fragmented (more than two DAPI-stained spots

positioned posterior to the nucleus) and ancillary kDNA phenotypes (Klingbeil et al., 2002). To

examine whether the POLA and UCR domains are necessary for either kDNA replication or

distribution, we coupled inducible RNAi against the POLIC 3’ untranslated region (UTR) with

concurrent inducible ectopic expression of epitope-tagged variants that contained an unrelated

3' UTR to avoid RNAi targeting for a structure-function analysis. We first tested whether POLIC

3¢-UTR (IC-UTR) RNAi is sufficient to deplete POLIC mRNA. To generate the IC-UTR cell line, a 517

bp fragment immediately downstream of POLIC coding sequence was cloned into a stem-loop

vector and subsequently introduced into the 29-13 cells (Wirtz et al., 1999). Tetracycline

induction of dsRNA synthesis resulted in LOF following 10 doublings compared to uninduced cells

(Fig 3.1A). RT-qPCR revealed a 65% reduction of POLIC mRNA following 48 hours of induction

with no off-target effects for the two paralogs (POLIB and POLID) or the gene downstream of

POLIC, glutathione synthetase (Fig 3.1B). DAPI-staining of uninduced and induced cells was used

to assess the effects of POLIC 3’ UTR silencing on kDNA networks. Parasites per time point were

classified according to kDNA size and distribution within the cell. Using the previously reported

categories, ten days of IC-UTR silencing resulted in cells that displayed small kDNA (74%),

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fragmented/ asymmetric networks (10.4%), normal kDNA (7.9%), and no kDNA (7.8%) (Fig 3.1C,

E). These percentages closely resembled those from POLIC ORF silencing (Klingbeil et al., 2002).

To further dissect the multiple kDNA-associated defects, cells that contained ancillary or

asymmetrically divided kDNA (progeny kDNA in which one is larger than the other) were also

quantified, as ancillary kDNA were not previously counted. Ancillary kDNA and asymmetric

network division can be classified as distribution defects and specifically quantifying them

independently would help identify potential non-replication defects (Grewal et al., 2016; Trikin

et al., 2016; Wang et al., 2002; Zhao et al., 2008). Due to the low frequency of fragmented

networks, we quantified them as part of the “other” category that included any cell with an

abnormal number of nuclei or kDNA.

By further subdividing the categories, IC-UTR RNAi still produced cells with a majority of small

kDNA (54.7%) vs normal kDNA (7.9%), no kDNA (7.5%) and other (3.7%), but also highlighted the

distribution defect signatures of asymmetric division (10.4%) and ancillary kDNA (15.6%) (Fig

3.1D, E). Based on the proximity of POLIC and TAC102 in the KFZ/ULF, we wanted to test whether

TAC102 localization is impacted during POLIC depletion. After 4 days of POLIC depletion, the

majority of TAC102 localized properly in close proximity to the basal body while some cells

showed TAC102 co-localizing with ancillary kDNA as previously reported (Trikin et al., 2016).

TAC102 protein appeared stable throughout the induction. (Fig 3.2). Lastly, by analyzing the free

minicircle population using Southern blotting and a minicircle-specific probe, both unreplicated

CC monomers and newly replicated N/G molecules increased when POLIC was depleted

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compared to the uninduced control, another indication of a defect not strictly related to

minicircle replication (Fig 3.1F). Taken together, IC-UTR silencing phenocopied the previously

reported LOF, and kDNA replication defect but also displayed clear signatures of a distribution

defect.

To determine whether the deficiencies caused by IC-UTR RNAi could be rescued by ectopic

expression of PTP-tagged wild-type POLIC (IC-WT, 201 kDa), we transfected the IC-UTR cells with

pLewIC-WT-PTPPuro (RNAi+OE). In this genetic complementation system, the tagged POLIC variant

utilizes an unrelated 3’UTR to avoid targeting by RNAi. IC-WT complementation alleviated the

RNAi-mediated defects with consistent expression of IC-WT throughout the induction (Fig 3.3A-

E). IC-WT levels were four-fold above endogenous POLIC protein detected in a cell line

engineered to endogenously express the epitope-tagged POLIC from a single allele (+C, Fig 3.3C)

(Concepción-Acevedo et al., 2018). Additionally, we analyzed the cell cycle localization of IC-WT

and found discrete antipodal POLIC-PTP foci formation in 35% of an unsynchronized population

almost exclusively in the 1N1K* stage, similar to endogenous 26% POLIC-PTP AS foci detected in

the single expresser cell line (Fig 3.3F) (Concepción-Acevedo et al., 2018).

We also transfected pLewIC-WT-PTPPuro into parental 29-13 cells generating the IC-WT

overexpression (OE) cell line to study the effects of increased IC protein in the absence of POLIC

3’UTR RNAi. IC-WT overexpression (8-fold) for 10 days did not interfere with fitness or kDNA

maintenance and IC-WT localization was similar to endogenous POLIC but IC-WT OE cells

displayed additional matrix localization (Fig 3.4). The localization patterns from IC-WT

overexpression and complementation were nearly identical (Fig 3.3F and 3.4C). Therefore, IC-

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UTR RNAi was specific based on the complete rescue using IC-WT and the unique pleiotropic

phenotype could be attributed solely to silencing of POLIC.

3.4.2 POLIC DNA polymerase activity is essential for kDNA replication and cell cycle localization. Mutational and structural analyses of Family A DNA polymerases have revealed two invariant Asp

residues essential for catalytic activity that coordinate two divalent metal ions for transition state

stabilization necessary for all nucleotidyl transferases (Sousa, 1996; Steitz, 1998). POLIC and the

other T. brucei mitochondrial DNA polymerases contain Family A conserved motifs A, B and C and

the non-variant Asp residues (Klingbeil et al., 2002). It is possible that the polymerase function

may not be the essential function of POLIC. In yeast, the DNA polymerase and 3ʹ-5ʹ exonuclease

activity of DNA Pol ε are nonessential for cell growth, whereas the protein’s non-catalytic C-

terminal domain is required (Dua et al., 1999; Kesti et al., 1999).

To address whether the nucleotidyl transferase activity is essential for POLIC in vivo functions,

we generated a full-length POLIC PTP-tagged variant with catalytic residue mutations (D1380A,

D1592A) to inactivate polymerase activity (IC-DEAD), and transfected it into the overexpression

(OE) and the IC-UTR RNAi backgrounds (RNAi+OE). To confirm the mutated Asp residues

successfully inactivated POLIC polymerase activity, IC-WT and IC-DEAD were overexpressed for

48 hours, immunoprecipitated and analyzed by western blot and an in vitro primer extension

assay. The POLIC variants were detected at their predicted sizes (IC-DEAD, 201 kDa; IC-POLA, 75

kDa, IC-UCR, 160 kDa), but only IC-WT demonstrated nucleotidyl transferase activity (Fig 3.5).

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Interestingly, IC-DEAD overexpression led to a dominant-negative LOF following just 4 days of

induction. The number of cumulative doublings after 10 days of IC-DEAD overexpression was 13.7

(Fig 3.6, left graph) compared to the 15.1 cumulative doublings following IC-UTR RNAi (Fig 3.1A).

However, complementation with the IC-DEAD variant exacerbated the LOF and reduced the

cumulative doublings to 9.1 after 10 days (Fig 3.6A, right graph). RT-qPCR confirmed depletion of

endogenous POLIC transcripts, and increased abundance of the variant transcript (Fig 3.6B). The

abundance of the paralog transcripts was also elevated compared to uninduced. IC-DEAD protein

levels were initially high compared to endogenous POLIC in both backgrounds (OE, 16.6-fold;

RNAi+OE, 12.5-fold) but were reduced to 3-fold by the end of the 10 day induction based on

ImageJ analyses from Western blot data (Fig 3.6C). IC-DEAD-PTP signal was concentrated on or

near the kDNA at all cell cycle stages in OE (98%) and RNAi+OE (99%) cell lines (Fig 3.6D-E) in

striking contrast to endogenous POLIC-PTP and IC-WT-PTP foci found exclusively in the 1N1K*

stage (compare Figs 3.6E and S2D). IC-DEAD complementation also occasionally displayed

ancillary kDNA co-localized with TAC102 (Fig 3.2).

Next, we examined the effects of IC-DEAD variant on kDNA maintenance in both backgrounds

(OE and RNAi+OE). Ten days of IC-DEAD overexpression in the absence of POLIC RNAi resulted in

a predominantly small kDNA phenotype (43.5%), followed by no kDNA (26.5%), ancillary (14.3%),

other (11%), and normal (4.7%) kDNA (Fig 3.8A). In striking contrast to both OE and the parental

RNAi phenotypes, IC-DEAD complementation displayed a majority of cells with no kDNA (59.1%)

that correlated with the exacerbated LOF. IC-DEAD also displayed small (16.1%), ancillary

(10.8%), other (9.6%), and normal (4.4%) kDNA, but did not display any asymmetric kDNA

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networks (Fig 3.8D). The IC-DEAD variant causes a more rapid onset of aberrant kDNA (after 2

days) in both genetic backgrounds and shifts from a predominantly small kDNA to no kDNA for

the IC-DEAD complementation (Figs 3.1D 3.8A, 3.8D).

