Journal article
Analysis of rad-51 separation of function allele suggests divergence of the SDSA and dHJ pathways prior to RAD-51 filament disassembly
Genetics (Austin), Vol.230(2), iyaf063
06/04/2025
DOI: 10.1093/genetics/iyaf063
PMCID: PMC12135186
PMID: 40192500
Appears in UI Libraries Support Open Access
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 chromosomes segregation. HR contains two sub pathways: synthesis dependent strand annealing (SDSA) that creates non-crossover, 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 ssDNA 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 two 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 non-crossover 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 non-crossovers vs crossovers have distinct recombination intermediates and diverge prior to RAD-51 disassembly.
Details
- Title: Subtitle
- Analysis of rad-51 separation of function allele suggests divergence of the SDSA and dHJ pathways prior to RAD-51 filament disassembly
- Creators
- Joseph Oberlitner - Department of Biology, The University of Iowa, Iowa City, IA 52242, USAMaggie Tinman - Department of Biology, The University of Iowa, Iowa City, IA 52242, USAAasthika Das - Department of Biology, The University of Iowa, Iowa City, IA 52242, USAEmily Koury - University of IowaNicola Silva - Masaryk UniversitySarit Smolikove - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Genetics (Austin), Vol.230(2), iyaf063
- DOI
- 10.1093/genetics/iyaf063
- PMID
- 40192500
- PMCID
- PMC12135186
- NLM abbreviation
- Genetics
- ISSN
- 1943-2631
- eISSN
- 1943-2631
- Publisher
- Oxford University Press
- Grant note
- Czech Science Foundation: P40 OD-010440 National Institutes of Health (NIH) Office of Research Infrastructure Programs
We are grateful to Jantsch V., for anti-phospho-SUN-1S8 antibody and the rad-51::degron strain. Some strains and clones were kindly provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD-010440), and the C. elegans Reverse Genetics Core Facility at the University of British Columbia, which is part of the International C. elegans Gene Knockout Consortium. We thank the National BioResource Project for the Experimental Animal "Nematode C. elegans," Japan for providing alleles for this study.
- Language
- English
- Electronic publication date
- 04/07/2025
- Date published
- 06/04/2025
- Academic Unit
- Biology
- Record Identifier
- 9984808530902771
Metrics
23 Record Views