Oberlitner Joseph, Tinman Maggie, Das Aasthika, Koury Emily, Silva Nicola, Smolikove Sarit
Department of Biology, The University of Iowa, Iowa City, IA 52242, USA.
Department of Biology, Faculty of Medicine, Masaryk University, Brno 625 00, Czech Republic.
Genetics. 2025 Jun 4;230(2). doi: 10.1093/genetics/iyaf063.
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.
DNA双链断裂（DSB）在减数分裂过程中形成，因此其在同源重组（HR）途径中的修复将导致交叉形成，这对于成功的染色体分离至关重要。HR包含2个子途径：产生非交叉的合成依赖链退火（SDSA）和产生交叉的双Holliday连接（dHJ）。RAD-51是HR所有产物形成所必需的蛋白质，因为它在加工后的DSB上组装，使单链DNA侵入同源区域。入侵后，RAD-54.L会将RAD-51移除，以便进行修复。在这里，我们研究了rad-51的功能分离等位基因rad-51::FLAG，与其他2个RAD-51等位基因rad-51::degron和GFP::rad-51进行比较。rad-51::FLAG显示出修复动力学减慢，导致RAD-51焦点积累。rad-51::FLAG线虫也会激活DSB检查点，但程度低于rad-51缺失突变体。在邻近连接分析中，RAD-54.L和RAD-51在rad-51::FLAG种系中显示出富集的共定位（但在rad-51::degron中没有），这与HR中链入侵步骤的停滞一致。rad-51::FLAG突变体中RAD-51拆卸的缺陷导致染色体片段的形成，其大小与在rad-51或rad-54.L缺失突变体中观察到的相似。然而，rad-51::FLAG突变体（与rad-51缺失、GFP::rad-51或rad-54.L缺失突变体不同）在交叉指定位点的形成（通过GFP::COSA-1定位）上没有缺陷。鉴于rad-51::FLAG线虫显示出检查点激活和染色体片段，这些结果表明交叉修复正常完成，而非交叉途径受到干扰。这与rad-51::degron和GFP::rad-51菌株显著不同，它们分别在这两个途径中 proficient 或 deficient。这些结果表明，非交叉与交叉具有不同的重组中间体，并且在RAD-51拆卸之前就有所不同。
