A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

English version

Autoři: Diedre Reitz ^aff001; Jennifer Grubb ^aff002; Douglas K. Bishop ^aff001
Působiště autorů: Committee on Genetics, Genomics, and Systems Biology, University of Chicago, Chicago, Illinois, United States of America ^aff001; Department of Radiation and Cellular Oncology, Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, United States of America ^aff002
Vyšlo v časopise: A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability. PLoS Genet 15(12): e32767. doi:10.1371/journal.pgen.1008217
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1008217

Souhrn

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.

Klíčová slova:

DNA-binding proteins – Saccharomyces cerevisiae – Meiosis – Recombinant proteins – DNA repair – Homologous recombination – Recombinase polymerase amplification – DNA recombination

Zdroje

1. Hunter N. Meiotic recombination: The essence of heredity. Cold Spring Harb Perspect Biol. 2015 Oct 28;7(12):a016618–35. doi: 10.1101/cshperspect.a016618 26511629

2. Bishop DK, Park D, Xu L, Kleckner N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell. 1992 May 1;69(3):439–56. doi: 10.1016/0092-8674(92)90446-j 1581960

3. Cloud V, Chan Y-L, Grubb J, Budke B, Bishop DK. Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science. 2012 Sep 7;337(6099):1222–5. doi: 10.1126/science.1219379 22955832

4. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–70. doi: 10.1016/0092-8674(92)90447-k 1581961

5. Sung P, Robberson DL. DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell. 1995 Aug 11;82(3):453–61. doi: 10.1016/0092-8674(95)90434-4 7634335

6. Keeney S, Giroux CN, Kleckner N. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell. 1997 Feb 7;88(3):375–84. doi: 10.1016/s0092-8674(00)81876-0 9039264

7. Symington LS. Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol. 2016 Apr 20;51(3):195–212. doi: 10.3109/10409238.2016.1172552 27098756

8. Bell JC, Kowalczykowski SC. RecA: Regulation and mechanism of a molecular search engine. Trends Biochem Sci. 2016 Jun;41(6):491–507. doi: 10.1016/j.tibs.2016.04.002 27156117

9. Jinks-Robertson S, Petes TD. High-frequency meiotic gene conversion between repeated genes on nonhomologous chromosomes in yeast. Proc Natl Acad Sci U S A. 1985 May;82(10):3350–4. doi: 10.1073/pnas.82.10.3350 3889906

10. Lichten M, Borts RH, Haber JE. Meiotic gene conversion and crossing over between dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae. Genetics. 1987 Feb;115(2):233–46. 3549449

11. Goldman AS, Lichten M. The efficiency of meiotic recombination between dispersed sequences in Saccharomyces cerevisiae depends upon their chromosomal location. Genetics. 1996 Sep;144(1):43–55. 8878672

12. Grushcow JM, Holzen TM, Park KJ, Weinert T, Lichten M, Bishop DK. Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice. Genetics. 1999 Oct;153(2):607–20. 10511543

13. Schwacha A, Kleckner N. Identification of joint molecules that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell. 1994 Jan 14;76(1):51–63. doi: 10.1016/0092-8674(94)90172-4 8287479

14. Brown MS, Bishop DK. DNA strand exchange and RecA homologs in meiosis. Cold Spring Harb Perspect Biol. 2015 Jan 5;7(1):a016659–31.

15. Wright WD, Shah SS, Heyer W-D. Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem. 2018 Jul 6;293(27):10524–35. doi: 10.1074/jbc.TM118.000372 29599286

16. McVey M, Khodaverdian VY, Meyer D, Cerqueira PG, Heyer W-D. Eukaryotic DNA polymerases in homologous recombination. Annu Rev Genet. 2016 Nov 23;50(1):393–421.

