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Abstract 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. 

Abstract Accumulation of DNA–RNA hybrids in the form of R-loops can result in replication–transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis. 

In meiotic prophase I, homologous chromosome pairing is promoted through chromosome movement mediated by nuclear envelope proteins, microtubules, and dynein. After proper homologue pairing has been established, the synaptonemal complex (SC) assembles along the paired homologues, stabilizing their interaction and allowing for crossing over to occur. Previous studies have shown that perturbing chromosome movement leads to pairing defects and SC polycomplex formation. We show that FKB-6 plays a role in SC assembly and is required for timely pairing and proper double-strand break repair kinetics. FKB-6 localizes outside the nucleus, and in its absence, the microtubule network is altered. FKB-6 is required for proper movement of dynein, increasing resting time between movements. Attenuating chromosomal movement in fkb-6 mutants partially restores the defects in synapsis, in agreement with FKB-6 acting by decreasing chromosomal movement. Therefore, we suggest that FKB-6 plays a role in regulating dynein movement by preventing excess chromosome movement, which is essential for proper SC assembly and homologous chromosome pairing.