Функция повторов аминокислотной последовательности белка Sfr1 в рекомбинационной репарации Schizosaccharomyces pombe тема диссертации и автореферата по ВАК РФ 03.01.07, кандидат биологических наук Хасанова, Ольга Сергеевна

• Хасанова, Ольга Сергеевна
• кандидат биологических наук
• 2010

Оглавление диссертации кандидат биологических наук Хасанова, Ольга Сергеевна

ГЛАВА 1. ВВЕДЕНИЕ.

ГЛАВА 2. ОБЗОР ЛИТЕРАТУРЫ.

2.1. Преимущества делящихся дрожжей для изучения репараци двухцепочечныхповреждений ДНК.

2.2. Гены рекомбинационной репарации S. Pombe.

2.2.1. Белки S. pombe, ответственные за образование 3' - выступающих однонитевых участков в сайтах повреждений ДНК.

2.2.2. Белки S. pombe, ответственные за образование Rad51 -нуклеопротеинового филамента.

2.3. Механизм рекомбинационной репарации в эукариотах.

2.4. Sfrl и толерантность к УФ-повреждениям ДНК.

2.5. Связь механизмов контроля клеточного цикла в репарации повреждений ДНК.

ГЛАВА 3. МАТЕРИАЛЫ И МЕТОДЫ.

3.1. Штаммы, использованные в работе.

3.2. Ростовые среды.

3.3. Генетические скрещивания.

3.4. Выделение плазмидной ДНК из клеток E.coli.

3.5. Электрофорез ДНК в агарозном геле.

3.6. Выделение ДНК из агароного геля с помощью набора Nucleospin (Macherey-nagel).

3.7. Получение компетентных клеток E.coli DH5a.

3.8. Трансформация клеток штамма E.coli DH5a.

3.9. Трансформация клеток S. pombe.

3.10. Тесты клеток дрожжей на генотоксический стресс. 3.11. Полимеразная цепная реакция.

3.12. Выделение белка Sfrl из клеток S.pombe.

3.13. Электрофорез белков в полиакриламидном геле.

3.14. Эксперимент с повышенной экспрессией белка 8&

3.15. Тест на эффективность мейотической внутригенной рекомбинации в клетках & ротЬе.

3.16. Тест на эффективность мейотической межгенной рекомбинации в клетках S. pombe.

3.17. Тесты на белок - белковые взаимодействия.

3.18. Манипуляции с ДНК.

3.19. Очистка His6 - меченого белка в денатурирующих условиях на Ni-NT^^ агарозе . ^

3.20. Приготовление хроматиновых спредов.^ ^^

3.21. Иммунофлуоресцентная микроскопия.

ГЛАВА 4. РЕЗУЛЬТАТЫ ИСЛЕДОВАНИЯ.^ ^

4.1. Белок S. pombe Sfr 1 взаимодействует с рекомбиназой Rad51.^ ^^

4.2. Идентификация нового мотива в белке Sfrl и его анализ.

4.3. Повышенная экспрессия PSA повторов приводит к Rad51 -зависимому доминант - негативному эффекту на выживаемость клеток.,

4.4. Мутации в PSA мотивах белка Sfrl приводят к ослабленной репара^^^ ДНК в клетках S. pombe. • *.

4.5. Мейотическая рекомбинация уменьшена в sfrl PSA — мутантных аллелях.

4.6. Sfrl-мутантные аллели дефектны для взаимодействия с Rad51.v

4.7. Образование фокусов Rad51, индуцированных ионизирующей радиацией, уменьшено в мутанте sfrl Л.^ * ■ * --.

ГЛАВА 5. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ.

••••-.

ВЫВОДЫ. •••■•.

Заключение диссертации по теме «Молекулярная генетика», Хасанова, Ольга Сергеевна

выводы

1. Функция белка Sfrl необходима для взаимодействия с ключевым белком гомологичной рекомбинации Rad51 в клетках эукариот и его доставку к сайтам повреждения ДНК.

