6. Identify forces responsible for chromosome movement Determine the repertoire of chromosome movement in segregation, repair and recombination What is the cellular response, including chromosome reorganization to DNA damage? Is chromosome movement integral for DNA repair?

16. Yeast chromosomes oscillate from pole to pole prior to anaphase onset. Centromeres separate prior to the chromosome arms Rate of separation of chromosome arms exceeds rate of centromere separation Mechanisms of cohesion and separation are likely to differ along the chromatin fiber

18. Centromeres on the same chromatid attach to opposite poles: Chromosome Breakage Centromeres on the same chromatid attach to same poles: NO Chromosome Breakage

19. 865bp 1100bp RAD + rad52 - or rad1 - Repair mutant Chromosome rearrangements visualized in time course from galactose to glucose Gal Glu

20. Single strand excision Repair via Single strand annealing Revealed by 1100 bp repair product

22. Activate Dicentric Chromosome mid-anaphase delay follows break Does mid-anaphase delay precede break ? or

25. Mid-anaphase pause is dependent upon RAD9 checkpoint gene Mid-anaphase pause is not dependent upon the spindle assembly checkpoint genes, MAD2 or BUB1 RAD9 recognizes DNA damage and/or chromatin alterations Does RAD9 “mobilize” chromosomes/repair machinery in response to DNA damage

27. Viability of cells upon activation of the dicentric chromosome Repair mutants (rad1, rad52 and Yku70), rad9 checkpoint and mt based motility mutants (dynein, kip3, kar9)

29. Cytoplasmic microtubule-based motor proteins contribute to the fidelity of chromosome repair How do “repair mutants” effect chromosome organization The mid-anaphase pause is “longer” in the absence of cytoplasmic dynein or kip3 Mt based motor proteins effect the DNA damage response

31. Ku- Non-homologous end-joining

32. Ku70, Ku80 knockouts in mice have similar phenotype to SCID –V(D)J defects arrest lymphocyte development Ku70, Ku80 -/- mice are runts compared to +/- littermates –Number of cell divisions in development limited by impaired ability to repair endogenously generated DNA damage –Ku-deficient cells might take longer to repair this damage Ku80 -/- dams fail to nurture their pups Physiological functions of Ku

33. Disruption of yKu70p and yKu80p genes affect telomeric silencing, telomere length maintenance and viability of cells containing dicentric chromosomes –inactivate Ku, lose telomeric silencing –inactivate Ku, shorten telomeres –inactivate Ku, decrease viability of cells containing dicentric plasmids Yeast Ku in telomere maintenance

34. Ku clusters yeast telomeres to peripheral sites in nucleus –In diploids, telomeres usually found in 6-7 clusters around nuclear periphery –In Ku subunit mutants, more clusters in random locations Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

35. Activation of dicentric chromosome in Ku mutants results in chromosome decondensation

36. Chromosome decondenses in stepwise fashion

37. Dicentric chromosome stretching in metaphase in a stepwise fashion and recoil upon spindle disassembly

39. Regularity of spacing upon chromatin decompaction

40. Chromosome breakage and retraction to spindle poles

42. Sir2p binds to stretched lacO DNA Chromatin stretching is recognized in vivo  Chromatin can act as an in vivo tensiometer MAT

43. RAD9+rad9 - Rad9 is required for chromosome unfolding in response to activation of the dicentric chromosome

47. Laboratory Projects Elaine Yeh- Asymmetric protein determinants Dale Beach- RNA localization Paul Maddox-Mt polarity and dynamics

48. THANKS !!!