DNA double-strand breaks (DSBs) are formed in meiosis, so their repair in the homologous recombination (HR) pathway will lead to crossover formation, which is essential for successful chromosome segregation. HR contains 2 subpathways: synthesis-dependent strand annealing (SDSA) that creates noncrossover and double Holliday junction (dHJ) that generates crossovers. RAD-51 is a protein essential to the formation of all products of HR, as it assembles on the processed DSB, allowing the invasion of the single-stranded DNA into a region of homology. RAD-51 is removed by RAD-54.L after invasion to allow for repair to occur. Here, we investigate a separation of function allele of rad-51, rad-51::FLAG, as compared to 2 other RAD-51 alleles: rad-51::degron and GFP::rad-51. rad-51::FLAG displays slowed repair kinetics, resulting in an accumulation of RAD-51 foci. rad-51::FLAG worms also activate the DSB checkpoint, but to a less extant than that of rad-51 null mutants. In a proximity ligation assay, RAD-54.L and RAD-51 show enriched colocalization in rad-51::FLAG germlines (but not in rad-51::degron), consistent with stalling at the strand invasion step in HR. The defects in RAD-51 disassembly in rad-51::FLAG mutants lead to formation of chromosomal fragments, similar in their magnitude to ones observed in rad-51 or rad-54.L null mutants. However, rad-51::FLAG mutants (unlike a rad-51 null, GFP::rad-51 or rad-54.L null mutants) displayed no defects in the formation of crossover-designated sites (via GFP::COSA-1 localization). Given that rad-51::FLAG worms show checkpoint activation and chromosomal fragments, these results suggest that crossover repair concludes normally, while the noncrossover pathway is perturbed. This is strikingly different from rad-51::degron and GFP::rad-51 strains, which are proficient or deficient in both pathways, respectively. These results suggest that noncrossovers vs crossovers have distinct recombination intermediates and diverge prior to RAD-51 disassembly.

Department of Biology Faculty of Medicine Masaryk University Brno 625 00 Czech Republic

Department of Biology The University of Iowa Iowa City IA 52242 USA

See more in PubMed

Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, et al. . 2024. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 630(8016):493–500. doi:10.1038/s41586-024-07487-w. PubMed DOI PMC

Afshar N, Argunhan B, Palihati M, Taniguchi G, Tsubouchi H, Tsubouchi H, Iwasaki H. 2021. A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors. eLife. 10:e64131. doi:10.7554/eLife.64131. PubMed DOI PMC

Allers T, Lichten M. 2001. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell. 106(1):47–57. doi:10.1016/S0092-8674(01)00416-0. PubMed DOI

Alpi A, Pasierbek P, Gartner A, Loidl J. 2003. Genetic and cytological characterization of the recombination protein RAD-51 in Caenorhabditis elegans. Chromosoma. 112(1):6–16. doi:10.1007/s00412-003-0237-5. PubMed DOI

Alt FW, Schwer B. 2018. DNA double-strand breaks as drivers of neural genomic change, function, and disease. DNA Repair (Amst). 71:158–163. doi:10.1016/j.dnarep.2018.08.019. PubMed DOI PMC

Andersen SL, Sekelsky J. 2010. Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes. Bioessays. 32(12):1058–1066. doi:10.1002/bies.201000087. PubMed DOI PMC

Berchowitz LE, Copenhaver GP. 2010. Genetic interference: don't stand so close to me. Curr Genomics. 11(2):91–102. doi:10.2174/138920210790886835. PubMed DOI PMC

Bhattacharya D, Sahoo S, Nagraj T, Dixit S, Dwivedi HK, Nagaraju G. 2022. RAD51 paralogs: expanding roles in replication stress responses and repair. Curr Opin Pharmacol. 67:102313. doi:10.1016/j.coph.2022.102313. PubMed DOI

Borde V. 2007. The multiple roles of the Mre11 complex for meiotic recombination. Chromosome Res. 15(5):551–563. doi:10.1007/s10577-007-1147-9. PubMed DOI

Crickard JB, Moevus CJ, Kwon Y, Sung P, Greene EC. 2020. Rad54 drives ATP hydrolysis-dependent DNA sequence alignment during homologous recombination. Cell. 181(6):1380–1394.e1318. doi:10.1016/j.cell.2020.04.056. PubMed DOI PMC