17. McMahill MS, Sham CW, Bishop DK. Synthesis-dependent strand annealing in meiosis. PLoS Biol. 2007 Nov 6;5(11):e299. doi: 10.1371/journal.pbio.0050299 17988174

18. Merker JD, Dominska M, Petes TD. Patterns of heteroduplex formation associated with the initiation of meiotic recombination in the yeast. Genetics. 2003 Sep;165(1):47–63. 14504217

19. Allers T, Lichten M. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell. 2001 Jul 13;106(1):47–57. doi: 10.1016/s0092-8674(01)00416-0 11461701

20. Hunter N, Kleckner N. The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell. 2001 Jul 13;106(1):59–70. doi: 10.1016/s0092-8674(01)00430-5 11461702

21. Schwacha A, Kleckner N. Identification of double Holliday junctions as intermediates in meiotic recombination. Cell. 1995 Dec 1;83(5):783–91. doi: 10.1016/0092-8674(95)90191-4 8521495

22. Zakharyevich K, Tang S, Ma Y, Hunter N. Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase. Cell. 2012 Apr 13;149(2):334–47. doi: 10.1016/j.cell.2012.03.023 22500800

23. Heyer W-D. Regulation of recombination and genomic maintenance. Cold Spring Harb Perspect Biol. 2015 Aug 3;7(8):a016501. doi: 10.1101/cshperspect.a016501 26238353

24. Krejci L, Altmannova V, Spirek M, Zhao X. Homologous recombination and its regulation. Nucleic Acids Res. 2012 Jul;40(13):5795–818. doi: 10.1093/nar/gks270 22467216

25. Kowalczykowski SC. An overview of the molecular mechanisms of recombinational DNA repair. Cold Spring Harb Perspect Biol. 2015 Nov 2;7(11).

26. Krejci L, Van Komen S, Li Y, Villemain J, Reddy MS, Klein H, et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature. 2003 May 15;423(6937):305–9. doi: 10.1038/nature01577 12748644

27. Veaute X, Jeusset J, Soustelle C, Kowalczykowski SC, Le Cam E, Fabre F. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature. 2003 May 15;423(6937):309–12. doi: 10.1038/nature01585 12748645

28. Veaute X, Delmas S, Selva M, Jeusset J, Le Cam E, Matic I, et al. UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli. EMBO J. 2005 Jan 12;24(1):180–9. doi: 10.1038/sj.emboj.7600485 15565170

29. Petrova V, Chen SH, Molzberger ET, Tomko E, Chitteni-Pattu S, Jia H, et al. Active displacement of RecA filaments by UvrD translocase activity. Nucleic Acids Res. 2015 Apr 30;43(8):4133–49. doi: 10.1093/nar/gkv186 25824953

30. Sasanuma H, Furihata Y, Shinohara M, Shinohara A. Remodeling of the Rad51 DNA strand-exchange protein by the Srs2 helicase. Genetics. 2013 Aug;194(4):859–72. doi: 10.1534/genetics.113.150615 23770697

31. Crickard JB, Kaniecki K, Kwon Y, Sung P, Greene EC. Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase. Proc Natl Acad Sci U S A. 2018 Oct 23;115(43):E10041–8. doi: 10.1073/pnas.1810457115 30301803

32. Bishop DK, Ear U, Bhattacharyya A, Calderone C, Beckett M, Weichselbaum RR, et al. Xrcc3 is required for assembly of Rad51 complexes in vivo. J Biol Chem. 1998 Aug 21;273(34):21482–8. doi: 10.1074/jbc.273.34.21482 9705276

33. Gasior SL, Olivares H, Ear U, Hari DM, Weichselbaum R, Bishop DK. Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8411–8. doi: 10.1073/pnas.121046198 11459983

34. Haaf T, Golub EI, Reddy G, Radding CM, Ward DC. Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2298–302. doi: 10.1073/pnas.92.6.2298 7892263

35. Gataulin DV, Carey JN, Li J, Shah P, Grubb JT, Bishop DK. The ATPase activity of E. coli RecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double stranded DNA. Nucleic Acids Res. 2018 Oct 12;46(18):9510–23. doi: 10.1093/nar/gky748 30137528

36. Brown MS, Grubb J, Zhang A, Rust MJ, Bishop DK. Small Rad51 and Dmc1 complexes often co-occupy both ends of a meiotic DNA double strand break. PLoS Genet. 2015 Dec;11(12):e1005653. doi: 10.1371/journal.pgen.1005653 26719980

37. Egelman EH, Stasiak A. Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP. J Mol Biol. 1986 Oct 20;191(4):677–97. doi: 10.1016/0022-2836(86)90453-5 2949085

38. Zakharyevich K, Ma Y, Tang S, Hwang PY-H, Boiteux S, Hunter N. Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions. Mol Cell. 2010 Dec 22;40(6):1001–15. doi: 10.1016/j.molcel.2010.11.032 21172664

39. Menetski JP, Bear DG, Kowalczykowski SC. Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc Natl Acad Sci U S A. 1990 Jan;87(1):21–5. doi: 10.1073/pnas.87.1.21 2404275

