Transcription Initiation Factor II H (TFIIH) and its Effects on the Recruitment of Rad50 to Double Strand Breaks in Yeast This study investigated the role of Transcription Initiation Factor II H (TFIIH) in the recruitment of repair proteins Rad50 and Rad52 to double strand breaks (DSBs) in yeast. The study found that in a mutant lacking the TFIIH subunit Rad3, recruitment of both Rad50 and Rad52 to DSBs was reduced. However, recruitment of the TFIIH subunit TFB1 still occurred in a mutant lacking Rad50. This suggests that TFIIH is involved in recruiting Rad50 to DSBs, rather than Rad50

1. 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 Zhu
Dr. 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
cs
HO x
x
x
x
HO
cs
xx
x
x
HO
x
x
cs
x
x
cs
x
x
HO
x
x
Induce DSB
Cross-link protein-DNA
IPIN
s3
s3hi
0
2
4
6
8
10
12
0 50 100 150 200 250
DegreeOccupancy
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
DegreeOccupancy
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
DegreeOccupancy
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 Genotype
TNX59- 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, H
IS3, 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