1. 5678 910 11 12 I II III Homologous Recombination (HR) Repair of DNA damage Genome instability: recombination between dispersed repetitive sequences Translocation 5678 9101112 II III 91078 5611 12 II/III III/II 14 Deletion 12 4 I I I 1 2 3 4 Inversion 13 24 I Genome instability correlates with the development of cancer 1234 3

2. Double strand break (DSB) repair Homologous recombination Non-homologous end-joining (NHEJ)

3. What are the factors that are involved in the control of Short Sequence Recombination (SSR)? Rad3 5’ to 3’ DNA helicase Ssl1 Zinc finger protein, may modulate the helicase activity of Rad3 Ssl2 3’ to 5’ DNA helicase Components of core TFIIH involved in basal transcription and Nucleotide Excision Repair (NER). XPDp44 XPB Mutants cause increase in SSR and increased stability of DNA ends

4. Why unwind DNA? Transcription (Tuteja N and Tuteja R, 2004) Nucleotide Excision Repair (NER)

5. Why study RAD3? Xeroderma Pigmentosum (XP)Trichothidystrophy (TTD) 11aIIIIIIVV 587613 589615 V 654671 656673 R658C (TTD) R660C A594P (TTD) A596P G595R XPD Rad3 RAD3 mutants –rad3(A596P) = XPD(A594P) –rad3(R660C) = XPD(R658C) –rad3(G595R) isolated in yeast Mutations in human homologue, XPD, causes:

7. DNA stability assay – Protocol Add Gal 60’ Filter, Add Dex 1hr TP 0 TP 30 TP 60 TP 120 TP 180 TP 240

8. DNA stability assay– Protocol cont. TP 0 TP 30 TP 60 TP 120 TP 180 TP 240 Isolate genomic DNA HO Cut with ClaI HO Southern blot with probe that anneals to both fragments

9. DNA stability assay - Hypothesis Exonucleases degrade ends of strands in Wt Functional Rad3 protein will permit degradation of DNA ends at the double strand break (Maines et al., 1998)

10. DNA stability assay– Results rad3-G595Rrad3-R660Crad3-A596PWT 0 3060120 180240 0 3060120 180240 0 3060120 180240 0 3060120 180240

11. DNA stability assay– Results cont.

12. Is rad3-G595R exerting its elevated SSR phenotype via a direct or indirect mechanism ? 1) Direct effect: Determine if Rad3 protein is physically present at DSBs using chromatin immunoprecipitation studies 2) Indirect effect: transcription defect. Microarrays studies to determine if the mutant Rad3-G595R can affect the expression of certain DNA repair genes.

13. rad3-G595R Degradation of DNA ends reduced because the endonuclease can not access ends or DNA ends are not compatible for nucleases to degrade. SSR Repair of double strand break by homologous SSR Hypothesis: Rad3 mutant or other mutants facilitate SSR by binding to DNA ends and stabilizing them after a DSB or by blocking DNA end degradation.

14. Tfb1 – FLAG-tagged Wild-type, X59-1D and X55-8D rad3-G595r mutant, X61-58A Strains used in CHIP assay for localizing proteins at a DSB

15. CHIP sample collection– Protocol Add Gal 60’ Filter, Add Dex 30’ 1 hr TP -60TP -30TP 0TP 30TP 60TP 120

16. CHIP assay TP - 60 TP - 30 TP 0TP 30 TP 60 TP 120 Hi s3 TFIIH HOcs His3

17. CHIP proteins bound to DSB -Induce DSB -Cross-link protein-DNA -lyse cells and sonicate DNA HO HOcs Sam1 cs HO cs IP IN Break region telomere IP IN -60 60

19. 0 0.1 0.2 0.3 0.4 0.5 0100200300400 length of HIS3 sequences (bp) His- /Leu- Wild Type rad27 Complementation Studies Insertion Deletion Assay

20. hi s3 ura3 LEU2 HIS3 XV his3ura3LEU2 his3 hi s3URA3 HIS3 hi s3URA3 hi s3ura3LEU2 HIS3 URA3 his3 HIS3 LEU2 hi s3 ur a3 hi s3URA3 his3 URA3 Insertion Deletion Assay

21. Time points: -60-30 03060 120 Start InductionStop Induction -60-30 03060 120 Start InductionStop Induction Time points SA Hocs M1Hoc s HO endonuclease SA Hocs M1HO cs TFII H Tfb1 flag TFII H Tfb1 flag

22. Stability of broken DNA ends cs HO cs HO Sonication InputPrecipitate HO cs HO cs Y Y Prot A

23. rad3-G595R WT Degradation of DNA ends reduced SSR Extensive degradation of DNA ends Aborted homologous recombination Repair of double strand break by homologous SSR

24. IP IN -60-3003060120 SAM1 TEL 0.5

25. Specific increase in the frequencies of recombination between short sequences The SSR phenotype is accompanied by an enhanced stability of broken DNA ends rad3-G595R and SSL1-T242I Stability of DNA ends in WT and mutant strains

26. Supriya Patel class 2004 WT vs rad3-G595R

27. DNA microarrays Rationale: The elevated SSR phenotype in a rad3- G595R mutant strain may be caused by differential gene expression Experimental Approach: DNA microarrays -compare gene expression in WT and rad3-G595R strains before and after the induction of a DSB

28. Aditi Bhargava class 2005 Example of some DNA repair genes that are downregulated after the induction of a DSB in the mutant strain: RAD55, RAD57, RAD59, DNA ligase IV, LIF1, NEJ1, RAD17, MUS81, MMS2,