2. Обнаружены повторы (PSA, pombe Sfrl associated) аминокислотной последовательности в белке Sfrl, ответственные за взаимодействие с рекомбиназой Rad51.

3. Повышенная экспрессия PSA повторов Sfrl приводит к Rad51-зависимому доминант - негативному эффекту на выживаемость клеток.

4. PSA повторы являются важной функциональной частью белка Sfrl. Замещение фенилаланина в коровых участках повторов приводит к строгому ослаблению репарации ДНК и рекомбинации в мейозе, что указывает на важность химической природы этого аминокислотного остатка.

5. Предложена модель полимеризации белка Rad51 на одноцепочечной ДНК с участием медиаторного комплекса Sfrl-Swi5 в клетках делящихся дрожжей.

Список литературы диссертационного исследования кандидат биологических наук Хасанова, Ольга Сергеевна, 2010 год

1. Klar, A J. (2007) Lessons learned from studies of fission yeast mating-type switching and silencing. Annu Rev Genet 41, 213-236.

2. Wray, J., Liu, J., Nickoloff, J.A. and Shen, Z. (2008) Distinct RAD51 associations with RAD52 and BCCIP in response to DNA damage and replication stress. Cancer Res 68, 2699-2707.

3. Franco, S., Alt, F.W. and Manis, J.P. (2006) Pathways that suppress programmed DNA breaks from progressing to chromosomal breaks and translocations. DNA Repair (Amst) 5, 1030-1041.

4. Ehmsen, K.T. and Heyer, W.D. (2008) Biochemistry of Meiotic Recombination: Formation, Processing, and Resolution of Recombination Intermediates. Genome Dyn Stab 3, 91-164.

5. Bosco, E.E., Mayhew, C.N., Hennigan, R.F., Sage, J., Jacks, T. and Knudsen, E.S. (2004) RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult. Nucleic Acids Res 32, 25-34.

6. Moore, J.K. and Haber, J:E. (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 16;,2164-2173.

7. Mao, Z., Bozzella, M., Seluanov, A. and Gorbunova, V. (2008) Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst) 7, 1765-1771.

8. Mortimer, R.K. (1958) Radiobiological and genetic studies on a polyploid series (haploid to hexaploid) of Saccharomyces cerevisiae. Radiat Res 9, 312-326.

9. Egel, R. (2005) Fission yeast mating-type switching: programmed damage and repair. DNA Repair (Amst) 4, 525-536.

10. Lisby, M. and Rothstein, R. (2004) DNA damage checkpoint and repair centers. Curr Opin Cell Biol 16, 328-334.

11. Thompson, L.H. and Schild, D. (2002) Recombinational DNA repair and human disease. Mutat Res 509, 49-78.

12. Scully, R.E. (1997) Hormonally active ovarian tumors. Verh Dtsch Ges Pathol 81,245-252.

13. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D.M. (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 88, 265-275.

14. Huysmans, E., Dams, E., Yandenberghe, A. and Wachter, R.D. (1983) The nucieotide sequences of the 5S rRNAs of four mushrooms and their use in studying the pnytogenetk position of bastdiomycetes among the eukaryotes. Nucl. Acids Res. 11, 2871-2880.

15. Prabhala, G., Rosenberg, G.H. and K/jufer, N.F. (1992) Architectural Features of Pre-mRNA Introns in the Fission Yeast Schizosaccharomyces pombe. Yeast 8, 171-182.

16. Mertins, P. and Gallwitz, D. (1987) Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element. Embo J 6, 1757-1763.

17. Nurse, P. (1990) Universal control mechanism regulating onset of M-phase. Nature 344, 503-508.

18. Davey, S., Nass, M.L., Ferrer, J.V., Sidik, K., Eisenberger, A., Mitchell, D.L. and Freyer, G.A. (1997) The fission yeast UVDR DNA repair pathway is inducible. Nucleic Acids Res 25, 1002-1008.

