Transcription Initiation Factor II H (TFIIH) and its Effects on the Recruitment of Rad50 to Double Strand Breaks in Yeast

Maria Arciniega, Graham Barlow, Samuel Du, Irene Hsiung, Muhammad Jalal, Daniel Dragon Phan, Elise Wolf, Nancy ZhuDr. Adam Bailis, Dr. Ruben Petreaca, and Dr. Tina Negritto

Pomona College, Molecular Biology Program, 175 West 6th Street, Claremont, CA 91711

Introduction

Methods

Acknowledgements

Abstract

Lyse cells Sonicate DNA(≈0.5 – 1 Kb)

HOcshi

csHO x

x

xx

HOcsxxx

x

HOxx

csxx

csxx

HOx

x

Induce DSBCross-link protein-DNA

IPIN

s3

s3hi

0

2

4

6

8

10

12

0 50 100 150 200 250

De

gre

e O

cc

up

an

cy

Time after DSB Induction (min)

Association of Rad50-FLAG with DSB

Rad3 Mutant

WT

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 50 100 150 200 250

Deg

ree

Oc

cu

pan

cy

Time after DSB Induction (min)

Association of Rad52-FLAG with DSB

Rad3 Mutant

WT

Results

Discussion

+Gal

TP 0

TP 30

TP 60

TP 90

TP 120

TP 180

TP 240

Figure 3. Illustration of chromatin immunoprecipitation (ChIP). A DSB was induced by an HO endonuclease with the addition of galactose and samples were taken at various time points following break induction. Our proteins of interest, including either Rad50, Rad52, and TFB1, were cross-linked to the DNA and were immunoprecipitated using a FLAG-tag. DNA was purified to be analyzed by qRT-PCR. Protein fractions at multiple stages of ChIP were kept for Western blotting.

DNA double-strand breaks (DSBs) are a particularly dangerous type of DNA damage. One mechanism of DSB repair is homologous recombination (HR), which has been previously shown to rely on sequential recruitment of different proteins to the break. Rad3 is a helicase associated with TFIIH, which is a multi-complex basal transcription factor. Previously, it has been shown that a rad3 mutant (rad3-G595R) affects repair of the break through a pathway involving proteins Rad50 and Rad52. We hypothesize that Rad3 may be involved in the recruitment of canonical established machinery. Previous chromatin immunoprecipitaton (ChIP) studies have shown that TFB1, a TFIIH subunit, is recruited to DSBs and that this recruitment is lost in a rad3-G595R mutant. This would suggest that TFIIH has an additional function in DSB repair. We attempt to confirm the role of TFIIH at DSBs by determining the recruitment of other known DSB repair proteins in various mutant backgrounds. Remarkably, we find that in a rad3-G595R mutant, both Rad50 and Rad52 recruitment is reduced. However, in a Δrad50 mutant, TFIIH recruitment still occurs. This finding challenges the central dogma that Rad50 is the first protein to be recruited at DSBs. We propose instead that TFIIH is necessary for Rad50 recruitment.

Figure 1. Possible model for DSB repair by homologous recombination in S. cerevisae. Rad50 and Rad52 are known to be important in this repair pathway. However, the role of TFIIH in this pathway and the necessary conditions for its recruitment are still unknown.

Figure 2. The Sam1 genomic locus with an inserted HO endonuclease cut site. The HO cut site is expressed in trans from the trp1 locus with a GAL promoter. For a Southern blot, genomic DNA was then cut with either the SspI or SalI restriction enzymes and probed with a labeled Sam1 probe created by PCR. Expected band sizes and probe annealing site are indicated.

Figure 4. Southern blots of samples taken at various time-points during ChIP were analyzed to confirm the presence of a DSB at the expected genomic locus. Using the probe indicated in Figure 2, we could monitor the induction of the DSB over the course of the experiment. Degree of occupancy was calculated by performing qRT-PCR on DNA recovered from the ChIP experiment. We used primers that amplify a region immediately to the right of the DSB, normalized to β-actin expression. This allows us to measure and quantify the amount of DNA that was cross-linked with our proteins of interest during ChIP: Rad50, Rad52, and TFB1.

Proposed Mechanism of DSB Repair

0

1

2

3

4

5

6

7

8

9

0 50 100 150 200 250

De

gre

e O

cc

up

an

cy

Time after DSB induction (min)

Association of TFB1-FLAG with DSB

WT

Δrad50

Figure 5. Western Blot performed with α-FLAG antibodies and chemiluminescence to ensure that Rad52-FLAG was pulled down during the ChIP procedure. Whole cell extract was compared with the FLAG fraction that was bound to the antibody beads and the fraction of FLAG proteins that were found in the unbound supernatant. All bands ran as expected according to molecular weight.

We thank the Pomona College Molecular Biology Program for funding this project. We also thank the Spring 2015 MOBI188 class, whose lab work contributed to this project.

Results

Strain GenotypeTNX59- 1D MAT::LEU2, ade2-1, can1-100, leu2-3, 112,

trp1::HIS3::GAL::HO, ura3-1, his3-11, sam1∆BglII::Hocs(117), TFBI-FLAG-KNMX

ABX1936-20D MAT::LEU2, ade2-1, can1-100, leu2-3, 112, trp1::GAL::HO::KNMX, ura3-1, his3-∆BglII::HOcs(117), RAD5, RAD52-FLAG-KNMX, RAD3

TNX47- 7D MAT::LEU2, ade2-1, can1-100, leu2-3, 112, trp1::HIS3::GAL::HO, ura3-1, his3-∆BglII::Hocs(117), RAD52-FLAG-KNMX, rad3-G595R

TNX126-18B MAT::LEU2, ade2-1, can1-100, leu2-3, ura3-1, sam1∆BglII::HOcs(117), trp1::Gal::HO::KNMX, HIS3, RAD50-3XFLAG::KNMX, RAD3

TNX126-39C MAT::LEU2, ade2-1, can1-100, leu2-3, ura3-1, sam1∆BglII::HOcs(117), trp1::Gal::HO::KNMX, HIS3, RAD50

TNX129-3A MAT::LEU2, ade2-1, can1-100, leu2-3, ura3-1, sam1∆BglII::HOcs(117), trp1::HIS3::Gal::HO, his3-11, rad50::HISG, rad3-G595R

TNX129-12D MAT::LEU2, ade2-1, can1-100, leu2-3, ura3-1, sam1∆BglII::HOcs(117), trp1::HIS3::Gal::HO, his3-11, rad50::HISG, rad3-G595R

We propose that TFIIH is the first element of the canonical pathway recruited to a DSB. TFBI, a TFIIH subunit, is recruited to the DSB in both the wild-type and a rad50 null mutant. By contrast, while Rad50 shows high degree occupancy in the wild-type strain, it shows greatly reduced recruitment in the rad3-G595R mutant. This data suggests that TFIIH is involved in the recruitment of Rad50, which is critical for exonucleolytic end resection, and that it may also act as a helicase to open the DNA for Rad50. Our data also confirms that TFIIH is essential for recruitment of Rad52, as we see no recruitment in the rad3-G595R mutant.

rad3-G595R

rad3-G595R