In addition to progressive loss of the kDNA network, hallmarks of perturbing kDNA replication

include a decrease in minicircle and maxicircle copy number as well as an increased ratio of

unreplicated covalently closed (CC) to nicked/gapped (N/G) progeny (Bruhn et al., 2010; Liu et

al., 2010; Scocca and Shapiro, 2008). Both IC-DEAD overexpression and complementation led to

a progressive decrease in free minicircle species (Fig 3.6B, E). In both genetic backgrounds, the

free minicircles initially increased (Days 2, 4) when the IC-DEAD variant was present. During the

later days of the inductions, the abundance of CC and N/G minicircles quickly declined, which

resembled the defect caused by RNAi of the paralog POLID (Chandler et al., 2008). When

comparing unreplicated CC to replicated N/G species, IC-DEAD overexpression showed a 1.7-fold

increase in the CC:N/G ratio and complementation displayed a 5.5-fold increase at day 10

compared to uninduced cells (Fig 3.6C, F).

These data indicate that IC-DEAD could not rescue any of the pleiotropic effects from POLIC RNAi.

Additionally, the variant resulted in disruption of cell cycle localization and displayed an overt

kDNA replication defect with a decrease in N/G progeny suggesting that the nucleotidyl

incorporation is essential for POLIC function.

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3.4.3 The POLA domain partially rescues minicircle replication defects. Next, we determined if the conserved POLA domain alone was sufficient to restore POLIC

function in vivo. We generated an N-terminal truncated POLIC PTP-tagged variant (IC-POLA, 437

aa), and transfected it into the overexpression (OE) and the IC-UTR RNAi backgrounds (RNAi+OE).

Consistent IC-POLA overexpression (5.4-fold compared to endogenous) did not affect parasite

fitness (Figs 3.8A, Fig 3.7A) or kDNA maintenance determined by both DAPI staining which

showed >90% normal cells at day 10, and by Southern blotting which indicated no perturbation

of free minicircle levels (Fig 3.7B, C). Surprisingly, immunoprecipitated IC-POLA did not display

nucleotidyl incorporation activity in the primer extension assay (Fig 3.5).

IC-POLA complementation closely resembled the parental IC-UTR LOF (15.1 cumulative

doublings, Day 10) with robust knockdown of endogenous POLIC mRNA (Fig 3.9A, B). Western

blot data showed consistent protein expression for 10 days (4.1-fold higher than endogenous)

(Fig 3.9C). IC-POLA showed mitochondrial matrix localization and weak signal near the kDNA

regardless of cell cycle stage, and no antipodal PTP foci were ever detected (Fig 3.9D, E). IC-POLA

complementation displayed cells with small (46.7%), no (9.8%), normal (7.5%), and other (3.1%)

kDNA (Fig 3.10A). Interestingly, IC-POLA cells with ancillary (19.5%) or asymmetric (13.3%) kDNA

were elevated compared to the combined percentage of fragmented/asymmetric (25.5%) kDNA

for the parental IC-UTR RNAi defect, suggesting that the polymerase domain alone could not

rescue the distribution defect. TAC102 was detected co-localizing with a portion of the ancillary

kDNA (Fig 3.2). Furthermore, in the presence of the IC-POLA variant, there was no detectable

accumulation of free minicircles over the course of the induction (Fig 3.10B, C), and there were

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no statistically significant changes in the CC:N/G ratio across the 10 day induction demonstrating

that POLA domain could rescue the minicircle replication defect.

Collectively, these data show that the C-terminal polymerase domain alone could partially

restore the free minicircle population in vivo even though it did not display strict cell cycle

localization to the AS possibly due to its ability to still localize near the kDNA disk. Additionally,

the increased distribution defect suggested that the UCR is necessary for kDNA distribution and

cell cycle dependent localization.

3.4.4 The UCR domain partially rescues IC-UTR RNAi defects. The N-terminal UCR (1291 amino acids) of POLIC has no conserved domains or motifs but does

contain a predicted hydrophobic patch and arginine methylation sites (R420, R1250, R1260) that

were detected in the T. brucei mitochondrial arginine methylproteome (Fisk and Read, 2011). To

evaluate a role in kDNA distribution, we generated a POLIC variant that contained only the UCR

region (IC-UCR), and transfected it into the overexpression (OE) and the IC-UTR RNAi backgrounds

(RNAi+OE). IC-UCR overexpression was consistent across the induction (5.3-fold increase

compared to endogenous), did not affect parasite fitness, and no kDNA replication defects were

detected (S4D-F).

IC-UCR complementation resulted in LOF with 15.7 cumulative doublings after 10 days of

induction (Fig 3.11A). RT-qPCR displayed robust POLIC knockdown and Western blot data

revealed initially high protein levels (18-fold increase over endogenous) that continually

decreased over 10 days (4-fold) (Fig 3.11B, C). There was clear formation of IC-UCR-PTP foci at AS

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in 17.6% of 1N1K* cells compared to IC-WT complementation (30%) and endogenous localization

(26%) and IC-UCR also displayed weak signal near the kDNA at other cell cycle stages (Fig 3.11D,

E). IC-UCR complementation displayed mostly cells with small (42%), normal (17%), ancillary

(15.3%), other (13.7%), no (7.7%), and asymmetric (4.3%) kDNA (Fig 3.11D). Interestingly, the IC-

UCR variant resulted in more cells with normal kDNA (17 vs. 7.8%), and fewer cells with

distribution defects when compared to IC-UTR RNAi alone (19.6 vs 25.5%) indicating a partial

rescue of the distribution phenotype. Despite the reduction in cells displaying asymmetric

networks, the percentage of cells displaying ancillary kDNA did not significantly improve and

TAC102 still co-localized with ancillary kDNA (Fig 3.2). In the presence of the IC-UCR variant, there

were no statistically significant changes in the CC:N/G ratio even though there was a decrease in

free minicircles after 8 days of complementation (Fig 3.10E, F). The POLIC variant effects are

summarized in Fig 3.12. Collectively, these data revealed POLIC has distinct functions in both

kDNA replication and distribution and that the UCR mediates antipodal site localization and the

distribution function.

3.5 Discussion Trypanosome kDNA replication presents an interesting model system in which replication of a

single nucleoid (the kDNA network) occurs once every cell cycle and segregation of daughter

networks is mediated by a structure linked to the flagellar basal body called the TAC. The current

kDNA replication model indicates spatial and temporal separation for early events occurring in

the KFZ and later Okazaki processing at the AS. Additionally, three Family A DNA polymerases

(Pol) are essential for kDNA replication. POLIB and POLID have demonstrated roles in minicircle

replication, but the precise role for POLIC in kDNA maintenance was unclear. Although the

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precise functions performed by the paralogs on minicircles and maxicircles have not yet been

defined, the trio of Pols superficially resembles the replicative polymerases Pol α, δ, and ɛ

required for nuclear DNA replication. Understanding catalytic and non-catalytic roles may

provide insight as to why trypanosomes utilize three distinct Pols for kDNA replication.

POLIC shares no similarity with other proteins outside of the conserved POLA domain at the C-

terminus. Consistent with an essential function in kDNA maintenance, POLIC and the overall

protein structure is conserved in all of the 41 currently sequenced trypanosomatid organisms

and some free-living relatives available at TriTrypDB.org (Aslett et al., 2010).

Interestingly, three arginine sites on POLIC are known to be methylated (Fisk and Read, 2011).

Arginine methylation is a reversible post-translational modification with regulatory roles in many

biological processes including signal transduction, transcription, RNA splicing and transport, and

facilitation of protein-protein interactions (Raposo and Piller, 2018). Although at this time it is

unknown whether POLIC from other trypanosomatids retain the arginine methylation sites.

To further understand the role of POLIC in kDNA replication, we investigated the structure–

function relationship by ectopically expressing several POLIC variants in trypanosome cells that

are deficient in endogenous POLIC. Tetracycline-inducible expression of a dsRNA targeting the 3’-

UTR of a gene and co-expression of an ectopic copy of the RNAi target is a validated approach for

in vivo complementation studies on flagellar motility, cytokinesis, mitochondrial protein import,

TAC function, and transferrin receptor trafficking in T. brucei (Käser et al., 2016; 2017; Ralston et

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al., 2011; Trikin et al., 2016; Weems et al., 2015; Yu et al., 2012). By using this approach we

demonstrated that IC-WT could rescue the replication and distribution defects and we defined

two functional domains: one for nucleotidyl incorporation and a non-catalytic domain for AS

localization and kDNA distribution. Two other dual-functioning proteins with roles in kDNA

maintenance have been described. UMSBP is a minicircle replication initiation protein (Abu-

Elneel et al., 1999). Interestingly, USMBP RNAi indicated impaired kDNA segregation through a

possible cell cycle checkpoint mechanism and not by a more classical mechanism via direct

inhibition of the TAC (Milman et al., 2007). The TCA cycle enzyme, α-ketoglutarate

dehydrogenase (α-KDE2), was reported to be an antipodal site protein with a role in distribution

of progeny networks but displayed no role in kDNA replication (Sykes and Hajduk, 2013). Unlike

UMSBP and α-KDE2, POLIC represents the clearest example bridging the replication and

segregation processes.