19. Yasuhira, S., Morimyo, M. and Yasui, A. (1999) Transcription dependence and the roles of two excision repair pathways for UV damage in fission yeast Schizosaccharomyces pombe. J Biol Chem 274, 26822-26827.

20. Phipps, J., Nasim, A. and Miller, D.R. (1985) Recovery, repair, and mutagenesis in Schizosaccharomyces pombe. Adv Genet 23, 1-72.

21. Paques, F. and Haber, J.E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63, 349-404.

22. Petrini, J.H., Bressan, D.A. and Yao, M.S. (1997) The RAD52 epistasis group in mammalian double strand break repair. Semin Immunol 9, 181-188.

23. Lim, D.S. and Hasty, P. (1996) A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol 16, 71337143.

24. Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y. and MoritaT (1996) Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc Natl Acad Sci USA 93, 6236-6240.

25. Xiao, Y. and Weaver, D.T. (1997) Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mrel 1 protein in murine embryonic stem cells. Nucleic Acids Res 25, 2985-2991.

26. Tavassoli, M., Shayeghi, M., Nasim, A. and Watts, F.Z. (1995) Cloning and characterization of the Schizosaccharomyces pombe rad.32 gene: a gene required for repair of double strand breaks and recombination. Nucleic Acids Res. 23, 383388.

27. Ajimura, M., Leem, S.H. and Ogawa, H. (1993) Identification of New Genes Required for Meiotic Recombination in Saccharomyces cerevisiae. Genetics 133, 51-66.

28. Eggleston, A.K. and West, S.C. (1997) Recombination initiation: easy as A, B, C, D. chi? Curr Biol 7, R745-749.

29. Sharpies, G.J. and Leach, D.R. (1995) Structural and functional similarities between the SbcCD proteins of Escherichia coli and the RAD50 and MRE11 (RAD32) recombination and repair proteins of yeast. Mol Microbiol 17, 12151217.

30. Wilson, S., Tavassoli, M. and Watts, F.Z. (1998) Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks. Nucleic Acids Res 26, 52615269.

31. Haber, J.E. (1998) The many interfaces of Mrel 1. Cell 95, 583-586.

32. Usui, T., Ohta, T., Oshiumi, H., Tomizawa, J., Ogawa, H. and Ogawa, T. (1998) Complex formation and functional versatility of Mrel 1 of budding yeast in recombination. Cell 95, 705-716.

33. Paull, T.T. and Gellert, M. (1998) The 3' to 5' exonuclease activity-of Mre 11 facilitates repair of DNA double-strand breaks. Mol Cell 1, 969-979.

34. Dong, Z., Zhong, Q. and Chen, P.L. (1999) The Nijmegen breakage syndrome protein is essential for Mrell phosphorylation upon DNA damage. J Biol Chem 274, 19513-19516.

35. Bianco, P.R., Tracy, R.B. and Kowalczykowski, S.C. (1998) DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci 3, D570-603.

36. Muris, D.F., Vreeken, K., Carr, A.M., Broughton, B.C., Lehmann, A.R., Lohman, P.H. and Pastink, A. (1993) Cloning the RAD51 homologue of Schizosaccharomyces pombe. Nucleic Acids Res 21, 4586-4591.

37. Jang, Y.K., Jin, Y.H., Kim, E.M., Fabre, F., Hong, S.H. and Park, S.D. (1994) Cloning and sequence analysis of rhp51+, a Schizosaccharomyces pombe homolog of the Saccharomyces cerevisiae RAD51 gene. Gene 142, 207-211.

38. Jang, Y.K., Jin, Y.H., Myung, K., Seong, R.H., Hong, S.H. and Park, S.D. (1996) Differential expression of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizosaccharomyces pombe. Gene 169, 125-130.

39. Shinohara, A., Ogawa, H. and Ogawa, T. (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457-470.

40. Sung, P. (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241-1243.

41. Sugiyama, T., Zaitseva, E.M. and Kowalczykowski, S.C. (1997) A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J Biol Chem 272, 7940-7945.

