Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm–/– spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.
|State||Published - 1 Dec 2020|
Bibliographical noteFunding Information:
We thank Todd Macfarlan, Mohamed Mahgoub, Michael Lichten, and Kevin Brick for comments on the manuscript; Francesca Cole, Todd Macfarlan, Mohamed Mahgoub, Scott Keeney, Agnieszka Lukaszewicz, Maria Jasin, Anjali Hinch, and Petko Petkov for sharing unpublished data and insightful suggestions; Petko Petkov for PRDM9 KO mice and Scott Keeney for DMC1 knockout mice; R. Daniel Camerini-Otero and Florencia Pratto for HOP2 KO mice; Keith W. Caldecott for purified human TDP2; and Jennifer Wise and Kelly Smith for assistance with animal work. The A.N. laboratory is supported by the Intramural Research Program of the NIH, an Ellison Medical Foundation Senior Scholar in Aging Award (AG-SS-2633-11), the Department of Defense Idea Expansion (W81XWH-15-2-006) and Breakthrough (W81XWH-16-1-599) Awards, the Alex Lemonade Stand Foundation Award, and an NIH Intramural FLEX Award.
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