How the POLIC polymerase domain participates at replication forks remains an open question.

Although the immunoprecipitated POLA domain did not have detectable activity in a primer-

extension assay (Fig. 3.5), IC-POLA complementation indicated that the polymerase domain alone

is sufficient to partially support minicircle replication even though IC-POLA localized mainly to

the mitochondrial matrix (Fig. 3.9D, 3.10B, 3.10C). IC-POLA includes all the conserved Family A

motifs, but this truncated fragment may lack important upstream residues required for binding

the primer-template in the in vitro assay. Despite IC-POLA failing to exhibit cell cycle-dependent

localization during complementation, abundance around the kDNA was detected and could

support the maintenance of the free minicircle population directly or indirectly. It is also possible

that IC-POLA in vivo has access to accessory factors that allow it to maintain the free minicircle

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population. This truncated version of POLIC still contains two of the three arginine residues that

were detected in a methylarginine proteome analysis that could promote protein interactions.

It is compelling that the IC-DEAD variant defective in nucletotidyl incorporation generated the

most striking kDNA replication defects in both overexpression and complementation (Fig. 3.12).

IC-DEAD overexpression caused a dominant negative LOF with a kDNA replication defect (Fig.

3.6A), and abolished POLIC spatiotemporal cell cycle localization. Unlike IC-POLA mitochondrial

matrix localization, IC-DEAD always localized with or around the kDNA disk (Fig. 3.6D, E).

Interestingly, there are now 250 pathogenic mutations in human mitochondrial DNA polymerase

gamma that interfere with dNTP selectivity, processivity or accessory subunit binding with many

of the dominant mutations located in the polymerase domain that result in loss of mtDNA

(Stumpf et al., 2013). It is possible that IC-DEAD was bound to minicircles and/or maxicircles

outcompeting endogenous POLIC for substrate, inhibited access of other replication proteins, or

sequestered replication factors. IC-DEAD complementation results support the idea that access

of other replication proteins was impacted. The dramatic loss of kDNA (Fig. 3.8D) and increase in

unreplicated free minicircles (Fig. 3.8E, F) are hallmarks of a kDNA replication defect that closely

resemble depletion of POLID, a known minicircle replication factor that localizes to AS only during

kDNA S phase (Chandler et al., 2008; Concepción-Acevedo et al., 2012). We previously showed

that POLID depletion disrupted localization of POLIC to AS. It is possible that overexpression of

IC-DEAD may be having a similar effect on POLID or other replication proteins by inhibiting access

to the kDNA (Concepción-Acevedo et al., 2018). However, the mechanisms(s) governing AS

localization and paralog interactions have not yet been determined.

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Despite lacking any recognizable catalytic domain, the UCR appears to contain a determinant for

localization to AS. IC-UCR complementation resulted in PTP foci formation that closely resembled

the localization pattern for endogenous POLIC where signal was detected in the KFZ in 1N1K cells,

IC-UCR-PTP foci at AS were detected in 1N1K* cells, and the signal was disperse in the other cell

cycle stages (Fig. 3.11D, E). Although two different methods were used to evaluate the

percentage of cells in kDNA S phase, the proportion of PTP foci positive cells in this stage were

equivalent (0.59) (Concepción-Acevedo et al., 2018). Our current model suggests that cell cycle

localization of POLIC may be a mechanism that discriminates between the two functional

domains of this protein. For example, nucleotidyl incorporation activity may be utilized when

localized in the KFZ, while the distribution function may be more important at AS. The IC-UCR

variant contains all three of the arginine methylated residues (Fisk and Read, 2011) that may

contribute to POLIC localization at AS through protein-protein interactions. The methylation

status of the full-length protein may also promote one of the dual functions over the other. For

example, the demethylated state may favor interactions with other replication proteins while the

fully methylated state may interact with AS proteins and distribution factors, or vice versa. Future

studies will be focused on understanding the impact of methylation on POLIC function. IC-UCR is

the first sequence from a kDNA protein shown to promote AS localization and further

experimentation is necessary to refine the specific sequences determinants involved.

Another non-catalytic role for the UCR is associated with kDNA distribution. IC-UCR

complementation partially rescued RNAi by reducing the number of cells with asymmetrically

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sized networks and increasing the number of cells with normal kDNA (Fig. 3.10D). In contrast,

complementation with IC-POLA (lacking the UCR) worsened the distribution defect (Fig. 3.10A,

8), perhaps due to the elimination of the spatiotemporal localization pattern where POLIC starts

in the KFZ and then is detected at the AS at later stages of kDNA replication (Concepción-Acevedo

et al., 2018). At this time we do not understand the mechanism on how the UCR domain alone

can partially rescue, since IC-DEAD complementation displayed an apparent rescue of

asymmetric networks (Fig. 3.8D, 8). It is possible that asymmetric networks were not observed

solely because the replication defect developed too rapidly which prevented a distribution defect

from being detected at the time points analyzed. The decline in IC-UCR abundance over the

course of the complementation provides one explanation for the incomplete rescue.

Alternatively, the POLA domain may also have a role in distribution through protein interactions.

Surprisingly, no POLIC variant complementation was able to reduce the appearance of ancillary

kDNA (Fig. 3.8D, 3.10A, 3.10D, 3.12) suggesting that a fully functional protein is critical to

suppress the ancillary kDNA phenotype. It is likely that POLIC is not playing a direct role in

distribution but is impacting other proteins essential for distribution.

POLIC is not a classical TAC component based on the criteria outlined by (Schneider and

Ochsenreiter, 2018). IC-UTR RNAi does not result in 1 cell with a large kDNA and the other lacking

kDNA. Instead, there is unequal partitioning of a reduced sized network due to the POLIC

replication defect. Additionally, ancillary kDNA is an unusual aberrant phenotype that appears

when elements related to the mitochondrial membrane or the TAC are perturbed as seen with

functional disruptions of PUF9, ACP, PNT1, HSP70/HSP40, and TAC102 (Archer et al., 2009;

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Clayton et al., 2011; Grewal et al., 2016; Trikin et al., 2016; Týč et al., 2015). The TAC component

most proximal to the kDNA is TAC102. When the C-terminus of this protein is obstructed, TAC102

cannot connect to the upstream TAC components properly (and subsequently to the basal body)

resulting in mislocalization with ancillary kDNA (Trikin et al., 2016). It is interesting that

complementation with all POLIC variants produced ancillary kDNA with mislocalization of TAC102

independent of their rescue status (Fig 3.2). POLIC is more proximal to the kDNA than TAC102

(Concepción-Acevedo et al., 2018) and our data suggest that a fully functional POLIC is important

for TAC102 to connect to upstream TAC components. At this time we do not know if the TAC is

required for proper localization of POLIC or how POLIC might interact with the TAC. The recently

described minicircle reattachment protein MiRF172 may provide a bridge between POLIC and

TAC102 since MiRF172 remains associated with isolated flagella following biochemical

fractionations, and its localization is more proximal to the kDNA than TAC102 (Amodeo et al.,

2018). The proximity of the replication and segregation machinery in T. brucei could suggest a

physical interaction of the components involved in the two processes. Thus far, the dual-

functioning DNA polymerase, POLIC, is the clearest example of a protein bridging these two

essential processes.

80

Gene Purpose Primer Name Primer Sequence Linker

TbPOLIC (927.7.3990)

3' UTR RNAi

construct

MK622 5'-TCA ATT ACG CGT GCG GAG GTG AGG AGT AGC GTC G-3' MluI

MK623 5'-TCA ATT AAG CTT GCG GAG GTG AGG AGT AGC GTC G-3' HindIII

MK624 5'-TCT ATT TCT AGA GTG TAG TAA TCA GGG CGA CG-3' XbaI

Subcloning MK653 5'-ATC ATC AAG CTT ATG CGA CGT ACT TTC

AGT C-3' HindIII

MK654 5'-ATC ATC TCT AGA CCT GA CAA CTC CCC TAG TG-3' XbaI

Gibson Assembly

MK840 5'-cac caa aaa gta aaa ttc aca agc ttA TGC GAC GTA CTT TCA G-3' -

MK842 5'- GC GCG TAC CGA GTT GGG AAG CAG GC-3' -

MK843 5'-GC GCG TAC CGA GTT GGG AAG CAG GC -3' -

MK844 5'-cca cct gat ctt cca gcg gtc tag aCC TGG ACA ACT CCC CTA G-3' -

MK841 5'-ACG TTA TCT AGA CAA CAC ATT GAT CCG C-3' -

Site directed mutagenesis

MK663 5'-TCG TAT GAT TGA GGC GGC CTA TAG TCA GTT GGA AG-3'

