![]() Signal summed from −2000 to −250 bp (bottom strand) and +250 to +2000 bp (top strand) around hot spot centers. Each point is a SPO11-oligo hot spot, with S1-seq ( D) Correlation (Pearson's r) of S1-seq read count with DSB intensity measured by SPO11-oligo sequencing. ( C) S1-seq reads averaged around 13,960 SPO11-oligo hot spot centers in wild type and Spo11 –/–. The baseline of the Y-axis for each plot is at 0. SPO11-oligo sequencingĭata here and throughout are from Lange et al. ExoT-seq uses an exonuclease instead of S1 endonuclease to remove ssDNA tails. Dmc1 +/+ is a Dmc1-proficient control from the same breeding colony as Dmc1 –/– null on a mixed background. ( B) Strand-specific S1-seq (reads per million mapped reads ) at a representative DSB hot spot. (Panel ii) In S1-seq, sequencing adaptors are linked to duplex ends generated by removal of ssDNA tails using S1 nuclease. Homologous duplex and carry out strand invasion. Resected ends have 3′-ssDNA ends that serve as substrates for strand-exchange proteins DMC1 and RAD51, which search for a MRE11 and associated factors, providing an entry point(s) for exonucleolytic resection and release of SPO11-oligo complexes. SPO11-bound strands are nicked (arrowheads) by (Panel i) SPO11 (magenta ellipses) cuts DNA via a covalent protein–DNA intermediate. ( A) Early recombination steps and S1-seq. Nucleotide-resolution maps of meiotic DSB resection in mice. In mice, for example, we do not even know whetherĮXO1 is required, and we have only a low-resolution population-average view of resection lengths that was deduced indirectlyįrom sequencing of ssDNA bound by the strand exchange protein DMC1 ( Lange et al. However, aside from yeast, meiotic resection mechanisms are unknown. 2011 Cannavo and Cejka 2014 Mimitou et al. 2005) and providing entry points for modest 3′ → 5′ Mre11 exonuclease and robust 5′ → 3′ Exo1 exonuclease ( Zakharyevich et al. Our current understanding, from the budding yeast Saccharomyces cerevisiae, is that endonucleolytic cleavage by Mre11–Rad50–Xrs2 (MRX) plus Sae2 nicks Spo11-bound strands, releasing Spo11 bound toĪ short oligonucleotide ( Keeney et al. 1A, panel i Lam and Keeney 2014 Hunter 2015). With DSBs made by SPO11 via a covalent protein–DNA intermediate ( Fig. Meiotic recombination, which ensures homologous chromosome pairing and segregation and enhances genetic diversity, initiates Issues here through genome-wide analysis of DSB resection during meiosis in mouse spermatocytes. Of resection mechanisms and the fine-scale structure of resected DNA ends in most species, including mammals. Despite a decades-long appreciation of resection's central role in DSB repair, however, we lack detailed understanding Is used for homology search and strand invasion during recombination ( Symington 2014). Nucleolytic processing of double-strand break (DSB) ends, termed resection, generates the single-stranded DNA (ssDNA) that Processing and repair in meiotic chromatin. Our findings give insight into the mechanisms of DSB Remodeling leading to increased accessibility at recombination sites. Finally, we provide evidence for PRDM9-dependent chromatin In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions,Ĭonsistent with joint molecules with incompletely invaded 3′ ends. Is not the major 5′ → 3′ exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly
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