AbdelRahman et al. PNAS 98: 2538 2001

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Genome instability in tumor cells Truncations Tr
anslocations Inversions Duplications Amplification
s Deletions Mutations
Abdel-Rahman et al. PNAS 98 2538 (2001)
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In vertebrates, repair of chromosome breaks is
essential for life. Double-strand breaks (DSBs)
arise during normal DNA replication.
DSBs arising in chicken DT40 cells after Rad51 is
depleted (Takeda lab)
Ionizing radiation Mechanical
rupture Programmed DSBs (VDJ recombination MAT
switching)
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DSB
HO, I-SceI
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Saccharomyces MAT switching a model for
DSB-induced recombination and repair
HO endonuclease
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Saccharomyces MAT switching a model for
DSB-induced recombination and repair
Galactose-induced HO endonuclease
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How are double-strand breaks repaired? In
vivo biochemistry what happens to the DNA?
what proteins are doing   what?
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0 1 2 4 hr
Physical monitoring of gene conversion by
Southern blot analysis
MATa

MATa
HO cut
Connolly, White and Haber 1988
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HMLa
MATa
5 to 3 resection (MRX and ?) Strand invasion
(RPA, Rad51 etc) Primer extension (PCNA and
Pold and e) 3 nonhomology removal
(Ercc1-XPF) Strand displacement (Srs2) Second
strand synthesis
HMLa
MATa
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Visualizing synapsis between MAT and HML by
chromatin immunoprecipitation of Rad51
Crosslink proteins to DNA with formaldehyde
shear chromatin immunoprecipitate with
anti-Rad51 antibody reverse crosslinks PCR
amplify regions of interest
Non MAT DNA
MAT IP
HML IP
Xuan Wang, Neal Sugawara
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MATa
Watching synapsis between MAT and HML
Debra Bressan
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60
50
40
Cells with a single GFP spot ()
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20
10
0
0
60
120
180
240
300
360
Time after HO induction (min)
Suvi Jain Debra Bressan
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How does the cell know which donor to
chose? donor preference
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Donor preference during MAT switching
85-90
MATa
HMRa
HMLa
HMRa
MATa
HMRa
HMLa
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Donor Preference is controlled by the
Recombination Enhancer that acts over 50 kb to
control accessiblity of the left arm of
chromosome III. RE binds both forkhead and SCF
transcription factors, but there are no
transcripts nearby!
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HMRa
RE
CEN
MATa
We believe Fkh1 and Sw14/Swi6 make contact with
tethering sites to change the mobility or
position of the left arm of chromosome III
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Biophysics of live chromosome movement!
In collaboration with Jané Kondev (Physics)
Opportunities for biophysics students!
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The DNA damage checkpoint Preventing cell
cycle progression to allow more time for DNA
repair
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Analysis of homologous recombination (HO on for
1 hr)
HML
MAT
When HO endonuclease is continuously on
HO
0.2
gt 99 cells have a single broken chromosome
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arrest
adaptation
HO endonuclease
unrepaired
DSB
1 DSB
recovery
DSB
repaired
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arrest
HO endonuclease
1 DSB
recovery
How is the checkpoint turned off when DNA is
repaired? (Recovery)
DSB
repaired
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Turning the checkpoint off requires PP2C
phosphatases Casein kinase II Srs2
helicase Removing the phosphorylation from
histone H2AX requires chromatin remodeling
  factors PP4C phosphatase
recovery
DSB
repaired
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Farokh Dotiwala Wade Hicks Suvi Jain Jung-Ae
Kim John Lydeard Debra Bressan Eric Coïc Jake
Harrison Neal Sugawara André Walther Miyuki
Yamaguchi Qiuqin Wu
Mechanisms of DNA repair nonhomologous
end-joining          homologous
recombination Regulation of chromosome
architecture donor preference
DNA damage checkpoint establishment
maintenance adaptation
recovery
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