MK664 5'-GTT AAT ACC GTT CAC GCT TGC GTT TGG ATT GAT GCC CAC GAA TC-3'

qPCR MK561 5'-ATG CTC TTT GTC CCA ACC CTC TCA-3' -

MK562 5'-ATG ATC CGG TTC CTC CCA CTG TTT-3' -

TbPOLIC 3' UTR qPCR

MK651 5'-TGG CGG ACC GAA TGT TAG TGG AAA-3' -

MK652 5'-TGT GTT ACG GGC AAC TCC TCG GA-3' -

TbPOLID (927.11.3260)

qPCR

MK559 5'-TGG CGA AAT CAT ACG GTG GTC AGA-3' -

MK560 5'-TGC GCG AGT GCT CGT TCT ATA TGT-3' -

TbPOLIB (927.11.4690)

qPCR

MK563 5'-GTG ATT GCA TTC ATG GCG ACG GAA-3' -

MK564 5'-AGT ACT TGG TCC ATG GCT CCA CAA-3' -

TERT (927.11.10190)

qPCR

MK804 5'-GAG CGT GTG ACT TCC GAA GG-3' -

MK805 5'-AGG AAC TGT CAC GGA GTT TGC-3' -

Glu Synth (927.7.4000)

qPCR

MK665 TCT GTT GTA TGC AGC CGC TAT GGA -

MK666 AGG AGC TTC CCA GTG AAG TTG ACA -

PTP PTP tag amplification

MK681 5'-TTA TTA TCT AGA CCG CTG GAA GAT CAG GTG G-3' XbaI

MK713 5'-TTG TTG TGA TCA TCA GGT TGA CTT CCC CGC-3' BclI

Table 3.1: Primers used in this study. Underline; restriction enzyme sites, lowercase letters; plasmid backbone sequence.

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Figure 3.1: Phenotype of POLIC 3’UTR-targeted depletion. (A) Growth curves of uninduced and RNAi induced procyclic cells. Error bars represent the standard deviation from the mean of three biological replicates. (B) RT-qPCR analysis uninduced (black) and tetracycline induced (blue/white hashed) IC-UTR cells after 48 hr of growth. Data are normalized to TERT and error bars represent the standard deviation from the mean of three biological replicates. IC UTR, POLIC 3’ untranslated region; IC ORF, POLIC open reading frame; IB ORF, POLIB open reading frame; ID ORF, POLID open reading frame; Glu Synth, Glutathione Synthetase. Asterisks represent statistical significance based on pairwise Student’s T-test. * p < 0.1; ** p < 0.01; *** p < 0.001. (C) Quantification of kDNA morphology utilizing categories from Klingbeil et al., 2002. Over 300 DAPI-stained cells were scored at each time point and error bars represent standard deviation from the mean of three biological replicates. (D) Quantification of kDNA morphology utilizing new categories. Over 300 DAPI-stained cells were scored at each time point and error bars represent standard deviation from the mean of three biological replicates. (E) Representative images for the various kDNA phenotypes. Scale bar, 5 µm. (F) Representative Southern blot showing the increase in free minicircle species during IC 3’ UTR silencing. k, kDNA network; N/G, nicked/gapped; L, linearized; CC, covalently closed; T, tubulin.

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Figure 3.2: TAC102 colocalizes with ancillary kDNA. (A) Representative images of TAC102 and ancillary kDNA co-localization in the IC-UTR, IC-DEAD RNAi + OE, IC-POLA RNAi + OE, and IC-UCR RNAi + OE cell lines. DAPI-staining (blue); anti-TAC102 (red); YL1/2 (green). The posterior end of the cell is the rounded end and the expected position of normal kDNA. White arrows indicate ancillary kDNA and purple arrowheads show TAC102 colocalized with ancillary kDNA. Scale bar, 5 μm. (B) Western blot detection of TAC102 and HSP70 protein levels following 10 days of induction for each of the listed cell lines. 1x106 cell equivalents was loaded into each well except for C where 5x106 cell equivalents was loaded. +C, single expresser control cell line.

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Figure 3.3: Rescue with ectopically expressed IC-WT in the IC-UTR RNAi cell line. (A) Schematic representation of full-length POLIC with the C-terminal PTP tag. Growth curves of uninduced, RNAi complementation with IC-WT, parental IC-UTR RNAi procyclic cells. Error bars represent the standard deviation from the mean of three biological replicates. (B) RT-qPCR analysis of uninduced (black) and induced (blue/white hashed) cells following 48 hours of tetracycline induction. Data are normalized to TERT and error bars represent the standard deviation from the mean of three biological replicates. IC UTR, POLIC 3’ untranslated region; IC ORF, POLIC open reading frame; IB ORF, POLIB open reading frame; ID ORF, POLID open reading frame. (C) Western blot detection of the PTP tag and HSP70 over 10 days of induced complementation. At each time point, 1x106 cell equivalents was loaded into each well except for +C where 5x106 cell equivalents was loaded. +C, single expresser control cell line. (D) Quantification of kDNA phenotypes. Over 300 DAPI-stained cells were scored at each time point and error bars represent standard deviation from the mean of three biological replicates. Blue bar, IC-WT complementation; gray bar, POLIC RNAi. Specific kDNA phenotype information can be found in Figure 1E. (E) Representative Southern blot showing no change in the free minicircle population. k, kDNA network; N/G, nicked/gapped; L, linearized; CC, covalently closed; T, tubulin. (F) Quantification of PTP foci formation at each cell cycle stage. Percentages refer to the fraction of total cells in the population. Green/white hashed, foci positive; gray, foci negative.

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Figure 3.4: Overexpression of ectopically expressed IC-WT in the absence of POLIC RNAi. (A) Growth curves of uninduced and tetracycline-induced procyclic cells. Error bars represent the standard deviation from the mean of three biological replicates. (B) Western blot detection of the PTP tag and HSP70 protein levels following 10 days of induced overexpression. 1x106 cell equivalents loaded except for +C where 5x106 cell equivalents was loaded. +C, POLIC-PTP single expresser cell line. (C) Representative images of OE IC-WT-PTP throughout the cell cycle. Scale bar, 5 μm. (D) Quantification of PTP foci formation at each cell cycle stage. Percentages refer to the fraction of total cells in the population. Green/white hashed, POLIC-PTP foci positive; gray, foci negative. (E) Representative Southern blot showing the effect on free minicircle population. k, kDNA network; N/G, nicked/gapped; L, linearized; CC, covalently closed; T, tubulin.

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Figure 3.5: IC-WT displays nucleotide incorporation activity. (A) Representative immunoblot analyses of 5 µL eluate from POLIC variant immunoprecipitation from three separate experiments. IP products were run on an 8% SDS-PAGE gel, blotted, and detected with PAP reagent (Sigma). 3X value indicates the number of total cells that were isolated for IP. Protein marker masses are indicated in kilodaltons (kDa). (B) Representative assay for DNA incorporation activity of Klenow fragment (Invitrogen) and POLIC variants in vitro as measured by counts per minute (CPM) in a scintillation counter from three separate experiments. K, Klenow, numbers underneath indicate enzyme units (U) input; WT, IC-WT; DEAD, IC-DEAD; POLA, IC-POLA, UCR, IC-UCR; numbers indicate µL of eluate input; 29-13, 29-13 cell lysate; Beads, beads only; Buffer, buffer only. Error bars represent standard deviation from two technical replicates.

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Figure 3.6: Overexpression of IC-DEAD results in a dominant-negative phenotype while complementation exacerbates loss of fitness. (A) POLIC-DEAD domain structure with the asp acid residues mutated to alanine indicated. Growth curves of uninduced and tetracycline-induced POLIC-DEAD (OE, RNAi+OE) procyclic cells. Error bars represent the standard deviation from the mean of three biological replicates. (B) RT-qPCR analysis of uninduced (black) and induced (blue/white hashed) cells following 48 hours of tetracycline induction. Data are normalized to TERT and error bars represent the standard deviation from the mean of three biological replicates. IC UTR, POLIC 3’ untranslated region; IC ORF, POLIC open reading frame; IB ORF, POLIB open reading frame; ID ORF, POLID open reading frame. Asterisks represent statistical significance based on pairwise Student’s T-test. * p < 0.1; ** p < 0.01; *** p < 0.001. (C) Western blot detection of the PTP tag and HSP70 protein levels following 10 days of induction. 1x106 cell equivalents was loaded into each well except for C where 5x106 cell equivalents was loaded. +C, single expresser control cell line. (D) Representative images of IC-DEAD at each cell cycle stage. DAPI-staining (blue); anti-protein A (green); YL1/2 (red). Scale bar, 5 μm. (E) Quantification of PTP foci at each cell cycle stage in the overexpression (OE, top) and complementation (RNAi+OE, bottom) cell lines after 48 hours of induction. Foci positive (Green/white hashed); foci negative (gray). Error bars represent the standard deviation from the mean of three biological replicates.

