1. Most UV lesions are repaired by Nucleotide Excision Repair (NER) Stalled replication forks may be bypassed by alternative (bypass) DNA polymerases (REV1, REV3, RAD30) Bypass polymerases have a cost They are “error prone” on normal sequences

2. Replication -Replication through ssDNA creates DSB -DSB can arise spontaneously or by artificial means -Ionizing radiation -Mechanical force i.e. mitosis -Incomplete action of topos -endonucleases -How does the cell deal with these DSB and what are possible outcomes???

3. Recombination as a source of genetic instability A hallmark of cancer cells is their genetic instability Most of these types of instability may be explained by various mechanisms of homologous and nonhomologous recombination In all probability the initiating lesion on DNA that leads to these changes in chromosomes can be attributed to a double-strand break (DSB) Loss of heterozygosity Reciprocal and nonreciprocal translocations Deletions Truncations Chromosome loss

4. Abdel-Rahman et al. PNAS 98: 2538 Genome instability in tumor cells Truncations Translocations Inversions Duplications Amplifications

5. When breaks in DNA cannot be repaired, bad things happen Here, DT40 cells lack the key recombination protein, Rad51

6. Stalled replication forks may “break” (probably they are cut by an endonuclease)

7. Replication forks often stall at highly repetative sequences

8. size shape Restriction fragment

9. Replication fork “regression” Formation of a Holliday junction Extension of leading strand Branch migration reverses Holliday junction and allows bypass of the UV-lesion Holliday junction formation Stabilize Replication Forks

10. Holliday Junctions All base pairs made Cross Point (Branch can move) Resolved with or without help of factors

14. Holliday Junction

15. Replication fork “regression” Formation of a Holliday junction RuvA, RuvB Extension of leading strand Branch migration reverses Holliday junction and allows bypass of the UV-lesion Holliday junction formation

16. B Shape Helicase

17. RuvA

18. GO TO: http://www.sdsc.edu/journals/mbb/ruva.htmlhttp://www.sdsc.edu/journals/mbb/ruva.html RuvB

19. RuvC Necessary for resolution RuvA/B sufficient to resolve HJ with an end nearby RuvC cleaves symmetrically on 2 homologous DNA segments The EMBO Journal (1997) 16, 1464–1472, doi: 10.1093/emboj/16.6.1464

20. Branch Migration Resolution of a Holliday Junction

21. Holliday Junction

22. Cleavage of a Holliday junction at a stalled replication fork produces an intact template and a broken-ended molecule BIR

23. Binding of Rad51 Strand invasion 5’ to 3’ exonuclease resection Homology search Re-establishment of a replication fork BIR

24. Branch migration allows gap to be filled in Another Holliday junction Ends flipped over for easy viewing

25. STRAND INVASION

26. Basic strand exchange

29. Replication Restart Can Lead to SCE

31. Recombination as a source of genetic instability A hallmark of cancer cells is their genetic instability Most of these types of instability may be explained by various mechanisms of homologous and nonhomologous recombination In all probability the initiating lesion on DNA that leads to these changes in chromosomes can be attributed to a double-strand break (DSB) Loss of heterozygosity Reciprocal and nonreciprocal translocations Deletions Truncations Chromosome loss

32. Abdel-Rahman et al. PNAS 98: 2538 Genome instability in tumor cells Truncations Translocations Inversions Duplications Amplifications

33. Eukaryotic recombination machinery complex Large number of proteins necessary –Rad51, Rad52, 5 Rad51 paralogs (load Rad51) –Rad54 – strand invasion –BRCA1 and BRCA2 Rad51, BRCA1/2 KOs lethal Rad51 paralogs – less SCE

34. Chromosomes and Chromatids break other times than during replication Repaired BIR and Gene Conversion

35. BIR – Non replicating cell

37. In humans. Telomerase is shut off at birth. Telomeres get shorter and shorter every time DNA replicates. People who have a deletion of one of the two telomerase RNA genes suffer from dyskeratosis congenita (haplo-insufficiency) Tumors arise by: (a) re-activating telomerase (b) ALT (alternative lengthening of telomeres)

38. ALT Extension telomeres by HR demonstrated yeast Deleted telomerase enzyme –Shorten 10nt/generation –Die 30-50 generations –Few telomerase independent survivors Eliminated Rad52

39. Gene Conversion - Synthesis dependent strand annealing (SDSA)

40. inducible Requires Rad51, Rad52, Rad54 etc. Also requires PCNA and DNA ploymerases Cell knows which donor to choose – requires recombination enhancer

41. Restriction size differences at a and alpha site

42. Can look for presence of HJs Studied using 2D Gels hotspots Looking for differences novel spots not part of replicating arc of DNA

43. Cut out and use denaturing gel –Each strand characterized

46. Chromosomes and Chromatids break other times than during replication Repaired BIR and Gene Conversion

48. Hin gene promoter H1 gene Rh2 gene OP H2 gene Hin gene promoter H1 gene Rh2 gene OP H2 gene P P No transcription

49. Site-specific Recombination Nicks on one pair of strands Reciprocal strand exchange (Holloiday junction) Nicks on second pair of strands Reciprocal strand exchange Two examples: Hin and FLP

50. Site-specific recombination One example: phase variation in Salmonella Flagella composed of H1 flagellin Anti H1 antibody eliminates 99.99% Survivors have alternative H2 flagellin