The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates

Abstract

Summary Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo , with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Genome-wide variability in recombination activity is associated with meiotic chromatin organization

“…Additionally, we note that genomic positions of DSBs and cohesin ChIP sites appear to have relatively low cis/total ratios, despite the fact that immunofluorescent microscopy shows DSB machinery and cohesin subunits enriched along the axes [47,48,49]. In general, genomic loci with axial positions in a brush-loop structure would be expected to display high cis/total ratios characteristic of low spatial accessibility.…”

“…Therefore, the Hi-C signals we observe at DSB or crossoverfavoured genomic coordinates are unlikely to directly reflect chromosomal configuration at individual DSB and crossover events, as even the most favoured coordinates are not sites of recombination in most cells. Additionally, we note that genomic positions of DSBs and cohesin ChIP sites appear to have relatively low cis/total ratios, despite the fact that immunofluorescent microscopy shows DSB machinery and cohesin subunits enriched along the axes [47, 48, 49]. In general, genomic loci with axial positions in a brush-loop structure would be expected to display high cis/total ratios characteristic of low spatial accessibility.…”

DNA Damaged Induced Cell Death in Oocytes

“…This massive induction of DNA DSBs, of course, requires an efficient repair mechanism. Following the exchange of genetic material between homologous chromosomes, lesions are repaired by the homologous recombination (HR) pathway that is dependent on DMC1 and RAD51 [18]. Approximately 10% of the lesions are thus repaired by a crossover and 90% by a non-crossover mechanism.…”

Programmed DNA Damage and Physiological DSBs: Mapping, Biological Significance and Perturbations in Disease States

“…Chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) was one of the earlier methods to incorporate NGS and has been applied in order to identify sites of protein-DNA interactions [86]. Work done by Hinch et al demonstrated the use of ChIP-seq in order to understand the roles of RPA, RAD51 and DMC1 in the strand exchange of mammalian meiosis [87]. ChIP-seq can also be utilized to study the chromatin state of genes, by immunoprecipitating and analyzing epigenetic modifications, as demonstrated by Grosselin et al [88].…”

“…Work done by Hinch et al demonstrated the use of ChIP-seq in order to understand the roles of RPA, RAD51 and DMC1 in the strand exchange of mammalian meiosis [87]. ChIP-seq can also be utilized to study the chromatin state of genes, by immunoprecipitating and analyzing epigenetic modifications, as demonstrated by Grosselin et al [88].…”