40. Rosselli W, Stasiak A. Energetics of RecA-mediated recombination reactions. Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle. J Mol Biol. 1990 Nov 20;216(2):335–52. doi: 10.1016/S0022-2836(05)80325-0 2147722

41. Sung P, Stratton SA. Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis. J Biol Chem. 1996 Nov 8;271(45):27983–6. doi: 10.1074/jbc.271.45.27983 8910403

42. Campbell MJ, Davis RW. Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation. J Mol Biol. 1999 Feb 19;286(2):417–35. doi: 10.1006/jmbi.1998.2456 9973561

43. Stasiak A, Egelman EH. Visualization of recombination intermediates. Kucherlapati R, Smith GR, editors. Genetic Recombination. 1988:265–307.

44. Sung P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science. 1994 Aug 26;265(5176):1241–3. doi: 10.1126/science.8066464 8066464

45. Zaitseva EM, Zaitsev EN, Kowalczykowski SC. The DNA binding properties of Saccharomyces cerevisiae Rad51 protein. J Biol Chem. 1999 Jan 29;274(5):2907–15. doi: 10.1074/jbc.274.5.2907 9915828

46. Hong EL, Shinohara A, Bishop DK. Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA. J Biol Chem. 2001 Nov 9;276(45):41906–12. doi: 10.1074/jbc.M105563200 11551925

47. Solinger JA, Kiianitsa K, Heyer W-D. Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell. 2002 Nov;10(5):1175–88. doi: 10.1016/s1097-2765(02)00743-8 12453424

48. Sheridan SD, Yu X, Roth R, Heuser JE, Sehorn MG, Sung P, et al. A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments. Nucleic Acids Res. 2008 Jul;36(12):4057–66. doi: 10.1093/nar/gkn352 18535008

49. Li X, Heyer W-D. RAD54 controls access to the invading 3'-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae. Nucleic Acids Res. 2009 Feb;37(2):638–46. doi: 10.1093/nar/gkn980 19074197

50. Chi P, Kwon Y, Seong C, Epshtein A, Lam I, Sung P, et al. Yeast recombination factor Rdh54 functionally interacts with the Rad51 recombinase and catalyzes Rad51 removal from DNA. J Biol Chem. 2006 Sep 8;281(36):26268–79. doi: 10.1074/jbc.M602983200 16831867

51. Holzen TM, Shah PP, Olivares HA, Bishop DK. Tid1/Rdh54 promotes dissociation of Dmc1 from nonrecombinogenic sites on meiotic chromatin. Genes Dev. 2006 Sep 15;20(18):2593–604. doi: 10.1101/gad.1447106 16980587

52. Shah PP, Zheng X, Epshtein A, Carey JN, Bishop DK, Klein HL. Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth. Mol Cell. 2010 Sep 24;39(6):862–72. doi: 10.1016/j.molcel.2010.08.028 20864034

53. Mason JM, Dusad K, Wright WD, Grubb J, Budke B, Heyer W-D, et al. RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells. Nucleic Acids Res. 2015 Mar 31;43(6):3180–96. doi: 10.1093/nar/gkv175 25765654

54. Hilario J, Amitani I, Baskin RJ, Kowalczykowski SC. Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc Natl Acad Sci U S A. 2009 Jan 13;106(2):361–8. doi: 10.1073/pnas.0811965106 19122145

55. Morgan EA, Shah N, Symington LS. The requirement for ATP hydrolysis by Saccharomyces cerevisiae Rad51 is bypassed by mating-type heterozygosity or RAD54 in high copy. Mol Cell Biol. 2002 Sep;22(18):6336–43. doi: 10.1128/MCB.22.18.6336-6343.2002 12192033

56. Fung CW, Fortin GS, Peterson SE, Symington LS. The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation. Mol Cell Biol. 2006 Dec;26(24):9544–54. doi: 10.1128/MCB.00599-06 17030607

57. Li X, Zhang X-P, Solinger JA, Kiianitsa K, Yu X, Egelman EH, et al. Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics. Nucleic Acids Res. 2007;35(12):4124–40. doi: 10.1093/nar/gkm412 17567608

58. Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, et al. Crystal structure of a Rad51 filament. Nat Struct Mol Biol. 2004 Aug;11(8):791–6. doi: 10.1038/nsmb795 15235592

