Summary The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots.

DNA double-strand breaks (DSBs) with protein covalently attached to 5′ strand termini are formed by Spo11 to initiate meiotic recombination 1,2 . The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal has been unclear 3 . We show here that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3′-OH. Surprisingly, two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-significantly earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.We previously proposed that Spo11 might be removed from DSB ends by either of two mechanisms: direct hydrolysis of the covalent protein-DNA linkage, or single-stranded endonucleolytic cleavage releasing Spo11 covalently attached to a short oligonucleotide (Fig. 1a) 1,4 . These mechanisms are distinguished by the presence or absence of an oligonucleotide bound to Spo11. We identified this predicted protein-DNA complex using a direct biochemical approach in S. cerevisiae. A strain expressing HA epitope-tagged Spo11 was induced to enter meiosis, denaturing extracts were prepared, then Spo11-HA was immunoprecipitated and treated with 32 P-labelled nucleotide and terminal deoxynucleotidyl transferase (TdT), which catalyzes untemplated addition of nucleotides to a free 3′-OH DNA end. A chain terminating nucleotide was used to limit incorporation to a single residue.Four radiolabelled bands were observed between ~60 and 110 kDa (Fig. 1b, lane 3). Two bands (asterisks) were non-specific because they were present when TdT and nucleotide were incubated alone (Fig. 1b, lane 1). Two bands (solid arrows) were specific for Spo11-HA because they were not seen with mock immunoprecipitation (Fig. 1b, lane 2), or untagged Spo11 (Fig.1b, lane 4). Labelling was not observed if DSBs were not formed, namely, when the catalytic tyrosine of Spo11 was mutated to phenylalanine 2 (Fig. 1b, lane 5) or in a mei4 mutant 4 (Fig. 1b, lane 6). Labelled Spo11 species were also not observed in rad50S or sae2Δ mutants (Fig. 1b, lanes 7-8), in which Spo11 remains covalently attached to DSB ends 1,5 . Thus, Spo11-oligonucleotide complexes did not arise from non-physiological disruption of covalent Spo11-DSB complexes.Correspondence and requests for materials should be addressed to S.K. (e-mail: keeneys@mskcc.or...

SUMMARY Heritability and genome stability are shaped by meiotic recombination, which is initiated via hundreds of DNA double-strand breaks (DSBs). The distribution of DSBs throughout the genome is not random, but mechanisms molding this landscape remain poorly understood. Here we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure—DSBs occur within narrow zones between methylated nucleosomes—and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide a genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features governing successive steps in mammalian meiotic recombination.

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes1. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation2. However, improperly repaired DSBs can cause meiotic arrest or mutation3,4, thus having too many DSBs is likely as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse5, and SPO11 and its accessory factors remain abundant long after most DSB formation ceases1, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signaling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least ten-fold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.