De novo deletions and duplications at recombination hotspots in mouse germlines.

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

The Landscape of Mouse Meiotic Double-Strand Break Formation, Processing, and Repair.

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.

Structure and DNA-bridging activity of the essential Rec114-Mei4 trimer interface.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher-order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C terminus of Rec114 with the N terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

Divergence and conservation of the meiotic recombination machinery.

Sexually reproducing eukaryotes use recombination between homologous chromosomes to promote chromosome segregation during meiosis. Meiotic recombination is almost universally conserved in its broad strokes, but specific molecular details often differ considerably between taxa, and the proteins that constitute the recombination machinery show substantial sequence variability. The extent of this variation is becoming increasingly clear because of recent increases in genomic resources and advances in protein structure prediction. We discuss the tension between functional conservation and rapid evolutionary change with a focus on the proteins that are required for the formation and repair of meiotic DNA double-strand breaks. We highlight phylogenetic relationships on different time scales and propose that this remarkable evolutionary plasticity is a fundamental property of meiotic recombination that shapes our understanding of molecular mechanisms in reproductive biology.

YTHDC2 control of gametogenesis requires helicase activity but not m6A binding.

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.

Temporospatial coordination of meiotic DNA replication and recombination via DDK recruitment to replisomes.

It has been long appreciated that, during meiosis, DNA replication is coordinated with the subsequent formation of the double-strand breaks (DSBs) that initiate recombination, but a mechanistic understanding of this process was elusive. We now show that, in yeast, the replisome-associated components Tof1 and Csm3 physically associate with the Dbf4-dependent Cdc7 kinase (DDK) and recruit it to the replisome, where it phosphorylates the DSB-promoting factor Mer2 in the wake of the replication fork, synchronizing replication with an early prerequisite for DSB formation. Recruiting regulatory kinases to replisomes may be a general mechanism to ensure spatial and temporal coordination of replication with other chromosomal processes.

Mechanistic Insight into Crossing over during Mouse Meiosis.

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.

The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Chromosome-autonomous feedback down-regulates meiotic DNA break competence upon synaptonemal complex formation.

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.

Molecular structures and mechanisms of DNA break processing in mouse meiosis.

Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Surprisingly, EXO1 is not the major 5' → 3' exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3' ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

The joy of six: how to control your crossovers.

Meiotic cells tightly regulate the number and distribution of crossovers to promote accurate chromosome segregation. Yokoo and colleagues uncover a metazoan-specific, cyclin-like protein that is crucial for crossover formation. They utilize this protein's unique properties to explore a remarkable example of biological numerology, whereby nearly every meiotic cell in C. elegans makes precisely six crossovers, one for each of its six chromosome pairs.

DNA-driven condensation assembles the meiotic DNA break machinery.

The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein1,2. The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures3-5, but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery2. Each subcomplex of Rec114-Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodispersed in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive this condensation. Mutations that weaken protein-DNA interactions strongly disrupt both condensate formation and DSBs in vivo, and thus these processes are highly correlated. In vitro, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centres of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of the biophysical properties of the condensates.

Concerted cutting by Spo11 illuminates meiotic DNA break mechanics.

Genetic recombination arises during meiosis through the repair of DNA double-strand breaks (DSBs) that are created by Spo11, a topoisomerase-like protein1,2. Spo11 DSBs form preferentially in nucleosome-depleted regions termed hotspots3,4, 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.

REC114 Partner ANKRD31 Controls Number, Timing, and Location of Meiotic DNA Breaks.

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.

A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation.

The nonrandom distribution of meiotic recombination influences 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 the accumulation of Spo11 protein covalently bound to small DNA fragments. By sequencing these fragments, we uncover a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore how DSB distribution is influenced by large-scale chromosome structures, chromatin, transcription factors, and local sequence composition. Our analysis offers mechanistic insight into DSB formation and early processing steps, supporting the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We also discuss implications for evolutionary dynamics of recombination hot spots.

Multilayered mechanisms ensure that short chromosomes recombine in meiosis.

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. 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 arbitrarily2. 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 chromosomes3-7 and are nearly universal in eukaryotes8-11. 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.

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region.

Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation1,2. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.

Essential roles of the ANKRD31-REC114 interaction in meiotic recombination and mouse spermatogenesis.

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine whether and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31-REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31-REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114 and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.

Triple-helix potential of the mouse genome.

Certain DNA sequences, including mirror-symmetric polypyrimidine•polypurine runs, are capable of folding into a triple-helix­containing non­B-form DNA structure called H-DNA. Such H-DNA­forming sequences occur frequently in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)­specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides an approach for studying DNA secondary structure genome-wide at high spatial resolution.

yama, a mutant allele of Mov10l1, disrupts retrotransposon silencing and piRNA biogenesis.

Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation, named yama, in the Mov10l1 N-terminal region. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5' ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development.