93

Figure 3.7: Characterization of IC-POLA and IC-UCR overexpression cell lines. (A) Western blot detection of the PTP tag and HSP70 or alpha tubulin protein levels following 10 days of induction in the OE IC-POLA cell line. (B) Quantification of kDNA morphology over the course of the induction in the OE IC-POLA cell line. Over 300 cells were scored at each time point and error bars represent standard deviation from the mean of three biological replicates. Gray, normal kDNA; yellow, small kDNA; red, no kDNA; purple, ancillary kDNA; orange, asymmetric size; green, other. (C) Representative Southern blot showing the effect on free minicircle population in the OE IC-POLA cell line. k, kDNA network; L, linearized; N/G, nicked/gapped; CC, covalently closed; T, tubulin. (D-F) Same as A-C but for the OE IC-UCR cell line.

95

Figure 3.8: kDNA replication phenotype analysis of IC-DEAD overexpression and complementation. (A) Quantification of kDNA morphology for OE IC-DEAD over the course of the induction demonstrating progressive loss of kDNA. Over 300 DAPI-stained cells were scored at each time point and error bars represent standard deviation from the mean of three biological replicates. Gray, normal kDNA; yellow, small kDNA; red, no kDNA; purple, ancillary kDNA; orange, asymmetric size; green, other. (B) Representative Southern blot showing the effect on free minicircle population. k, kDNA network; N/G, nicked/gapped; L, linearized; CC, covalently closed; T, tubulin. (C) Ratio of CC:N/G over the course of the induction. Phosphoimager quantification was used to plot the relative abundance of unreplicated CC and newly replicated N/G intermediates. Error bars represent the standard deviation from the mean of three biological replicates. (D-F) Same as A-C but for the RNAi + IC-DEAD cell line.

97

Figure 3.9: IC-POLA complementation is not sufficient to rescue the RNAi phenotype (A) Growth curves of uninduced and tetracycline-induced IC-POLA (OE, RNAi+OE) procyclic cells. (B) RT-qPCR analysis of uninduced (black) and induced (blue/white hashed) cells following 48 hours of tetracycline induction. Data are normalized to TERT and error bars represent the standard deviation from the mean of three biological replicates. IC UTR, POLIC 3’ untranslated region; IC ORF, POLIC open reading frame; IB, POLIB; ID, POLID. Asterisks represent statistical significance based on pairwise Student’s T-test. * p < 0.1; ** p < 0.01; *** p < 0.001. (C) Western blot detection of the PTP tag and alpha tubulin protein levels following 10 days of induction. (D) Representative images of IC-POLA at each cell cycle stage. Scale bar, 5 μm. (E) Quantification of foci formation at each cell cycle stage. Green, foci positive; gray, foci negative.

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Figure 3.10: kDNA replication phenotype analysis of IC-POLA and IC-UCR complementation. (A) Quantification of RNAi + IC-POLA kDNA morphology over the course of the induction. Over 300 cells were scored at each time point and error bars represent standard deviation from the mean of three biological replicates. Gray, normal kDNA; yellow, small kDNA; red, no kDNA; purple, ancillary kDNA; orange, asymmetric size; green, other. (B) Representative Southern blot showing the effect on free minicircles of RNAi + IC-POLA. k, kDNA network; L, linearized; N/G, nicked/gapped; CC, covalently closed; T, tubulin. (C) Quantification of the CC:N/G ratio over the course of the induction. Phosphoimager quantification was used to plot the relative abundance of unreplicated CC and newly replicated N/G intermediates. Error bars represent the standard deviation from the mean of three biological replicates. (D-F) Same as A-C but for the RNAi + IC-UCR cell line.

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Figure 3.11: IC-UCR complementation is not sufficient to rescue the RNAi phenotype. (A) Schematic representation of IC-UCR variant. Growth curves of uninduced and tetracycline-induced IC-UCR (OE, RNAi+OE) procyclic cells. (B) RT-qPCR analysis of uninduced (black) and induced (blue/white hashed) cells following 48 hours of tetracycline induction. Data are normalized to TERT and error bars represent the standard deviation from the mean of three biological replicates. IC UTR, POLIC 3’ untranslated region; IC ORF, POLIC open reading frame; IB ORF, POLIB; ID ORF, POLID. (C) Western blot detection of the PTP tag and HSP70 protein levels following 10 days of induction. (D) Representative images of IC-UCR at each cell cycle stage. Scale bar, 5 μm. (E) Quantification of PTP foci formation at each cell cycle stage. Green/white hashed, foci positive; gray, foci negative. Error bars represent the standard deviation from the mean of three biological replicates.

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CHAPTER 4

4. LOOKING FORWARD

4.1 Looking backwards (briefly) The body of work contained in this thesis demonstrates one key philosophical principle of

trypanosomatid organisms: they defy many conventional understandings of biology (it has also

been argued that such “bizarre and cumbersome” mechanisms like RNA editing suggest a

disbelief in intelligent design. Englund, 2014). What seems counterintuitive prima facie is that T.

brucei also serves as a great model organism to study mitochondrial biology because of its

divergence to mammalian mitochondria, but the single-unit nature of both the mitochondrion

organelle and the mtDNA nucleoid allow careful investigation into mtDNA replication,

maintenance, and inheritance. The advances in molecular and cell biology techniques, most

notably genome sequencing and experimentally induced RNAi have allowed T. brucei researchers

to discover and assign function to proteins involved in these processes.

Searching for the mitochondrial DNA pol γ (the sole replicative DNA polymerase for mitochondria

in animals) from fractionated mitochondrial extracts of Crithidia fasiculata did not yield a

homolog. Instead, genome sequencing of T. brucei revealed four bacterial Pol-I like genes that all

localized to the mitochondrion (Klingbeil et al., 2002). This initial discovery of the three essential

kDNA polymerases in T. brucei, the original paper’s final line stated, “Further studies are

underway to determine the precise role of this enzyme [POLIC]”, though the following

investigations in subsequent years revealed roles for POLIB and POLID in kDNA replication with

no clearly-defined role for POLIC (Bruhn et al., 2010; Bruhn et al., 2011; Chandler et al., 2008;

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Concepción-Acevedo et al., 2012; Concepción-Acevedo et al., 2018). Identification of a role

beyond replication for POLIC as this thesis demonstrate helps complete the picture of kDNA

transactions occurring in this remarkable organism.

4.2 Aligning our data with the kDNA field Two key takeaways from this thesis are the well-defined kDNA S phase POLIC localization pattern

in relation to the genome inheritance factor TAC102 (from Chapter 2) and the discovery of an

antipodal site determinant in POLIC’s primary structure. Taken together, this suggests a model

where POLIC’s multiple roles are compartmentalized via cell cycle-dependent localization: that is

to say that POLIC may participate in direct kDNA transactions while in the KFZ early in kDNA S

phase, and then participate in directing symmetrical network division at the antipodal sites (see

Figure 2.4 for this localization/function model).

4.2.1 Further defining role of POLIC utilizing the cell lines generated To further bolster the work of this thesis, several key experiments would be beneficial to more

precisely address the biological role of POLIC utilizing the cell lines generated from this work.

Transmission electron microscopy of thin sections of kDNA networks would show impacts on its

ultrastructure (size and topology) to determine if networks are still intact and properly

compacted. Furthermore, nanoparticle labeling would reveal the distribution of mutant protein

and/or TAC protein(s) of interest. Fluorescence in-situ hybridization would give us greater

specificity as to what is being impacted in our mutant POLIC cell lines utilizing probes that are

specific for minicircles or maxicircles under denaturing or non-denaturing conditions to

distinguish between covalently closed and nicked/gapped molecules. Quantification of minicircle

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replication defects by TdT labelling would tell us if the POLA or UCR variants are involved

specifically in minicircle replication. A major hurdle encountered during this time was the use of

Southern blotting to examine specific effects on the minicircle and maxicircle populations and

further troubleshooting may help understand the specific impacts on kDNA these variants

produce. The best TAC marker that was most proximal is TAC102 but using an antibody against

MiRF172 and fluorescently labeling would help further define the function of POLIC as MiRF172

may also play a role between kDNA replication and segregation. MiRF172 shares no overall

similarities with other proteins except for low complexity regions at the C-terminus with weak

sequence similarity to a putative kinase from Dictyostelium and transcription corepressor (trfA),

but was found to be involved in minicircle reattachment and could be involved in both replication

and segregation (Amodeo et al., 2018).

4.2.2 Interconnections among kDNA polymerases The strong replication defect from POLIB or POLID silencing (and mild defect from POLIC

silencing), the phenotype seen from inducing expression of additional POLIC during POLIB or

POLID silencing, and the presence of aberrant kDNA phenotypes exclusively with POLIC silencing

further support the model of essential and distinct kDNA polymerases with similar but

nonredundant functions. However, some level of interdependency must exist as they all

participate together to choreograph kDNA maintenance. Generating cell lines to knock down one

polymerase and tag the other to examine effects on cell cycle dependent localization would help

elucidate how these proteins may be acting in a sequential manner during kDNA S phase.

