DNA Replication 1-General Principles2-The enzymology of DNA polymerases

3- Prokaryotic DNA Replication

4- Eukaryotic DNA Replication

A-General Properties:B-Bidirectionality and Priming problemsC-Catalytic Properties of DNA polymerase I

Polymerase5’->3’ exonuclease3’->5’ exonuclease and proofreading

A - The prokaryotic Replisome

A-The eukaryotic ReplisomeB- Dealing with Chromatin C-Dealing with linear chromosomes: Telomeres and Telomerase

B - Interference between Replication and Transcription• Not treated:Old experiments(Meselson & Stahl, Cairns)

Replication is bidirectional from theOrigin of Replication:(Cairns exp.)

• 1 origin of replication in most eubacterial chromosomes• several origins of replications inarchea, some eubacteria, and in eukaryotes

Replication is Semi-Conservative:1 parental strand is transmitted into each

daughter DNA molecule(Meselson & Stahl exp.)

DNA Replication by DNA polymerases : Copying the genetic material to prepare for cell division

Parental DNA

2 Copies

Fundamental properties of DNA Polymerases

O BaseO

P

OH

O-O

O Base nO

OH

5’

OO

P

O

O-O

OO

OH

5’

O

O-

PO

O

O-

PHO

O

Base n

Base n+1

:

+

O-

O-

PO

O

O-

P

O

HO

1) Catalyze the polymerization of deoxyribonucleotides in the 5’->3’ direction: (dNMP)n+dNTP -> (dNMP)n+1 + PPi

2) Require a template (usually DNA)

3) Require a primer (DNA or RNA)

dNTPs

5’3’ 5’

3’primer

template

5’3’ 5’

3’New strand

PPi

The bidirectionality problem

Synthesis of DNAis semi-discontinuous

Synthesis of DNA on thelagging strand requirescontinuous synthesis of primers

OH

BOH

PPP

rNTPs

OH

B

PPP

a32P-dNTPs

DNA synthesis

P

BOH

P

BOH

P

BOH

P

B

P

B

P

B

P

B

Alkaline hydrolysis

P

BOH

OH

P

BOH

OH

B

P

B

P

B

P

B

OHP

BOH

OH

Ribonucleotide with a 3’P-

: Diagnostic of a 5’RNA-3’DNA junction

Where do the primers used during DNA replication come from ?

The primers are made of RNA since RNA polymerases do not require primers. The existence of joint RNA-DNAmolecules was demonstrated by alkaline hydrolysis of Okazaki fragments.

alkaline hydrolysis

DNA Polymerase I = the prototype DNA polymerase

- Discovered in the late ‘50s by Arthur Kornberg (1959 Nobel Prize in Medicine)

- First DNA polymerase discovered

- 3 Enzymatic activities associated to three distinct active sites on a single polypeptide chainThe activities can be artificially separated by experimental treatment with the trypsin protease ( these fragments have no physiological relevance)

+NH3

COO-109 kD

34 kD 75 kD = Klenow Fragment

5’ -> 3’ Exonuclease

5’ -> 3’ Polymerase 3’ -> 5’ Exonuclease

Limited Tryptic proteolysis

- Me=divalent metal ion(usually Mg++)

Two metal ion mechanism for

DNA Polymerase I(T.Steitz)

-MeA=activates the 3’OH forattack on the a phosphate of theincoming dNTP (lowers pKa of 3’O)

-MeB=plays the dual role of stabili-zing the neg.charge that builds upon the leaving oxygen and chelatingthe and phosphates

-MeA and B stabilize both the structureand charge of the pentacovalenttransition state

http://www.doe-mbi.ucla.edu/CHEM125/0532Movie1.mov

How does DNA Polymerase I select correct nucleotides from incorrect nucleotides ?

Nucleotides formingWatson-Crick base pairs fit the active site(BLUE SQUARE)

Nucleotides formingnon Watson-Crick base pairs do not fit the active site andare ejected

Suggests that H-bonding per sedoes not contributeto nt selection byDNA polymerases

5’ 3’XTemplate = 24 nt

5’Labeled primer = 23 ntAfter extension = 24 nt

A,C,G,Tor F ?

How do DNA polymerasesselect the proper nucleotide:

Testing the importance of H-bonding in base pairs

for the fidelity of nucleotide

incorporation

Thymine Di-fluorotolueneCan H-bond with A Same size as

Thymine but cannot H-bond with A

Biochemistry, 41, 10256–10261

Steric Clashwith the Y416residue if a 2’-OHis present

Use riboNTP

Use riboNTP

Use dNTP

Use dNTP

The aromatic/large side chain found in DNA polymerases close to the dNTP binding site provides a steric gate against riboNTPs

CatalyticAsp

Discrimination for deoxynucleotides vs ribonucleotides by DNA polymerases

Alignment of sequences of polymerases active site

This reaction is not the reversal of the 5’->3’ polymerization: The attacking group is water rather than pyrophosphate(a hydrolysis rather than a pyrophosphorolysis). For this reason, the active sites of the polymerization and of the 3’->5’ exonuclease reactions must be different. This is essential forthe biological role of the 3’->5’ exonucleolytic reaction, which is toedit newly polymerized sequences.