42. Kowalczykowski, S.C., Dixon, D.A., Eggleston, A.K., Lauder, S.D. and Rehrauer, W.M. (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58, 401-465.

43. Ogawa, T., Yu, X., Shinohara, A. and Egelman, E.H. (1993) Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259, 1896-1899.

44. Conway, A.B., Lynch, T.W., Zhang, Y., Fortin, G.S., Fung, C.W., Symington, L.S. and Rice, P.A. (2004) Crystal structure of a Rad51 filament. Nat Struct Mol Biol 11,791-796.

45. Zaitseva, E.M., Zaitsev, E.N. and Kowalczykowski, S.C. (1999) The DNA binding properties of Saccharomyces cerevisiae Rad51 protein. J Biol Chem 274, 2907-2915.

46. Johnson, R.D. and Symington, L.S. (1995) Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol Cell Biol 15,4843-4850.

47. Hays, S.L., Firmenich, A.A. and Berg, P. (1995) Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc Natl Acad Sci U S A 92, 6925-6929.

48. New, J.H., Sugiyama, T., Zaitseva, E. and Kowalczykowski, S.C. (1998) Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391, 407-410.

49. Shinohara, A. and Ogawa, T. (1998) Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391, 404-407.

50. Benson, F.E., Baumann, P. and West, S.C. (1998) Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature 391, 401-404.

51. Sung, P. (1997) Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev 11, 1111-1121.

52. Khasanov, F.K., Savchenko, G.V., Bashkirova, E.V., Korolev, V.G., Heyer, W.D. and Bashkirov, V.l. (1999) A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA. Genetics 152, 1557-1572.

53. Haruta, N., Kurokawa, Y., Murayama, Y., Akamatsu, Y., Unzai, S., Tsutsui, Y. and Iwasaki, H. (2006) The Swi5-Sfrl complex stimulates Rhp51/Rad51- and Dmcl-mediated DNA strand exchange in vitro. Nat Struct Mol Biol 13, 823-830.

54. Salakhova, A.F., Savchenko, G.V., Khasanov, F.K., Chepurnaia, O.V., Korolev, V.G. and Bashkirov, V.l. (2005) The dds20+ gene controls a novel

55. Rad51 Sp-dependent pathway of recombinational repair in Schizosaccharomyces pombe. Genetika 41, 736-745.

56. Tsutsui, Y., Khasanov, F.K., Shinagawa, H., Iwasaki, H. and Bashkirov, V.I. (2001) Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. Genetics 159, 91-105.

57. Lehmann, A.R., Walicka, M., Griffiths, D.J., Murray, J.M., Watts, F.Z., McCready, S. and Carr, A.M. (1995) The radl8 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Mol Cell Biol 15, 7067-7080.

58. Verkade, H.M., Bugg, S.J., Lindsay, H.D., Carr, A.M. and O'Connell, M.J. (1999) Radl8 is required for DNA repair and checkpoint responses in fission yeast. Mol Biol Cell 10, 2905-2918.

59. Hartsuiker, E., Vaessen, E., Carr, A.M. and Kohli, J. (2001) Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair. Embo J 20, 6660-6671.

60. Kaykov, A. and Arcangioli, B. (2004) A programmed strand-specific and modified nick in S. pombe constitutes a novel type of chromosomal imprint. Curr Biol 14, 1924-1928.

61. Vengrova, S. and Dalgaard, J.Z. (2004) RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching. Genes Dev 18, 794-804.

62. Ostermann, K., Lorentz, A. and Schmidt, H. (1993) The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae. Nucleic Acids Res 21, 5940-5944.

63. Grishchuk, A.L., Kraehenbuehl, R., Molnar, M., Fleck, O. and Kohli, J. (2004) Genetic and cytological characterization of the RecA-homologous proteins Rad51 and Dmcl of Schizosaccharomyces pombe. Curr Genet 44, 317-328.