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4.2.3 Narrowing in on the antipodal site determinant Importantly, this thesis has also demonstrated the existence of an antipodal site determinant

existing in the UCR of POLIC. Post translational modifications such as the experimentally

determined arginine methylation of POLIC may have an effect by its methylation state (Fisk and

Read, 2011). Since POLIC shows primary sequence-dependent localization to the antipodal sites,

other AS proteins may have sequence conservation in their respective non-catalytic primary

structure. POLID shows a very similar AS localization phenotype and may also contain a conserved

AS determinant (Concepción-Acevedo et al., 2012). A genetics approach by mutating the UCR to

observe ablation of AS localization could be done through a few rational approaches, each relying

on bioinformatics data (except the first):

A. A blind approach of random mutations targeted to the UCR region of POLIC.

B. Mutations of conserved regions found in the POLIC UCR among kinetoplastid organisms.

C. Mutations of conserved regions found in the POLIC UCR among AS proteins.

Populations of transfected cell lines could be dilution cloned and then quickly screened to detect

loss of AS localization. Positive clones would then be further characterized and sequenced.

4.2.4 Addressing ancillary kDNA Ancillary kDNA was originally described to be non-replication intermediate minicircle-containing

structures in T. cruzi with confirmed mitochondrial origin using mitochondrial heat shock protein

mtp70 (Miyahira and Dvorak, 1994). Furthermore, treatment with a topoisomerase I and II

inhibitor did not affect the frequency of these structures in stocks of different developmental

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stages of the organism. Ancillary kDNA structures are also induced due to perturbations with

expression of a variety of kDNA-related proteins with no clear connection between them. The

simplest explanation for this is that the “ancillary phenotype” detected in these experiments is

more heterogeneous than we understand, especially considering multiple ancillary kDNAs are

detected upon perturbing TAC102 and PNT1 but is not seen in all cases. It would be helpful to

know if TAC102 is also detected with ancillary kDNA in these other experimental cell lines, or if

kDNA FISH probes tell us the genetic makeup is similar, i.e. that they are minicircles. FISH

experiments showed that ancillary DNA was not nuclear but originated from the kinetoplast (ref)

and specifically minicircle DNA, whereas there was no detection with a maxicircle FISH probe

(ref). However, the discovery of ancillary kDNA in T. cruzi demonstrated that under

nondenaturing conditions, it could not be detected meaning that replication intermediates could

not be detected. When acyl carrier protein (ACP) is depleted in BSF T. brucei, ancillary kDNA could

be detected and maxicircles could not. This could be due to differences in replication between

the two organisms, or that the species of ancillary kDNA is different depending on how kDNA

maintenance is affected. FISH analysis may help pinpoint the type of defect underlying their

presence. Additionally, promastigote-like cells in T. brucei undergo sexual reproduction and

display either one or two kinetoplasts at both anterior and posterior positions relative to the

nucleus, with only one being associated with the flagellum (Peacock et al., 2014). Presence of

ancillary kDNA could be due to messing up the meiosis pathway.

What might be the connection between seemingly disparate proteins whose inhibited functions

result in the presence of ancillary kDNA? Ancillary kDNA appears to arise when mitochondrial

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structure is perturbed in some fashion. Impacts to kDNA distribution (POLIC), TAC structure

(TAC102), protein import (Tim17), mitochondrial membrane phospholipid content (ACP), or a

cysteine peptidase either directly (PNT1) or indirectly (PUF9), all result in the appearance of

ancillary kDNA.

The unusual connection between POLIC and ancillary kDNA in conjunction with pleiotropic

defects exhibited from POLIC silencing indicated a role beyond kDNA replication. Our data

demonstrate a clear distinction between the role of POLIC and the other replicative kDNA

polymerases in kDNA maintenance. However, the underlying mechanisms of how POLIC

functions in both replication and distribution are still not well understood. Taking all the data

together, we hypothesize that POLIC has a limited role in kDNA replication, leading to a more

gradual shrinking of networks than with what is seen in POLIB or POLID silencing. Small networks

undergoing scission may not correctly line up at the scission plane, thereby producing

asymmetrically sized networks in the cell. Additionally, the role of POLIC in distribution could be

due to interacting partners such as MiRF172 which is involved in minicircle reattachment. Though

currently unknown, factors connecting kDNA to the TAC could be impacted by POLIC silencing,

where they are weakened and disconnect from the segregation machinery, leading to the

appearance of ancillary kDNA.

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Experiment FeaturePercent of population

Nuclear probe shows negative.Conserved minicircle probe containing 120 bp of minirepeat region specific for T. cruzi .Detection requires denaturing conditions.Appeared to move freely in the cytoplasm (video).mitochondrial localization confirmed by mtp70 co-localization.All stocks of the order displayed aDNA.No effect on topoI inhibitor (nDNA replication is not involved in their production).Unclear effect from topoII inhibitor.May have been liberated prior to or following kDNA replication.Result of random kDNA replication errors and is of no consequence to the organism.

POLIC RNAi in PCFCells with multiple DAPI-staining spots on occasion showed a morphology similar to that of ancil lary kDNA (not shown).

PUF9 RNAi in BSF, TAP of PUF9

PUF9 depletion destabilizes its target RNAs (LIGKA, PNT1, PNT2) and results in accumulation of G2-phase cells. Extra nuclei or kinetoplasts were seen after 24 hours. PCF experiments showed no obvious phenotype.33% of cells showed small and faintly staining kinetoplasts after 8 hours, relative to 5% in uninduced cells.myc formed a double closely flanking the kDNA or overlapping it.extra structures could represent degenerating kinetoplasts retained from a sister cell during division, fragmented kinetoplasts, or semi-synthesized kinetoplasts formed by aborted, late re-replication.1-3% lacked a normal kineoplast and only contained ancil lary or none at all. In these cells, total myc signal was dramatically increased and detected throughout the mitochondrion.

Six days: 25% no kDNA, 19% big kDNA, 6% small.Networks became more heterogenous but rarely beyond double size.Two types of segregation defects: complete block and cell with double network and cell with none. Asymmetric division with one progeny with larger and one progeny with smaller than normal.18% of cells had asymmetrically dividing kinetoplasts after six days.After 4 days: 13% had ancil lary kDNA.

FISH under conditional

knockout

Detectable with minicircle probe, not with maxicircle probe (either not present or below detection threshold). They are not covalently closed molecules since the probe would not hybridize.

Conclusions ACP loss may promote network fragmentation and those fragments may disconnect from TAC.

ConclusionsWhy seen in BSF but not PCF? PCF reliance on conventional respiratory chain die of that before emergence of kDNA segregation phenotype.

Morphology of gamete-liked trypanosomes 1K1N and 2K1N promastigote-like cells (PL) discovered.YFP::p166 localization p166 in 2K1N PL cells show intensity usually greater in anterior than posterior TAC.

Light microscopy2K1N PL cells had two kinetoplasts widely separated at two poles of the cell, on either side of the nucleus.

YFP::PFR1 localization

PFR1 in 23 of 26 (88%) 2K1N PL cells showed single flagellum arising near anterior, with the 3 remaining cells showing two flagella, the posterior flagellum appearing as a tiny dot near the posterior kinetoplast.

DNA content measurement 12 of ~600 examined from SG spil lout were PL dividing cells.

Conclusions

Both 1K1N and 2K1N PL cells have enlarged kinetoplasts. 1K1N and 2K1N PL cells might arise due to unequal division of a 3K1N cell, since both were found in equal numbers. Both 3K1N and 3K2N cells were observed.

up to 10% incidencefragmentation

recoded HA tagged PNT1 Localized within or near AP sites, more prominent in dividing kinetoplasts.PNT1 RNAi 46% 1N0K after 4 days.

36% of cells had a mislocated kinetoplast after 2 days.PNT1 was also localized on these incorrectly positioned kinetoplasts.PNT1 OE cells showed mislocated kinetoplasts anterior to the nucleus at all cell cycle stages.

25% of cells show additional foci after 5 days of OE.Some TAC102 foci co-localize with small ancil lary kinetoplasts.More than 80% of cells with ancil lary kDNAs have one "extra" kDNA per cell.Partial rescue (2 day delay).>98% of cells after 5 days sti l l have kDNA, in many cases additionally to the proper posterior location also at non-conventional positions within the mitochondrion.Ancil lary kinetoplasts co-localized with additional TAC102 punctae.Of ancil lary kDNA cells, >60% had just one "extra".No rescue.After five days, >60% of cells have lost their kDNA, while 14% showed ancil lary kDNAs.Of ancil lary kDNA cells, they contained one, two, or three with similar frequencies (and a few with more).N-terminus of TAC102 is important for the connection to upstream components of the TAC (closer to the basal body) and its deletion or obstruction by a tag leads to partial loss of function (proper localization).The c-terminal portion is sufficient to either directly connect to kDNA or initiate the assembly of downstream components that connect to the kDNA and thus initiate the appearance of ancil lary kinetoplasts that have lost the connection to the upstream components of the TAC, and consequently, the basal body.