Day NJ, Wang X, Voronina E. 2020. In situ detection of ribonucleoprotein complex assembly in the C. elegans germline using proximity ligation assay. J Vis Exp. (159):10.3791/60982. doi:10.3791/60982https://pubmed.ncbi.nlm.nih.gov/32449701/. PubMed DOI PMC

Dello Stritto RM, Bauer B, Barraud P, Jantsch V. 2021. DNA topoisomerase 3 is required for efficient germ cell quality control. J Cell Biol. 220(6):e202012057. doi:10.1083/jcb.202012057. PubMed DOI PMC

Diagouraga B, Tambones I, Carivenc C, Bechara C, Nadal M, de Massy B, le Maire A, Robert T. 2024. The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization. Nucleic Acids Res. 52:8930–8946. doi:10.1093/nar/gkae587. PubMed DOI PMC

Game JC, Zamb TJ, Braun RJ, Resnick M, Roth RM. 1980. The role of radiation (rad) genes in meiotic recombination in yeast. Genetics. 94(1):51–68. doi:10.1093/genetics/94.1.51. PubMed DOI PMC

Hegazy M, Cohen-Barak E, Koetsier JL, Najor NA, Arvanitis C, Sprecher E, Green KJ, Godsel LM. 2020. Proximity ligation assay for detecting protein–protein interactions and protein modifications in cells and tissues in situ. Curr Protoc Cell Biol. 89(1):e115. doi:10.1002/cpcb.115. PubMed DOI PMC

Heyer WD. 2004. Recombination: Holliday junction resolution and crossover formation. Curr Biol. 14(2):R56–R58. doi:10.1016/j.cub.2003.12.043. PubMed DOI

Hicks T, Trivedi S, Eppert M, Bowman R, Tian H, Dafalla A, Crahan C, Smolikove S, Silva N. 2022. Continuous double-strand break induction and their differential processing sustain chiasma formation during Caenorhabditis elegans meiosis. Cell Rep. 40(13):111403. doi:10.1016/j.celrep.2022.111403. PubMed DOI

Hinman AW, Yeh HY, Roelens B, Yamaya K, Woglar A, Bourbon H-MG, Chi P, Villeneuve AM. 2021. Caenorhabditis elegans DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks. Proc Natl Acad Sci U S A 118(33):e2109306118. doi:10.1073/pnas.2109306118. PubMed DOI PMC

Hoppe MM, Jaynes P, Wardyn JD, Upadhyayula SS, Tan TZ, Lie S, Lim DGZ, Pang BNK, Lim S, P S Yeong J, et al. . 2021. Quantitative imaging of RAD51 expression as a marker of platinum resistance in ovarian cancer. EMBO Mol Med. 13(5):e13366. doi:10.15252/emmm.202013366. PubMed DOI PMC

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(1):59–70. doi:10.1016/S0092-8674(01)00430-5. PubMed DOI

Hwang HY, Wang J. 2021. Fast genetic mapping using insertion-deletion polymorphisms in Caenorhabditis elegans. Sci Rep. 11(1):11017. doi:10.1038/s41598-021-90190-x. PubMed DOI PMC

Ito M, Fujita Y, Shinohara A. 2024. Positive and negative regulators of RAD51/DMC1 in homologous recombination and DNA replication. DNA Repair (Amst). 134:103613. doi:10.1016/j.dnarep.2023.103613. PubMed DOI

Koury E, Harrell K, Smolikove S. 2018. Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation. Nucleic Acids Res. 46(2):748–764. doi:10.1093/nar/gkx1243. PubMed DOI PMC

La Volpe A, Barchi M. 2012. Meiotic double strand breaks repair in sexually reproducing eukaryotes: we are not all equal. Exp Cell Res. 318(12):1333–1339. doi:10.1016/j.yexcr.2012.03.014. PubMed DOI

Lemmens BBLG, Tijsterman M. 2011. DNA double-strand break repair in Caenorhabditis elegans. Chromosoma. 120(1):1–21. doi:10.1007/s00412-010-0296-3. PubMed DOI PMC

Liu S, Mine-Hattab J, Villemeur M, Guerois R, Pinholt HD, Mirny LA, Taddei A. 2023. In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search. Nat Struct Mol Biol. 30(10):1582–1591. doi:10.1038/s41594-023-01065-w. PubMed DOI

Luke-Glaser S, Pintard L, Tyers M, Peter M. 2007. The AAA-ATPase FIGL-1 controls mitotic progression, and its levels are regulated by the CUL-3MEL-26 E3 ligase in the C. elegans germ line. J Cell Sci. 120(18):3179–3187. doi:10.1242/jcs.015883. PubMed DOI

