Molecular Genetics and Genomics

, Volume 275, Issue 2, pp 204–214

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI)

Original Paper

Abstract

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

Keywords

Meiotic recombination RecA homolog Rad51p Dmc1p VDE 

References

  1. Bergerat A, de Massy B, Gadelle D, Varoutas PC, Nicolas A, Forterre P (1997) An atypical topoisomerase II from Archaea with implications for meiotic recombination. Nature 386:414–417CrossRefPubMedGoogle Scholar
  2. Bishop DK (1994) RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis. Cell 79:1081–1092PubMedCrossRefGoogle Scholar
  3. Bishop DK, Park D, Xu L, Kleckner N (1992) DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69:439–456CrossRefPubMedGoogle Scholar
  4. Borner GV, Kleckner N, Hunter N (2004) Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis. Cell 117:29–45CrossRefPubMedGoogle Scholar
  5. Chen YK, Leng CH, Olivares H, Lee MH, Chang YC, Kung WM, Ti SC, Lo YH, Wang AH, Chang CS, Bishop DK, Hsueh YP, Wang TF (2004) Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation. Proc Natl Acad Sci USA 101:10572–10577CrossRefPubMedGoogle Scholar
  6. Dresser ME, Ewing DJ, Conrad MN, Dominguez AM, Barstead R, Jiang H, Kodadek T (1997) DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway. Genetics 147:533–544PubMedGoogle Scholar
  7. Fukuda T, Nogami S, Ohya Y (2003) VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner. Genes Cells 8:587–602CrossRefPubMedGoogle Scholar
  8. Fukuda T, Nagai Y, Ohya Y (2004) Molecular mechanism of VDE-initiated intein homing in yeast nuclear genome. Adv Biophys 38:215–232CrossRefGoogle Scholar
  9. Gasior SL, Wong AK, Kora Y, Shinohara A, Bishop DK (1998) Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes. Genes Dev 12:2208–2221PubMedGoogle Scholar
  10. Gasior SL, Olivares H, Ear U, Hari DM, Weichselbaum R, Bishop DK (2001) Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis. Proc Natl Acad Sci USA 98:8411–8418CrossRefPubMedGoogle Scholar
  11. Gerton JL, DeRisi JL (2002) Mnd1p: an evolutionarily conserved protein required for meiotic recombination. Proc Natl Acad Sci USA 99:6895–6900PubMedCrossRefGoogle Scholar
  12. Gimble FS, Thorner J (1992) Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae. Nature 357:301–306PubMedCrossRefGoogle Scholar
  13. Hayase A, Takagi M, Miyazaki T, Oshiumi H, Shinohara M, Shinohara A (2004) A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1. Cell 119:927–940CrossRefPubMedGoogle Scholar
  14. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59PubMedCrossRefGoogle Scholar
  15. Hong EL, Shinohara A, Bishop DK (2001) Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA. J Biol Chem 276:41906–41912CrossRefPubMedGoogle Scholar
  16. Hunter N, Kleckner N (2001) The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination. Cell 106:59–70CrossRefPubMedGoogle Scholar
  17. Kane SM, Roth R (1974) Carbohydrate metabolism during ascospore development in yeast. J Bacteriol 118:8–14PubMedGoogle Scholar
  18. Keeney S, Giroux CN, Kleckner N (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88:375–384CrossRefPubMedGoogle Scholar
  19. Klein HL (1997) RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis. Genetics 147:1533–1543PubMedGoogle Scholar
  20. Leu JY, Chua PR, Roeder GS (1998) The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes. Cell 94:375–386CrossRefPubMedGoogle Scholar
  21. Malkova A, Klein F, Leung WY, Haber JE (2000) HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events. Proc Natl Acad Sci USA 97:14500–14505CrossRefPubMedGoogle Scholar
  22. McKee AH, Kleckner N (1997) Mutations in Saccharomyces cerevisiae that block meiotic prophase chromosome metabolism and confer cell cycle arrest at pachytene identify two new meiosis-specific genes SAE1 and SAE3. Genetics 146:817–834PubMedGoogle Scholar
  23. Miyazaki T, Bressan DA, Shinohara M, Haber JE, Shinohara A (2004) In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J 23:939–949CrossRefPubMedGoogle Scholar