TAC102 Trikin et al 2016PCF T. brucei

mycΔN-TAC102 OE

Experiment-dependent but 25% after 5 days of OE, 14% after five days

of complementation.

myc-TAC102 complementation

mycΔN-TAC102 complementation

Conclusions

PCF 29-13

Tim17 RNAi Up to 10% of cellsPNT1 Grewal et al 2016

BSF T. brucei

PNT1-Myc OE 25-35%

conditional knockout

PL-liked cells Peacock et al 2014tsetse-transmissible, 2K1N PL cells

12 of 600 (~2%) of SG spil lout were PL

dividing cells.Tim17 Jirí et al 2015

13% ancil lary after 4 days in BSF

conditional KO.

PUF9 Archer et al 2009Derived from Lister 427 (BSF), PCF

PNT1::myc OE in PCF

ACP Clayton et al 2011BSF

33% after 8 hours in PCF

Miyahira and Dvorak 1994L. major, L. donovani, L. infantum, T. cruzi, T. rangeli, C lucil iae

FISH experiments and low light-level

video

Varied between members of the

Order. T cruzi showed a stock-

dependent positive correlation between

aDNA and population

doubling time (between single digit and 16%).

POLIC Klingbeil et al 200229-13 procyclic T. brucei with PZJM RNAi vector

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Table 4.1: Table summary of experiments that induce ancillary kDNA.

Experiment FeaturePercent of population

Nuclear probe shows negative.Conserved minicircle probe containing 120 bp of minirepeat region specific for T. cruzi .Detection requires denaturing conditions.Appeared to move freely in the cytoplasm (video).mitochondrial localization confirmed by mtp70 co-localization.All stocks of the order displayed aDNA.No effect on topoI inhibitor (nDNA replication is not involved in their production).Unclear effect from topoII inhibitor.May have been liberated prior to or following kDNA replication.Result of random kDNA replication errors and is of no consequence to the organism.

POLIC RNAi in PCFCells with multiple DAPI-staining spots on occasion showed a morphology similar to that of ancil lary kDNA (not shown).

PUF9 RNAi in BSF, TAP of PUF9

PUF9 depletion destabilizes its target RNAs (LIGKA, PNT1, PNT2) and results in accumulation of G2-phase cells. Extra nuclei or kinetoplasts were seen after 24 hours. PCF experiments showed no obvious phenotype.33% of cells showed small and faintly staining kinetoplasts after 8 hours, relative to 5% in uninduced cells.myc formed a double closely flanking the kDNA or overlapping it.extra structures could represent degenerating kinetoplasts retained from a sister cell during division, fragmented kinetoplasts, or semi-synthesized kinetoplasts formed by aborted, late re-replication.1-3% lacked a normal kineoplast and only contained ancil lary or none at all. In these cells, total myc signal was dramatically increased and detected throughout the mitochondrion.

Six days: 25% no kDNA, 19% big kDNA, 6% small.Networks became more heterogenous but rarely beyond double size.Two types of segregation defects: complete block and cell with double network and cell with none. Asymmetric division with one progeny with larger and one progeny with smaller than normal.18% of cells had asymmetrically dividing kinetoplasts after six days.After 4 days: 13% had ancil lary kDNA.

FISH under conditional

knockout

Detectable with minicircle probe, not with maxicircle probe (either not present or below detection threshold). They are not covalently closed molecules since the probe would not hybridize.

Conclusions ACP loss may promote network fragmentation and those fragments may disconnect from TAC.

ConclusionsWhy seen in BSF but not PCF? PCF reliance on conventional respiratory chain die of that before emergence of kDNA segregation phenotype.

Morphology of gamete-liked trypanosomes 1K1N and 2K1N promastigote-like cells (PL) discovered.YFP::p166 localization p166 in 2K1N PL cells show intensity usually greater in anterior than posterior TAC.

Light microscopy2K1N PL cells had two kinetoplasts widely separated at two poles of the cell, on either side of the nucleus.

YFP::PFR1 localization

PFR1 in 23 of 26 (88%) 2K1N PL cells showed single flagellum arising near anterior, with the 3 remaining cells showing two flagella, the posterior flagellum appearing as a tiny dot near the posterior kinetoplast.

DNA content measurement 12 of ~600 examined from SG spil lout were PL dividing cells.

Conclusions

Both 1K1N and 2K1N PL cells have enlarged kinetoplasts. 1K1N and 2K1N PL cells might arise due to unequal division of a 3K1N cell, since both were found in equal numbers. Both 3K1N and 3K2N cells were observed.

up to 10% incidencefragmentation

recoded HA tagged PNT1 Localized within or near AP sites, more prominent in dividing kinetoplasts.PNT1 RNAi 46% 1N0K after 4 days.

36% of cells had a mislocated kinetoplast after 2 days.PNT1 was also localized on these incorrectly positioned kinetoplasts.PNT1 OE cells showed mislocated kinetoplasts anterior to the nucleus at all cell cycle stages.

25% of cells show additional foci after 5 days of OE.Some TAC102 foci co-localize with small ancil lary kinetoplasts.More than 80% of cells with ancil lary kDNAs have one "extra" kDNA per cell.Partial rescue (2 day delay).>98% of cells after 5 days sti l l have kDNA, in many cases additionally to the proper posterior location also at non-conventional positions within the mitochondrion.Ancil lary kinetoplasts co-localized with additional TAC102 punctae.Of ancil lary kDNA cells, >60% had just one "extra".No rescue.After five days, >60% of cells have lost their kDNA, while 14% showed ancil lary kDNAs.Of ancil lary kDNA cells, they contained one, two, or three with similar frequencies (and a few with more).N-terminus of TAC102 is important for the connection to upstream components of the TAC (closer to the basal body) and its deletion or obstruction by a tag leads to partial loss of function (proper localization).The c-terminal portion is sufficient to either directly connect to kDNA or initiate the assembly of downstream components that connect to the kDNA and thus initiate the appearance of ancil lary kinetoplasts that have lost the connection to the upstream components of the TAC, and consequently, the basal body.

TAC102 Trikin et al 2016PCF T. brucei

mycΔN-TAC102 OE

Experiment-dependent but 25% after 5 days of OE, 14% after five days

of complementation.

myc-TAC102 complementation

mycΔN-TAC102 complementation

Conclusions

PCF 29-13

Tim17 RNAi Up to 10% of cellsPNT1 Grewal et al 2016

BSF T. brucei

PNT1-Myc OE 25-35%

conditional knockout

PL-liked cells Peacock et al 2014tsetse-transmissible, 2K1N PL cells

12 of 600 (~2%) of SG spil lout were PL

dividing cells.Tim17 Jirí et al 2015

13% ancil lary after 4 days in BSF

conditional KO.

PUF9 Archer et al 2009Derived from Lister 427 (BSF), PCF

PNT1::myc OE in PCF

ACP Clayton et al 2011BSF

33% after 8 hours in PCF

Miyahira and Dvorak 1994L. major, L. donovani, L. infantum, T. cruzi, T. rangeli, C lucil iae

FISH experiments and low light-level

video

Varied between members of the

Order. T cruzi showed a stock-

dependent positive correlation between

aDNA and population

doubling time (between single digit and 16%).

POLIC Klingbeil et al 200229-13 procyclic T. brucei with PZJM RNAi vector

117

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118

APPENDIX

GENETIC COMPLEMENTATION WITH IC-WT IN A POLIB OR POLID RNAI BACKGROUND

A.1 Introduction

Original studies on the kDNA polymerases in Trypanosoma brucei demonstrated that of the four

identified (POLIA, POLIB, POLIC, POLID), three are essential for parasite viability (Bruhn et al.,

2010; Bruhn et al., 2011; Chandler et al., 2008; Klingbeil et al., 2002). However, the single

expresser cell line analysis has revealed that POLIC is the least abundant of the three essential

kDNA polymerases. To determine if the role of POLIC is truly redundant, we generated cells lines

to induce expression of epitope-tagged POLIC under POLIB, POLID, or dual-gene silencing

conditions to see if additional POLIC could rescue the RNAi phenotype. These data show that

induced expression of additional POLIC is unable to rescue the loss of fitness from POLIB or POLID

silencing amd several clones that appeared to show a LOF rescue did not show any knockdown.

Taken together, these data demonstrate the non-redundant function of kDNA polymerases in T.

brucei.

A.2 Methods

A.2.1 RNAi.

The SLIB RNAi clonal cell line used was from Bruhn et al 2010. Briefly, 500 bp of POLIB sequence

was PCR amplified from TREU 927 gDNA using primers with appropriate restriction enzyme

linkers (forward: 5ʹ-AAGATG AGCGTGTCAACGAGG-3ʹ and reverse: 5ʹ-GGTAAACCG

TGGCGCGACGGAGG-3ʹ) to generate the two fragments for subsequent cloning steps, using the

same region for the pZJMIB vector previously reported (Klingbeil et al 2002). The resulting vector,

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pStLIC3¢UTR was EcoRV-linearized for genome integration. The SLID RNAi clonal cell line used was

from Chandler et al 2008. Briefly, the same region of the POLID coding sequence was used for

the construction of the stem-loop vector pSLID, as previously described by Wang et al. The

resulting stem-loop plasmid contains two copies of the POLID fragment in opposite orientations

separated by the stuffer fragment. pSLID was linearized with EcoRV for genome integration. To

generate the stemloop construct pSLDB, nucleotides 694 – 1193 of the TbPOLID coding sequence

(Gene ID Tb11.02.0770) were PCR amplified with forward (5’- GAG TCT AGA CGT GAT TGC TTA

GTA AGT TGG -3’) and reverse (5’- TAT GAG CCA TGG GTA CGA ATC AGT GCC CAA GTG G) primers

containing XbaI and NcoI linker (underlined). The resulting product was purified, restriction

enzyme digested with XbaI and NcoI, and ligated into vector pL4440 to generate pL4440-ID.