Macaisne N, Kessler Z, Yanowitz JL. 2018. Meiotic double-strand break proteins influence repair pathway utilization. Genetics. 210(3):843–856. doi:10.1534/genetics.118.301402. PubMed DOI PMC

Penkner AM, Fridkin A, Gloggnitzer J, Baudrimont A, Machacek T, Woglar A, Csaszar E, Pasierbek P, Ammerer G, Gruenbaum Y, et al. . 2009. Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1. Cell. 139(5):920–933. doi:10.1016/j.cell.2009.10.045. PubMed DOI

Ranjha L, Howard SM, Cejka P. 2018. Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes. Chromosoma. 127(2):187–214. doi:10.1007/s00412-017-0658-1. PubMed DOI

Rijkers T, Van Den Ouweland J, Morolli B, Rolink AG, Baarends WM, Van Sloun PP, Lohman PH, Pastink A. 1998. Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation. Mol Cell Biol. 18(11):6423–6429. doi:10.1128/MCB.18.11.6423. PubMed DOI PMC

Rinaldo C, Bazzicalupo P, Ederle S, Hilliard M, La Volpe A. 2002. Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development. Genetics. 160(2):471–479. doi:10.1093/genetics/160.2.471. PubMed DOI PMC

Rosu S, Zawadzki KA, Stamper EL, Libuda DE, Reese AL, Dernburg AF, Villeneuve AM. 2013. The C. elegans DSB-2 protein reveals a regulatory network that controls competence for meiotic DSB formation and promotes crossover assurance. PLoS Genet. 9(8):e1003674. doi:10.1371/journal.pgen.1003674. PubMed DOI PMC

Roy U, Greene EC. 2021. The role of the Rad55-Rad57 complex in DNA repair. Genes (Basel). 12(9):1390. doi:10.3390/genes12091390. PubMed DOI PMC

Spirek M, Taylor MRG, Belan O, Boulton SJ, Krejci L. 2021. Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function. Nat Commun. 12(1):5545. doi:10.1038/s41467-021-25830-x. PubMed DOI PMC

Stamper EL, Rodenbusch SE, Rosu S, Ahringer J, Villeneuve AM, Dernburg AF. 2013. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint. PLoS Genet. 9(8):e1003679. doi:10.1371/journal.pgen.1003679. PubMed DOI PMC

Takanami T, Mori A, Takahashi H, Horiuchi S, Higashitani A. 2003. Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination. Chromosome Res. 11(2):125–135. doi:10.1023/A:1022863814686. PubMed DOI

Tischler JD, Tsuchida H, Bosire R, Oda TT, Park A, Adeyemi RO. 2024. FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress. Nat Commun. 15(1):866. doi:10.1038/s41467-024-45139-9. PubMed DOI PMC

Ward JD, Muzzini DM, Petalcorin MIR, Martinez-Perez E, Martin JS, Plevani P, Cassata G, Marini F, Boulton SJ. 2010. Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair. Mol Cell. 37(2):259–272. doi:10.1016/j.molcel.2009.12.026. PubMed DOI

Williams AB, Michael WM. 2010. Eviction notice: new insights into Rad51 removal from DNA during homologous recombination. Mol Cell. 37(2):157–158. doi:10.1016/j.molcel.2010.01.009. PubMed DOI

Yamaya K, Wang B, Memar N, Odiba AS, Woglar A, Gartner A, Villeneuve AM. 2023. Disparate roles for C. elegans DNA translocase paralogs RAD-54.L and RAD-54.B in meiotic prophase germ cells. Nucleic Acids Res. 51(17):9183–9202. doi:10.1093/nar/gkad638. PubMed DOI PMC

Yu Z, Kim Y, Dernburg AF. 2016. Meiotic recombination and the crossover assurance checkpoint in Caenorhabditis elegans. Semin Cell Dev Biol. 54:106–116. doi:10.1016/j.semcdb.2016.03.014. PubMed DOI PMC

Zhang L, Köhler S, Rillo-Bohn R, Dernburg AF. 2018. A compartmentalized signaling network mediates crossover control in meiosis. eLife. 7:245. doi:10.7554/eLife.30789. PubMed DOI PMC

Loss of Meiotic Double Strand Breaks Triggers Recruitment of Recombination-independent Pro-crossover Factors in C. elegans Spermatogenesis