64. Arcangioli, B. (1998) A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast. Embo J 17, 4503-4510.

65. Game, J.C. (1993) DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol 4, 73-83.

66. Suto, K., Nagata, A., Murakami, H. and Okayama, H. (1999) A double-strand break repair component is essential for S phase completion in fission yeast cell cycling. Mol Biol Cell 10, 3331-3343.

67. Wold, M.S. (1997) Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66, 61-92.

68. Parker, A.E., Clyne, R.K., Carr, A.M. and Kelly, T.J. (1997) The Schizosaccharomyces pombe radll+ gene encodes the large subunit of replication protein A. Mol Cell Biol 17, 2381-2390.

69. Lee, S.E., Moore, J.K., Holmes, A., Umezu, K., Kolodner, R.D. and Haber, J.E. (1998) Saccharomyces Ku70, mrell/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399-409.

70. Brush, G.S., Morrow, D.M., Hieter, P. and Kelly, T.J. (1996) The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast Proc Natl Acad Sci U S A 93, 15075-15080.

71. Brush, G.S. and Kelly, T.J. (2000) Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response. Nucleic Acids Res 28, 3725-3732.

72. Carty, M.P., Zernik-Kobak, M., McGrath, S. and Dixon, K. (1994) UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein. Embo J 13, 2114-2123.

73. Hays, S.L., Firmenich, A.A., Massey, P., Banerjee, R. and Berg, P. (1998) Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA. Mol Cell Biol 18, 4400-4406.

74. Sugiyama, T., New, J.H. and Kowalczykowski, S.C. (1998) DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc Natl Acad Sci USA 95, 6049-6054.

75. Sung, P. and Klein, H. (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7, 739-750.

76. Eggler, A.L., Inman, R.B. and Cox, M.M. (2002) The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A. J Biol Chem 277, 39280-39288.

77. Beernink, H.T. and Morrical, S.W. (1999) RMPs: recombination/replication mediator proteins. Trends Biochem Sci 24, 385-389.

78. Lisby, M., Barlow, J.H., Burgess, R.C. and Rothstein, R. (2004) Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699-713.

79. Gasior, S.L., Wong, A.K., Kora, Y., Shinohara, A. and Bishop, D.K. (1998) Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes. Genes Dev 12, 2208-2221.

80. Sugawara, N., Wang, X. and Haber, J.E. (2003) In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol Cell 12, 209219.

81. Shinohara, A., Shinohara, M., Ohta, T., Matsuda, S. and Ogawa, T. (1998) Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing. Genes Cells 3, 145-156.

82. Singleton, M.R., Wentzell, L.M., Liu, Y., West, S.C. and Wigley, D.B. (2002) Structure of the single-strand annealing domain of human RAD52 protein. Proc Natl Acad Sci U S A 99, 13492-13497.

83. Sharan, S.K., Morimatsu, M., Albrecht, U., Lim, D.S., Regel, E., Dinh, C., Sands, A., Eichele, G., Hasty, P. and Bradley, A. (1997) Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386, 804-810.

84. Amitani, I., Baskin, RJ. and Kowalczykowski, S.C. (2006) Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 23, 143-148.

85. Mazin, A.V., Alexeev, A.A. and Kowalczykowski, S.C. (2003) A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J Biol Chem 278, 14029-14036.

86. Wolner, B. and Peterson, C.L. (2005) ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break. J Biol Chem 280, 10855-10860.

87. Heyer, W.D., Li, X., Rolfsmeier, M. and Zhang, X.P. (2006) Rad54: the Swiss Army knife of homologous recombination? Nucleic Acids Res 34, 41154125.

88. Tan, T.L., Kanaar, R. and Wyman, C. (2003) Rad54, a Jack of all trades in homologous recombination. DNA Repair (Amst) 2, 787-794.

89. Petukhova, G., Stratton, S. and Sung, P. (1998) Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393, 91-94.

90. Solinger, J.A., Kiianitsa, K. and Heyer, W.D. (2002) Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell 10, 1175-1188.

