ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Abstract

Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations ("hotspots") are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate ("SPO11-RI") present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3' resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm -/spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing. MainRecombination between homologous chromosomes during meiosis requires DNA double-strand break (DSB) formation by the topoisomerase-like protein SPO11 1 . After cutting, SPO11 remains covalently bound to a two-nucleotide, 5' overhang at both ends of the DNA via phosphotyrosyl linkage. Recombination then begins with the processing of SPO11-bound DSBs into resected 3' single-stranded DNA (ssDNA) tails that preferentially invade the homologous chromosome by the recombinases DMC1 and RAD51. Studies in budding yeast Saccharomyces cerevisiae determined that the MRE11/RAD50/NBS1 (MRN) complex detects SPO11 and cooperates with Sae2 to produce a nick on the SPO11-bound strand via MRE11 endonuclease activity 2 . The nick serves as an entry point for both short-range MRE11 3'-5' exonuclease activity to degrade back to the DSB, thereby removing covalently bound SPO11 attached to a ssDNA oligonucleotide, as well as for more extensive long-range processing of 5' strands (Extended Data Fig. 1a) 2 . In budding yeast, Exo1 nuclease is uniquely responsible for this long-range 5'-3' resection 3 .Moreover, short-and long-range resection are tightly coupled in a single processive reaction (Extended Data Fig. 1a). As a result, meiotic DSBs are maximally resected as soon as they appear and unresected SPO11-bound DSBs are extremely rare 4-6 . While ATM has been shown to regulate DSB numbers and locations 7,8 , its remains unclear whether it also functions downstream in regulating SPO11 processing and resection.Distinct from yeast, DSB hotspots in mice and humans are determined by the DNA binding specificity of the PRDM9 methyltransferase 9 . Besides positioning DSBs, PRDM9...

DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

“…In addition, one or both DSB ends might remain embedded within the condensate via the persistence of Spo11-oligos that might cap the ends after resection. End-capping by Spo11 (Neale et al, 2005) has recently received support from patterns of recombination intermediates detected in mice (Paiano et al, 2019;Yamada et al, 2019), and is consistent with the observation that Spo11 binds tightly to DNA ends in vitro, even in the absence of a covalent link (Claeys Bouuaert et al, in preparation).…”

Structural and functional characterization of the Spo11 core complex

“…The remarkably tight end-binding activity of the core complex suggests that Spo11-oligo complexes may in fact cap most DSB ends, potentially affecting subsequent repair. In support for this hypothesis, recent mapping of ssDNA-dsDNA junctions in mouse spermatocytes revealed patterns of recombination intermediates consistent with scenarios where the 3'-end remains annealed to the Spo11 oligo (Paiano et al, 2019;Yamada et al, 2019). Since the core complex itself is tethered to the chromosome axis, probably embedded within Rec114-Mei4-Mer2 nucleoprotein condensates, end-capping would therefore maintain a physical connection between the two broken ends during recombination, facilitating their repair and potentially reducing the risks of gross chromosomal rearrangements.…”

The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

“…The role of BRCA1 in loading RAD51 can be partly compensated for by RNF168 60 , whereas PALB2 and BRCA2 are absolutely required 25,35 . Productive assembly of the recombinase has been shown to limit the extent of end resection 61,62 . Therefore, it is possible that deletion of THAP1/SHLD1 or 53BP1 would lead to excessive single-strand DNA in BRCA2-deficient cells, which in turn could increase genomic instability by engaging highly mutagenic RAD51independent single strand annealing pathways 52 .…”