Nucleotides 275 - 774 of the TbPOLIB coding sequence (Gene ID Tb11.02.2300) were PCR

amplified by forward (5’- TAT GAG CCA TGG AAG ATG AGC GTG TCA ACG AGG -3’) and reverse

(5’- TAT GAG AAG CTT GGT AAA CCG TGG CGC GAC GAG G -3’) primers containing NcoI and HindIII

linkers (underlined). This fragment was digested and ligated into pL4440-ID to create pL4440-DB.

The 1020 bp region containing both TbPOLID and TbPOLIB fragments, DB, was PCR amplified from

pL4440-DB using forward (5’-GAG TCT AGA CGT GAT TGC TTA GTA AGT TGG-3’) and reverse (5’-

TAT GAG ACG CGT GGT AAA CCG TGG CGC GAC GAG G-3’) primers containing XbaI and MluI

linkers (underlined). Resulting fragment DB was restriction digested with XbaI and MluI and then

ligated into pLEW100 to generate pLEW100-DB. Separately, fragment DB was digested with

HindIII and NheI and ligated into pJM326 to create pJM-DB. To create the final stemloop vector,

pLew100-DB was digested with XbaI and HindIII, and the vector backbone (6316bp) was gel

extracted. pJM326-DB was digested with XbaI and HindIII, the insert (DB+stuffer ~1500bp) was

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gel extracted. The final pSLDB was acquired by ligation of vector backbone and insert. The

fragments used to make the dual gene construct are the same regions that were used for the

single gene silencing constructs (Chandler 2008, Bruhn 2010).

A.2.2 Inducible expression of epitope-tagged POLIC.

To create pLew100-PTPPuro for inducible expression of PTP tagged POLIC variants, the Myc tag of

pLew100-MycPuro (gift from Dr. Laurie Read, SUNY Buffalo) was replaced with the PTP tag. Briefly,

the PTP tag was PCR amplified from pC-PTP-NEO (Schimanski et al., 2005) and cloned into pCR®-

Blunt II-TOPO vector. Site-directed mutagenesis was used to introduce a silent mutation and

eliminate an internal BamHI site generating pTOPO-PTP-ΔBamHI. The Myc tag was replaced with

PTP-ΔBamHI to generate pLew100-PTPPuro. The full-length open reading frames of wild-type

POLIC was cloned into pLew100-PTPPuro (pLewIC-WT-PTPPuro) and verified by direct sequencing.

A.2.3 Trypanosome cell culture and transfection.

Procyclic form T. brucei strain 29-13 was cultured at 27˚C in SDM-79 medium supplemented with

FBS (15%), G418 (15 µg/mL) and hygromycin (50 µg/mL) (Wirtz et al., 1999). pLewIC-WT-PTPPuro

(15 µg) was transfected by nucleofection using the Amaxa Nucleofection Parasite Kit (Lonza) into

parental RNAi cells to generate complementation cell lines. Following selection with 1 µg/mL

puromycin, cell lines were cloned by limiting dilution. All cell lines were maintained between

1x105 – 1x107 and were not continuously cultured for more than three weeks. Cell density was

determined using a Beckman Coulter Z2 particle counter. RNAi and overexpression were induced

by the addition of tetracycline (1 µg/mL). Cultures were supplemented with 0.5 µg/mL Tet on

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non-dilution days to maintain IC-WT expression and POLIB, POLID, or dual RNAi (Rusconi et al.,

2005).

A.2.4 RNA isolation and RT-qPCR analysis.

Total RNA was isolated from 5x107 cells with Trizol (Thermo Fisher) using standard procedures.

RNA concentration was determined by Nanodrop and 100 ng of total RNA was reverse

transcribed to cDNA with random primers and RNase inhibitor (Applied Biosystems). qPCR was

performed using QuantiNova SYBR Green PCR (Qiagen) with 1 µg of cDNA and 0.2 µM of primer

per reaction with a Stratagene MxPro 3000x thermocycler. All reactions were performed in

triplicate and qPCR results were determined from three biological replicates. Gene knockdown

was normalized using TERT (Brenndörfer and Boshart, 2010).

A.2.5 SDS-PAGE and Western blot analysis.

Cells were harvested and washed once with PBS supplemented with protease inhibitor cocktail.

Samples were fractionated by SDS-PAGE and transferred overnight onto a PVDF membrane in 1X

transfer buffer containing 1% methanol. Membranes were blocked in Tris-buffered saline (TBS)

+ 5% non-fat dry milk for at least one hour. Peroxidase-Anti-Peroxidase soluble complex (PAP)

reagent (1:2000, 30 min) (Sigma, P1291) was used for PTP tag detection. For subsequent

detections where stripping was required because proteins of interest were similarly sized to the

loading control, membranes were stripped with 0.1 M glycine (pH 2.5, 15 min, 37 °C), washed in

TBST (0.1 % Tween-20), blocked and re-probed with specific Crithidia fasciculata anti-Hsp70

(1:5000, 1 h) (Johnson and Englund, 1998) and secondary chicken anti-rabbit-HRP (1:2000, 1 h)

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(Santa Cruz, sc-516087). Signal was detected with Clarity™ ECL Blotting Substrate (NEB) and on a

GE Imagequant LAS 4000 mini. Quantification of Western blot intensities was performed using

ImageJ software (http://imagej.nih.gov/ij/).

A.3 Results and Discussion

To determine if increased abundance could rescue the RNAi phenotype of POLIB, POLID, or dual-

gene silencing, genetic complementation cell lines were generated and clonal cell lines were

screened for growth inhibition. Four clones for the SLIB+IC-WT cell line where characterized by

growth curve analysis and in all but one case showed no rescue from LOF (Fig A.1 A). These clones

were subsequently checked by Western blot analysis to determine if IC-WT was being expressed.

The results show that expression after 48 hours is variable among each clone, and was almost

undetectable for clone P2D6, with P1A3 showing the highest levels of expression and P1A1 and

P2D2 showing relatively equivalent expression (Fig A.1 B). Because clone P1A3 appeared to show

rescue from LOF, qPCR was performed to see if POLIB was actually being knocked down. Relative

to uninduced cells, all three kDNA polymerases appear to show mild overexpression, and

revealed that POLIB was no longer being knocked down.

In similar fashion, three clones for the SLID+WT-IC were screened for growth inhibition. P1A2 and

P1C2 showed no rescue, while P2B4 appeared to demonstrate a rescue from LOF (Fig A.2 A).

Western blot analysis showed that all three clones were expressing with P1A2 and P2B4 being

relatively stronger expressers than P1C2 (Fig A.2 B). The apparent growth phenotype rescue in

clone P2B4 was shown to be due to the loss of POLID RNAi (Fig A.2 C).

http://imagej.nih.gov/ij/

123

Lastly, three clones for the SLDB+IC-WT cell line were screened to see if full-length IC-WT could

rescue loss of both POLIB and POLID. No rescue was observed and Western blot analysis revealed

that all three clones were expressing IC-WT, albeit at varying levels (Fig A.3).

Why were apparent rescues of LOF due to loss of RNAi? Genome integration of the stem-loop

RNAi plasmid utilizes that same genomic regions as the pLEW100PTPPURO plasmid. Namely,

there are nine copies of the rRNA genes throughout different chromosomes, and integration at

different non-transcribed spacer regions likely do not provide equal genetic control. During

transfection of the full-length IC-WT expression construct, there is a chance for the already-

transfected inducible RNAi machinery to be excised and replace with IC-WT which is also targeted

for the rRNA spacer region. These results show that increasing expression levels of POLIC cannot

compensate for loss of the other essential kDNA polymerases.

124

Figu

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.1: O

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n of

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an

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ackg

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) Gro

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ells

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Wes

tern

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125

Figure A.2: Overexpression of IC-WT in an SLID RNAi background. (A) Growth curves of uninduced and tetracycline-induced IB-RNAi + IC-WT procyclic cells. (B) Western blot detection of the PTP tag and HSP70 protein levels following 2 days of induction. (C) RT-qPCR analysis of uninduced (blue) and induced (orange) cells of clone P2B4 following 48 hours of tetracycline induction. Data are normalized to TERT.

126

Figure A.3: Overexpression of IC-WT in an SLDB RNAi background. (A) Growth curves of uninduced and tetracycline-induced IB-RNAi + IC-WT procyclic cells. (B) Western blot detection of the PTP tag and HSP70 protein levels following 2 days of induction.

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