91. Li, X. and Heyer, W.D. (2009) RAD54 controls access to the invading 3'-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae. Nucleic Acids Res 37, 638-646.

92. Khasanov, F.K., Salakhova, A.F., Chepurnaja, O.V., Korolev, V.G. and Bashkirov, V.I. (2004) Identification and characterization of the rlpl+., the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe. DNA Repair (Amst) 3, 1363-1374.

93. Lambert, S. and Lopez, B.S. (2001) Role of RAD51 in sister-chromatid exchanges in mammalian cells. Oncogene 20, 6627-6631.

94. Shivji, M.K. and Venkitaraman, A.R. (2004) DNA recombination, chromosomal stability and carcinogenesis: insights into the role of BRCA2. DNA Repair (Amst) 3, 835-843.

95. Baumann, P., Benson, F.E. and West, S.C. (1996) Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757-766.

96. Yasui, A. and McCready, S.J. (1998) Alternative repair pathways for UV-induced DNA damage. Bioessays 20, 291-297.

97. Murray, J.M., Lindsay, H.D., Munday, C.A. and Carr, A.M. (1997) Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance. Mol Cell Biol 17, 6868-6875.

98. Boddy, M.N., Lopez-Girona, A., Shanahan, P., Interthal, H., Heyer, W.D. and Russell, P. (2000) Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cdsl. Mol Cell Biol 20, 8758-8766.

99. Michel, B., Ehrlich, S.D. and Uzest, M. (1997) DNA double-strand breaks caused by replication arrest. Embo J 16, 430-438.

100. Kogoma, T. (1997) Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription. Microbiol Mol Biol Rev 61, 21-2-238.

101. Martinho, R.G., Lindsay, H.D., Flaggs, G., DeMaggio, A.J., Hoekstra, M.F., Carr, A.M. and Bentley, N.J. (1998) Analysis of Rad3 and Chkl protein kinases defines different checkpoint responses. Embo J 17, 7239-7249.

102. McCready, S.J., Osman, F. and Yasui, A. (2000) Repair of UV damage in the fission yeast Schizosaccharomyces pombe. Mutat Res 451, 197-210.

103. Myung, K., Chen, C. and Kolodner, R.D. (2001) Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae. Nature 411, 1073-1076.

104. Tercero, J.A. and Diffley, J.F. (2001) Regulation of DNA replication fork progression through damaged DNA by the Mecl/Rad53 checkpoint. Nature 412, 553-557.

105. Bashkirov, V.l., King, J.S., Bashkirova, E.V., Schmuckli-Maurer, J. and Heyer, W.D. (2000) DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints. Mol Cell Biol 20, 4393-4404.

106. Weichselbaum, R.R., Nove, J. and Little, J.B. (1978) Deficient recovery from potentially lethal radiation damage in ataxia telengiectasia and xeroderma pigmentosum. Nature 271, 261-262.

107. Siede, W., Friedberg, A.S. and Friedberg, E.C. (1993) RAD9-dependent Gl arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. Proc Natl Acad Sei U S A 90, 7985-7989.

108. Kolodner, R.D., Putnam, C.D. and Myung, K. (2002) Maintenance of genome stability in Saccharomyces cerevisiae. Science 297, 552-557.

109. Tercero, J.A., Longhese, M.P. and Diffley, J.F. (2003) A central' role for DNA replication forks in checkpoint activation and response. Mol Cell 11, 13231336.

110. Broomfield, S., Hryciw, T. and Xiao, W. (2001) DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat Res 486, 167-184.

111. Krogh, B.O. and Symington, L.S. (2004) Recombination proteins in yeast. Annu Rev Genet 38, 233-271.

112. Waddell, S., Jenkins, J.R. and Proikas-Cezanne, T. (2001) A "no-hybrids" screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast. Oncogene 20, 6001-6008.