3’-> 5’ exonuclease

activity of DNA

Polymerase I

OO

P

OH

O-

O Base n(now last base)

OH

5’

O

OO

P

O

O-O

OO

OH

Base n

Base n+1(Last Base of DNA)

O

O

H

H:

Base n+1

5’

O

O

P O-O P O-O

O-

Not making mistakes during polymerization is thermodynamically impossible

5’3’ 5’

templateA

5’3’ 5’

templateA

P1 P2

T5’

templateAC5’

3’

P1 = probability of incorporating the right nucleotideA:T base pair

P2 = probability of incorporating one incorrect nucleotidee.g. A C mismatch

P2

P1

= e(DG

AxC DG

A:T) /

RT(derived from the Boltzman distribution)

DGAxC DG

A:T= 3 kcal/mol. P2

P1

=0.01 or 1% - at least

DNA polymerases are much more accurate than 0.01 - HOW ??

Editing of newly synthesized DNA by the 3’->5’ exonuclease activity

5’3’ 5’

template

5’3’ 5’

Polymerization

5’3’ 5’

3’->5’ exo triggeredby the mistake

5’3’ 5’

3’->5’ exo

5’3’ 5’

Polymerization

template

Newstrand

Editing of mistakes require a switch between :polymerization mode and editing mode

Switch between Polymerizing and Editing Modesin DNA Polymerase: Structural Basis for “Proofreading”

Biological significance:-Allows the replacement of damaged or abnormal DNA sequences by “Nick translation”(important for DNA Repair Chapter)

-Also allows the removal of RNA sequences embedded in DNA

(removal of replication primers).

5’3’ 5’

5’3’ 5’

dNTPs PPi

3’3’New strand

nick“bad”DNA

The nick has moved

5’-> 3’ exonuclease activity of DNA

Polymerase I

OO

P

OH

O-

O Base 1O

O

O

OO

P

O

O-O

OO

O

Base 1

Base 2

5’end

P O-

O

-O

O

H

H:

Base 2(now base 1)

P O-

O

-O

New 5’end-O

The replisome of E.coli

1) Helicases

Unwind DNA at the replication forkin a reaction coupled to ATP Hydrolyis

2) Single-stranded DNA binding proteins (SSB)

Bind and stabilize the DNA in a singlestranded conformation after the meltingby helicases

3) The Primosome

Synthesizes RNA primers of the lagging strandContains Primase

4) DNA Polymerase III :The replicase

6) DNA topoisomerase II

Relaxes supercoiled DNA that forms ahead of the replication fork. Decatenates the final product

7) Rnase H Removes RNA primers

8) DNA Polymerase I

Removes RNA primersReplaces RNA primers with DNA by nick translation

8) DNA Ligase

Joins the Okazaki fragments

The Replisome of E.coli in action

“old model”

DNA Polymerase III Core

t Subunit

a- 130 kD = Catalytic site for polymerization

q - 10 kD = structural role ?

b Subunit = Sliding Clamp

A homodimer of 2 X 41 kDATP-dependent processivity factor = b clampPol.III Core is poorly processive by itself

gComplex = Clamp loader

4 polypeptidesATP-dependent conformational changesFacilitates the loading of the b clamp onto DNA

e- 27.5 kD = 3’->5’ editingexonuclease

Trends in Microbiology

15, 155-64 (2007)

Dimerization factorHolds two Pol. III cores together

Sliding b clamps provide processivity to DNA polymerase III

5’

3’

3’

Template Strand

NewlyReplicatedStrand

Polymerase III

b- Clamps

homodimer of 2 X 41 kDwrapped around dsDNA

The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (1)

Trends in Microbiology 15, 155-64 (2007)

Step1: The primase synthesizes a new RNA primer upstream in the lagging strand; the two polymerase

replicate DNA

Step2: A sliding clamp is assembledaround the new RNA primer;

primase dissociated

Trends in Microbiology 15, 155-64 (2007)

Step2: A sliding clamp is assembledaround the new RNA primer;

primase dissociated

Step3: the lagging strand polymerase detaches and associates with the newly

deposited sliding clamp

The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (2)

Step3: the lagging strand polymerase detaches and associates with the newly

deposited sliding clamp

Step4: the lagging strand polymerase start to synthesize the next Okazaki

fragment; primase will reinitiate synthesis of the next RNA primer

back to Step1Trends in Microbiology 15, 155-64 (2007)

The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (3)

Kelch et al. - Science 23 December 2011: Vol. 334 no. 6063 pp. 1675-1680

The Clamp loader use cycles of ATP hydrolysis to open and load

the sliding clamps around the primed DNA

Binding of the Clamp loaderto b-clamps;

Opening ofthe b-clamps;

Loading theb-clamps ona primer-templateduplex

Recyclingof the clamploader

What is the advantage of 3 Polymerase replisomes vs 2 Polymerases ?

