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Genetic recombination arises during meiosis through the repair of DNA double-strand breaks (DSBs) that are created by Spo11, a topoisomerase-like protein.sup.1,2. Spo11 DSBs form preferentially in nucleosome-depleted regions termed hotspots.sup.3,4, yet how Spo11 engages with its DNA substrate to catalyse DNA cleavage is poorly understood. Although most recombination events are initiated by a single Spo11 cut, here we show in Saccharomyces cerevisiae that hyperlocalized, concerted Spo11 DSBs separated by 33 to more than 100 base pairs also form, which we term 'double cuts'. Notably, the lengths of double cuts vary with a periodicity of 10.5 base pairs, which is conserved in yeast and mice. This finding suggests a model in which the orientation of adjacent Spo11 molecules is fixed relative to the DNA helix--a proposal supported by the in vitro DNA-binding properties of the Spo11 core complex. Deep sequencing of meiotic progeny identifies recombination scars that are consistent with repair initiated from gaps generated by adjacent Spo11 DSBs. Collectively, these results revise our present understanding of the mechanics of Spo11-DSB formation and expand on the original concepts of gap repair during meiosis to include DNA gaps that are generated by Spo11 itself. A small proportion of Spo11-dependent DNA double-strand breaks are 'double cuts'--adjacent breaks that occur in concert--revealing that gap repair during meiosis includes that of DNA gaps generated by Spo11 itself., Author(s): Dominic Johnson [sup.1] , Margaret Crawford [sup.1] , Tim Cooper [sup.1] , Corentin Claeys Bouuaert [sup.2] [sup.3] , Scott Keeney [sup.2] , Bertrand Llorente [sup.4] , Valerie Garcia [sup.1] [...]

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis.sup.1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily.sup.2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes.sup.3-7 and are nearly universal in eukaryotes.sup.8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes. Several mechanisms regulate the distribution of double-strand breaks during meiosis in Saccharomyces cerevisiae, ensuring that the shortest chromosomes are able to successfully recombine., Author(s): Hajime Murakami [sup.1] , Isabel Lam [sup.1] [sup.2] [sup.5] , Pei-Ching Huang [sup.1] [sup.3] , Jacquelyn Song [sup.1] , Megan van Overbeek [sup.1] [sup.6] , Scott Keeney [sup.1] [sup.2] [...]

Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. Here we test the hypothesis that DSB control involves a network of intersecting negative regulatory circuits. Using multiple complementary methods, we show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. ZMM-dependent DSB control is genetically distinct from a pathway tying break formation to meiotic progression through the Ndt80 transcription factor. These counterintuitive findings suggest that homologous chromosomes that have successfully engaged one another stop making breaks. Genome-wide DSB maps uncover distinct responses by different subchromosomal domains to the ZMM mutation zip3 (also known as cst9), and show that Zip3 is required for the previously unexplained tendency of DSB density to vary with chromosome size. Thus, feedback tied to ZMM function contributes in unexpected ways to spatial patterning of recombination., DSBs are hazardous genomic damage that most cells avoid but that each meiotic cell introduces in large numbers; therefore the activity of Spo 11, the protein that makes DSBs, must [...]

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes (1). Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation (2). However, improperly repaired DSBs can cause meiotic arrest or mutation (3,4); thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse (5) and SPO11 and its accessory factors remain abundant long after most DSB formation ceases (1), 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 signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold 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., SPO11 creates DSBs via a covalent protein-DNA intermediate that is endonucleolytically cleaved to release SPO11 attached to a short oligonucleotide, freeing DSB ends for further processing and recombination (5) (Fig. [...]

Author(s): Matthew J. Neale [1]; Scott Keeney (corresponding author) [1] Sexual organisms must halve the chromosome number in gametes to maintain genome size with each generation. This goal is achieved [...]

Author(s): Matthew J. Neale [1]; Jing Pan [1]; Scott Keeney (corresponding author) [1, 2] DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 [...]