Molecular structures and mechanisms of DNA break processing in mouse meiosis

Abstract

Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genomewide at nucleotide resolution. Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Surprisingly, EXO1 is not the major 5 ′ → 3 ′ exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3 ′ ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

A pathway for error-free non-homologous end joining of resected meiotic double-strand breaks

“…In some cases, these DSBs may be repaired through non-allelic homologous recombination with a nearby copy of the same transposable element ( 42 ), but it is reasonable to anticipate that the NHEJ repair mechanism we propose is not infrequently invoked in normal meiosis. The discovery that Spo11-oligos remain annealed in mouse meiosis ( 24 , 25 ), together with results presented here, suggests that this precise NHEJ backup pathway may be conserved in diverse eukaryotes.…”

“…We propose a model for precise post-resection NHEJ based on the recent reports that Spo11-oligonucleotides remain annealed to DSB ends after resection in mouse meiosis ( 24 , 25 ) (Figure 5 ; Supplementary Figure S1 ). We hypothesize that Spo11-oligonucleotides remain annealed during the homology search in Drosophila (Figure 5A – C ).…”

“…Rad51 and associated proteins (the meiosis-specific Rad51 paralog Dmc1 is not shown because it is not present in Drosophila ) assembles on the single-stranded DNA exposed by resection ( C ). Recent observations suggest that a short Spo11-oligonucleotide remains annealed at the end of each 3′ overhanging strand ( 24 , 25 ). A homology search and strand exchange produces a D-loop ( D ).…”

“…We propose that this is possible because of retention of Spo11-bound oligonucleotides at the ends of the DSBs. In male meiosis in mice, only a small fraction of DSB ends appeared to retain binding of Spo11-oligonucleotides ( 24 , 25 ). Although the tools to detect this intermediate in Drosphila do not exist, we speculate that this intermediate may be more prevalent.…”

“…We propose a model in which Spo11-bound oligonucleotides annealed to the ends of resected DSBs can function as primers for synthesis to fill in resected regions, allowing error-free repair by NHEJ. Recent studies provide evidence for a similar intermediate in mouse meiosis ( 24 , 25 ), suggesting that this may be a conserved mechanism for repair of meiotic DSBs when the homologous chromosome is unavailable.…”