59. Galkin VE, Wu Y, Zhang X-P, Qian X, He Y, Yu X, et al. The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity. Structure. 2006 Jun;14(6):983–92. doi: 10.1016/j.str.2006.04.001 16765891

60. Chan Y-L, Zhang A, Weissman BP, Bishop DK. RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination. Nucleic Acids Res. 2019 Jan 25;47(2):747–61. doi: 10.1093/nar/gky1160 30462332

61. Bishop DK. RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis. Cell. 1994 Dec 16;79(6):1081–92. doi: 10.1016/0092-8674(94)90038-8 7528104

62. Hayase A, Takagi M, Miyazaki T, Oshiumi H, Shinohara M, Shinohara A. A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1. Cell. 2004 Dec;119(7):927–40. doi: 10.1016/j.cell.2004.10.031 15620352

63. Tsubouchi H, Roeder GS. The budding yeast Mei5 and Sae3 proteins act together with Dmc1 during meiotic recombination. Genetics. 2004 Nov;168(3):1219–30. doi: 10.1534/genetics.103.025700 15579681

64. Zierhut C, Berlinger M, Rupp C, Shinohara A, Klein F. Mnd1 Is Required for Meiotic Interhomolog Repair. Curr Biol. 2004 May;14(9):752–62. doi: 10.1016/j.cub.2004.04.030 15120066

65. Henry JM, Camahort R, Rice DA, Florens L, Swanson SK, Washburn MP, et al. Mnd1/Hop2 facilitates Dmc1-dependent interhomolog crossover formation in meiosis of budding yeast. Mol Cell Biol. 2006 Apr;26(8):2913–23. doi: 10.1128/MCB.26.8.2913-2923.2006 16581767

66. Argunhan B, Murayama Y, Iwasaki H. The differentiated and conserved roles of Swi5-Sfr1 in homologous recombination. FEBS Lett. 2017 May 8;591(14):2035–47. doi: 10.1002/1873-3468.12656 28423184

67. Haruta N, Kurokawa Y, Murayama Y, Akamatsu Y, Unzai S, Tsutsui Y, et al. The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro. Nat Struct Mol Biol. 2006 Sep;13(9):823–30. doi: 10.1038/nsmb1136 16921379

68. Yuan J, Chen J. The role of the human SWI5-MEI5 complex in homologous recombination repair. J Biol Chem. 2011 Mar 18;286(11):9888–93. doi: 10.1074/jbc.M110.207290 21252223

69. Su G-C, Chung C-I, Liao C-Y, Lin S-W, Tsai C-T, Huang T, et al. Enhancement of ADP release from the RAD51 presynaptic filament by the SWI5-SFR1 complex. Nucleic Acids Res. 2014 Jan;42(1):349–58. doi: 10.1093/nar/gkt879 24078249

70. Ferrari SR, Grubb J, Bishop DK. The Mei5-Sae3 protein complex mediates Dmc1 activity in Saccharomyces cerevisiae. J Biol Chem. 2009 Apr 24;284(18):11766–70. doi: 10.1074/jbc.C900023200 19270307

71. Bugreev DV, Mazin AV. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):9988–93. doi: 10.1073/pnas.0402105101 15226506

72. Lu C-H, Yeh H-Y, Su G-C, Ito K, Kurokawa Y, Iwasaki H, et al. Swi5-Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation. Proc Natl Acad Sci U S A. 2018 Oct 23;115(43):E10059–68. doi: 10.1073/pnas.1812753115 30297419

73. McKee AH, Kleckner N. 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. 1997 Jul;146(3):817–34. 9215889

74. Akamatsu Y, Tsutsui Y, Morishita T, Siddique MSP, Kurokawa Y, Ikeguchi M, et al. Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes. EMBO J. 2007 Mar 7;26(5):1352–62. doi: 10.1038/sj.emboj.7601582 17304215

75. Akamatsu Y, Jasin M. Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination. PLoS Genet. 2010 Oct 14;6(10):e1001160. doi: 10.1371/journal.pgen.1001160 20976249

76. Tsubouchi H, Roeder GS. Budding yeast Hed1 down-regulates the mitotic recombination machinery when meiotic recombination is impaired. Genes Dev. 2006 Jul 1;20(13):1766–75. doi: 10.1101/gad.1422506 16818607