Nature Structural & Molecular BiologyVolume: 19, Pages: 113–116 (2012)

Newsflash – Recent works shows that There are 3 core DNA polymerases associated with most replisomes in vivo

Science. 2010 328(5977): 498–501

Why is a triPolymerase replisome ?

• TriPol.III replisome is more processive

Nature Structural & Molecular BiologyVolume: 19, Pages: 113–116 (2012)

TriPol.III diPol.III

• TriPol.III replisome leaves less gaps tobe filled by Pol.I

-> DNA Replication is overall more efficient

Problem of Coordinated Nucleic Acids Synthesis in vivo: What happens when DNA Polymerases and RNA polymerases collide ?(speed of DNA polymerase >> speed of RNA polymerase )

Nature456, 762-66

(2008)

Nature 456, 762-66 (2008)

Collision between DNA Polymerases and RNA polymerases result in polymerases dissociation and in the use of the RNA synthesized by RNA

Polymerase as a primer for replication

Eukaryotic DNA Replication: it’s similar to bacterial DNA replication….but it’s different !

• Machinery is overall similar to that use for bacterial DNA replication(names are different…)

• Idiosyncraties of eukaryotic DNA replication are linked to the size and organization of eukaryotic genomes:

• large size of eukaryotic chromosomes and limited time for DNA synthesis requires multiple origins of replication

• Replication machinery needs to deal with nucleosome packaging of eukaryotic DNA

• Problem of linear chromosomes

Architecture of the Eukaryotic Replisomeis similar to that of the bacterial Replisome

• Pol e - Replicates leading strand

• Pol d - Replicates lagging strand

•Pol -primase

Complex containing both primase

and DNA polymerase a activities

PCNA:(proliferating cells Nuclear antigen):= trimeric sliding clamp

• Replication Protein A (RPA) = SSB

• MCM =

heterohexameric helicase

Mol.Cell30, 137-44

(2008)

• FEN1 = nuclease that

removes RNA primers

The eukaryotic cell cycle

Eukaryotic DNA Replication:The limited time for DNA replication (6-8 hours) combined to the increased size of the genomes (>107 base pairs) explain the requirement for multiple replication origins

Eukaryotic Replication needs to deal with the nucleosome packaging of eukaryotic DNA

Science 21 December 2007:Vol. 318. no. 5858, pp. 1928 - 1931

H3/H4

Example of Asf1, an H3/H4 histone chaperone that interacts with MCM

Asf1 remove H3/H4 histones upstream from MCM and reloadsthem after replication has proceeded

MCM

• Needs to Remove Histones upstream from the replication fork such that replication is not impeded• Needs to reassemble histones/nucleosomes on newly replicated DNA tomaintain chromatin structure and epigenetic marks

Histone chaperones

Replication

Asf1 can also add “new” H3/H4 histones since there is only ½ of the histones needed on the parental DNA

Eukaryotic DNA Replication: Problem of maintaining the ends of linear chromosomes is linked to the degradation of RNA primers

last primers on each 5’-end are removed but the gaps cannot be filled because of the lack of 3’-OH group

http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/1915.jpg

Each cycle of replication would result inprogressive chromosome shortening

Preservation of Telomeres by the telomerase

Telomerase (TERT):1 RNA subunit (template)several proteins:1 “reverse transcriptase”

After extension of the upper strand by telomerase,the replication machinery can now use this strandto make a new RNA primer using primase, then a new okazaki fragment and fill in the lower strand to “elongate” this strand.

The EMBO Journal21 January 2010/emboj 2009409

© 2010 European Molecular Biology Organization

The problem of Processivity in Telomerase : Telomerase is poorly processive in vitro - this cause issues because of the repeated nature of telomeric repeats

Pot1-Ttp1 form a proteincomplex that binds to ss telomeric DNA

Pot1-Ttp1 Complex increase processivity by:-decreasing telomerase dissociation from tel. DNA

-increase effciency of thetranslocation rate

The Pot1-Ttp1 complex provides processivity to telomerase in vivo

Pot1-Ttp1

Connections between telomerase and aging

Humans• Somatic tissues lack telomerase, possibly contributing to the normal physiological

symptoms of aging. Shortening of telomeres may lead to senescence in cultured human cells

• Stem cells, and cancer cells all contain telomerase activity possibly explaining their ability to divide indefinitely. Germ cells and early embryos also contain telomerase.

Mice• Telomerase protein or RNA mutant mice are fine for a few generations, perhaps

because of the extraordinarily long telomeres in laboratory mice

• After a few generations, the telomerase-mutant mice exhibit reduced fertility, signs of premature aging, and shortened life-span

Prematurely grey, balding

Accelerated telomere shortening in response to life stressPNAS 101 (49), 17312-15 (2004)

“Women with the highest levels of perceived stress have telomeres shorter on average by the equivalent of at least one decade of additional aging compared to low stress women”.