  24. Ogawa T, Yu X, Shinohara A, Egelman EH (1993) Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259:1896–1899PubMedCrossRefGoogle Scholar
  25. Petronczki M, Siomos MF, Nasmyth K (2003) Un menage a quatre: the molecular biology of chromosome segregation in meiosis. Cell 112:423–440CrossRefPubMedGoogle Scholar
  26. Petukhova G, Stratton S, Sung P (1998) Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393:91–94CrossRefPubMedGoogle Scholar
  27. Petukhova G, Sung P, Klein H (2000) Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1. Genes Dev 14:2206–2215CrossRefPubMedGoogle Scholar
  28. Petukhova GV, Romanienko PJ, Camerini-Otero RD (2003) The Hop2 protein has a direct role in promoting interhomolog interactions during mouse meiosis. Dev Cell 5:927–936CrossRefPubMedGoogle Scholar
  29. Roeder GS (1997) Meiotic chromosomes: it takes two to tango. Genes Dev 11:2600–2621PubMedGoogle Scholar
  30. Sakumoto N, Mukai Y, Uchida K, Kouchi T, Kuwajima J, Nakagawa Y, Sugioka S, Yamamoto E, Furuyama T, Mizubuchi H, Ohsugi N, Sakuno T, Kikuchi K, Matsuoka I, Ogawa N, Kaneko Y, Harashima S (1999) A series of protein phosphatase gene disruptants in Saccharomyces cerevisiae. Yeast 15:1669–1679CrossRefPubMedGoogle Scholar
  31. Schwacha A, Kleckner N (1997) Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway. Cell 90:1123–1135CrossRefPubMedGoogle Scholar
  32. Shinohara A, Ogawa T (1998) Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391:404–407CrossRefPubMedGoogle Scholar
  33. Shinohara A, Ogawa H, Ogawa T (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69:457–470CrossRefPubMedGoogle Scholar
  34. Shinohara M, Shita-Yamaguchi E, Buerstedde JM, Shinagawa H, Ogawa H, Shinohara A (1997) Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis. Genetics 147:1545–1556PubMedGoogle Scholar
  35. Shinohara M, Gasior SL, Bishop DK, Shinohara A (2000) Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination. Proc Natl Acad Sci USA 97:10814–10819CrossRefPubMedGoogle Scholar
  36. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  37. Soustelle C, Vedel M, Kolodner R, Nicolas A (2002) Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae. Genetics 161:535–547PubMedGoogle Scholar
  38. Strahl-Bolsinger S, Hecht A, Luo K, Grunstein M (1997) SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 11:83–93PubMedCrossRefGoogle Scholar
  39. Sugawara N, Wang X, Haber JE (2003) In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol Cell 12:209–219CrossRefPubMedGoogle Scholar
  40. Sung P (1997a) Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J Biol Chem 272:28194–28197CrossRefGoogle Scholar
  41. Sung P (1997b) Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev 11:1111–1121CrossRefGoogle Scholar
  42. Symington LS (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66:630–670, table of contentsGoogle Scholar
  43. Tanaka T, Knapp D, Nasmyth K (1997) Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 90:649–660CrossRefPubMedGoogle Scholar
  44. Tsubouchi H, Roeder GS (2002) The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair. Mol Cell Biol 22:3078–3088CrossRefPubMedGoogle Scholar
  45. Tsubouchi H, Roeder GS (2003) The importance of genetic recombination for fidelity of chromosome pairing in meiosis. Dev Cell 5:915–925CrossRefPubMedGoogle Scholar
  46. Tsubouchi H, Roeder GS (2004) The budding yeast Mei5 and Sae3 proteins act together with Dmc1 during meiotic recombination. Genetics 168:1219–1230PubMedCrossRefGoogle Scholar
  47. Wolner B, van Komen S, Sung P, Peterson CL (2003) Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast. Mol Cell 12:221–232PubMedCrossRefGoogle Scholar
  48. Zierhut C, Berlinger M, Rupp C, Shinohara A, Klein F (2004) Mnd1 is required for meiotic interhomolog repair. Curr Biol 14:752–762CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Department of Integrated Biosciences, Graduate School of Frontier SciencesUniversity of TokyoChiba PrefectureJapan
  2. 2.Genetic Dynamics Research Unit LaboratoryRIKENSaitamaJapan

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