77. Busygina V, Sehorn MG, Shi IY, Tsubouchi H, Roeder GS, Sung P. Hed1 regulates Rad51-mediated recombination via a novel mechanism. Genes Dev. 2008 Mar 15;22(6):786–95. doi: 10.1101/gad.1638708 18347097

78. Shinohara A, Gasior S, Ogawa T, Kleckner N, Bishop DK. Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination. Genes Cells. 1997 Oct;2(10):615–29. doi: 10.1046/j.1365-2443.1997.1480347.x 9427283

79. Schwacha A, Kleckner N. Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway. Cell. 1997 Sep 19;90(6):1123–35. doi: 10.1016/s0092-8674(00)80378-5 9323140

80. Campbell MJ, Davis RW. On the in vivo function of the RecA ATPase. J Mol Biol. 1999 Feb 19;286(2):437–45. doi: 10.1006/jmbi.1998.2457 9973562

81. Fortin GS, Symington LS. Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51-DNA complexes. EMBO J. 2002 Jun 17;21(12):3160–70. doi: 10.1093/emboj/cdf293 12065428

82. Peck R, Olsen C. Statistics: Learning from Data (AP Edition). 1st ed. Boston: Cengage Learning.

83. Gasior SL, Wong AK, Kora Y, Shinohara A, Bishop DK. Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes. Genes Dev. 1998 Jul 15;12(14):2208–21. doi: 10.1101/gad.12.14.2208 9679065

84. Cao L, Alani E, Kleckner N. A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae. Cell. 1990 Jun 15;61(6):1089–101. doi: 10.1016/0092-8674(90)90072-m 2190690

85. Shinohara M, Shinohara A. Multiple pathways suppress non-allelic homologous recombination during meiosis in Saccharomyces cerevisiae. PLoS ONE. 2013;8(4):e63144. doi: 10.1371/journal.pone.0063144 23646187

86. Oh SD, Jessop L, Lao JP, Allers T, Lichten M, Hunter N. Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae. Methods Mol Biol. Totowa, NJ: Humana Press; 2009;557(Chapter 14):209–34. doi: 10.1007/978-1-59745-527-5_14 19799185

87. Hong S, Sung Y, Yu M, Lee M, Kleckner N, Kim KP. The logic and mechanism of homologous recombination partner choice. Mol Cell. 2013 Aug 22;51(4):440–53. doi: 10.1016/j.molcel.2013.08.008 23973374

88. Lao JP, Cloud V, Huang C-C, Grubb J, Thacker D, Lee C-Y, et al. Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation. PLoS Genet. 2013 Dec 19;9(12):e1003978–22. doi: 10.1371/journal.pgen.1003978 24367271

89. Dresser ME, Ewing DJ, Conrad MN, Dominguez AM, Barstead R, Jiang H, et al. DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway. Genetics. 1997 Oct;147(2):533–44. 9335591

90. Bishop DK. Rad51, the lead in mitotic recombinational DNA repair, plays a supporting role in budding yeast meiosis. Cell Cycle. 2012 Nov 15;11(22):4105–6. doi: 10.4161/cc.22396 23075494

91. Masson JY, Davies AA, Hajibagheri N, Van Dyck E, Benson FE, Stasiak AZ, et al. The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51. EMBO J. 1999 Nov 15;18(22):6552–60. doi: 10.1093/emboj/18.22.6552 10562567

92. Tarsounas M, Morita T, Pearlman RE, Moens PB. RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes. J Cell Biol. 1999 Oct 18;147(2):207–20. doi: 10.1083/jcb.147.2.207 10525529

93. Siaud N, Dray E, Gy I, Gérard E, Takvorian N, Doutriaux M-P. Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1. EMBO J. 2004 Mar 24;23(6):1392–401. doi: 10.1038/sj.emboj.7600146 15014444

94. Shinohara M, Gasior SL, Bishop DK, Shinohara A. Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination. Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10814–9. doi: 10.1073/pnas.97.20.10814 11005857

95. Shan Q, Bork JM, Webb BL, Inman RB, Cox MM. RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins. J Mol Biol. 1997 Feb 7;265(5):519–40. doi: 10.1006/jmbi.1996.0748 9048946

96. Sung P. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 1997 May 1;11(9):1111–21. doi: 10.1101/gad.11.9.1111 9159392

97. Liu J, Renault L, Veaute X, Fabre F, Stahlberg H, Heyer W-D. Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation. Nature. 2011 Oct 23;479(7372):245–8. doi: 10.1038/nature10522 22020281