113. Fousteri, M.I. and Lehmann, A.R. (2000) A novel SMC protein complex in Schizosaccharomyces pombe contains the Radl8 DNA repair protein. EMBO J 19, 1691-1702.

114. Santos-Rosa, H., Clever, B., Heyer, W.D. and Aguilera, A. (1996) The yeast HRS1 gene encodes a polyglutamine-rich nuclear protein required for spontaneous and hprl-induced deletions between direct repeats. Genetics 142, 705-716.

115. Pringle, J.R., Adams, A.E., Drubin, D.G. and Haarer, B.K. (1991) Immunofluorescence methods for yeast. Methods Enzymol 194, 565-602.

116. Egel, R., Beach, D.H. and Klar, A.J. (1984) Genes required for initiation and resolution steps of mating-type switching in fission yeast. Proc Natl Acad Sci U S A 81, 3481-3485.

117. Combet, C., Blanchet, C., Geourjon, C. and Deleage, G. (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25, 147-150.

118. Mitchell, T.G. and Perfect, J.R. (1995) Cryptococcosis in the era of AIDS--100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 8, 515-548.

119. Symington, L.S. (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66, 630-670, table of contents.

120. Pellegrini, L., Yu, D.S., Lo, T., Anand, S., Lee, M., Blundell, T.L. and Venkitaraman, A.R. (2002) Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature 420, 287-293.

121. Prinz, S., Amon, A. and Klein, F. (1997) Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae. Genetics 146, 781-795.

122. Penkner, A., Portik-Dobos, Z., Tang, L., Schnabel, R., Novatchkova, M., Jantsch, V. and Loidl, J. (2007) A conserved function for a Caenorhabditis elegans Coml/Sae2/CtIP protein homolog in meiotic recombination. EMBO J 26, 50715082.

123. Tsubouchi, H. and Roeder, G.S. (2004) The budding yeast mei5 and sae3 proteins act together with dmcl during meiotic recombination. Genetics 168, 1219-1230.

124. Hayase, A., Takagi, M., Miyazaki, T., Oshiumi, H., Shinohara, M. and Shinohara, A. (2004) A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmcl. Cell 119, 927-940.

125. Young, J.A., Hyppa, R.W. and Smith, G.R. (2004) Conserved and nonconserved proteins for meiotic DNA breakage and repair in yeasts. Genetics 167,593-605.

126. Li, X. and Heyer, W.D. (2008) Homologous recombination in DNA repair and DNA damage tolerance. Cell Res 18, 99-113.

127. Yang, H., Li, Q., Fan, J., Holloman, W.K. and Pavletich, N.P. (2005) The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNAssDNA junction. Nature 433, 653-657.

128. Bork, P., Blomberg, N. and Nilges, M. (1996) Internal repeats in the BRCA2 protein sequence. Nat Genet 13, 22-23.

129. Takahashi, M. (1989) Analysis of DNA-RecA protein interactions involving the protein self-association reaction. J Biol Chem 264, 288-295.

130. Forget, A.L., Kudron, M.M., McGrew, D.A., Calmann, M.A., Schiffer, C.A. and Knight, K.L. (2006) RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments. Biochemistry 45, 13537-13542.

131. Donovan, J.W., Milne, G.T. and Weaver, D.T. (1994) Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair. Genes Dev 8, 2552-2562.

132. Tanaka, K., Hiramoto, T., Fukuda, T. and Miyagawa, K. (2000) A novel human rad54 homologue, Rad54B, associates with Rad51. J Biol Chem 275, 26316-26321.

133. Liu, J., Doty, T., Gibson, B. and Heyer, W.D. (2010) Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA. Nat Struct Mol Biol.

134. Kurokawa, Y., Murayama, Y., Haruta-Takahashi, N., Urabe, I. and Iwasaki, H. (2008) Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators. PLoS Biol 6, e88.

135. Tarsounas, M., Davies, D. and West, S.C. (2003) BRCA2-dependent and independent formation of RAD51 nuclear foci. Oncogene 22, 1115-1123.