98. Shinohara M, Shita-Yamaguchi E, Buerstedde JM, Shinagawa H, Ogawa H, Shinohara A. Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis. Genetics. 1997 Dec;147(4):1545–56. 9409820

99. Oh SD, Lao JP, Hwang PY-H, Taylor AF, Smith GR, Hunter N. BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules. Cell. 2007 Jul 27;130(2):259–72. doi: 10.1016/j.cell.2007.05.035 17662941

100. Kaur H, De Muyt A, Lichten M. Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates. Mol Cell. 2015 Feb 19;57(4):583–94. doi: 10.1016/j.molcel.2015.01.020 25699707

101. Tang S, Wu MKY, Zhang R, Hunter N. Pervasive and essential roles of the Top3-Rmi1 decatenase orchestrate recombination and facilitate chromosome segregation in meiosis. Mol Cell. 2015 Feb 19;57(4):607–21. doi: 10.1016/j.molcel.2015.01.021 25699709

102. Cejka P, Plank JL, Bachrati CZ, Hickson ID, Kowalczykowski SC. Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nat Struct Mol Biol. 2010 Nov;17(11):1377–82. doi: 10.1038/nsmb.1919 20935631

103. Fasching CL, Cejka P, Kowalczykowski SC, Heyer W-D. Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism. Mol Cell. 2015 Feb 19;57(4):595–606. doi: 10.1016/j.molcel.2015.01.022 25699708

104. Piazza A, Shah SS, Wright WD, Gore SK, Koszul R, Heyer W-D. Dynamic processing of displacement loops during recombinational DNA repair. Mol Cell. 2019 Mar 21;73(6):1255–1266.e4. doi: 10.1016/j.molcel.2019.01.005 30737186

105. Piazza A, Wright WD, Heyer W-D. Multi-invasions are recombination byproducts that induce chromosomal rearrangements. Cell. 2017 Aug 10;170(4):760–773.e15. doi: 10.1016/j.cell.2017.06.052 28781165

106. Piazza A, Heyer W-D. Multi-invasion-induced rearrangements as a pathway for physiological and pathological recombination. Bioessays. 2018 May;40(5):e1700249. doi: 10.1002/bies.201700249 29578583

107. Oh SD, Lao JP, Taylor AF, Smith GR, Hunter N. RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination. Mol Cell. 2008 Aug 8;31(3):324–36. doi: 10.1016/j.molcel.2008.07.006 18691965

108. Jessop L, Lichten M. Mus81/Mms4 endonuclease and Sgs1 helicase collaborate to ensure proper recombination intermediate metabolism during meiosis. Mol Cell. 2008 Aug 8;31(3):313–23. doi: 10.1016/j.molcel.2008.05.021 18691964

109. De Muyt A, Jessop L, Kolar E, Sourirajan A, Chen J, Dayani Y, et al. BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism. Mol Cell. 2012 Apr 13;46(1):43–53. doi: 10.1016/j.molcel.2012.02.020 22500736

110. Piazza A, Heyer W-D. Moving forward one step back at a time: reversibility during homologous recombination. Curr Genet. 2019 May 23:1–8.

111. Forget AL, Kowalczykowski SC. Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature. 2012 Feb 5;482(7385):423–7. doi: 10.1038/nature10782 22318518

112. Wright WD, Heyer W-D. Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D-loop formation. Mol Cell. 2014 Feb 6;53(3):420–32. doi: 10.1016/j.molcel.2013.12.027 24486020

113. Grubb J, Brown MS, Bishop DK. Surface spreading and immunostaining of yeast chromosomes. J Vis Exp. 2015 Aug 9;(102):e53081. doi: 10.3791/53081 26325523

114. Lao JP, Tang S, Hunter N. Native/Denaturing two-dimensional DNA electrophoresis and its application to the analysis of recombination intermediates. Methods Mol Biol. Totowa, NJ: Humana Press; 2013;1054(Pt 2):105–20.

115. Arora C, Kee K, Maleki S, Keeney S. Antiviral protein Ski8 is a direct partner of Spo11 in meiotic DNA break formation, independent of its cytoplasmic role in RNA metabolism. Mol Cell. 2004 Feb 27;13(4):549–59. doi: 10.1016/s1097-2765(04)00063-2 14992724

116. James P, Halladay J, Craig EA. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 1996 Dec;144(4):1425–36. 8978031