RESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Meiose , Proteínas de Ligação a Fosfato/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Troca Genética , DNA de Cadeia Simples/metabolismo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , TestículoRESUMO
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' â 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.
Assuntos
Reparo do DNA/fisiologia , Meiose , Animais , Cromatina/química , Cromatina/metabolismo , DNA/química , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Estrutura Molecular , Recombinação GenéticaRESUMO
Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks, which requires choreographed binding of RPA, DMC1, and RAD51 to single-stranded DNA substrates. High-resolution in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the chromosome used as template for repair, revealing more mechanistic detail and enabling the structure of the recombination intermediates to be inferred. By applying CRISPR-Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this approach to explore the molecular detail of recombination when a key component is knocked out. As a proof of concept, we have generated hybrid biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in them. DMC1 is essential for meiotic recombination, and comparison of these maps with those from wild-type mice is informative about the structure and timing of critical recombination intermediates. We observe redistribution of RAD51 binding and complete abrogation of D-loop recombination intermediates at a molecular level in Dmc1 mutants. These data provide insight on the configuration of RPA in D-loop intermediates and suggest that stable strand exchange proceeds via multiple rounds of strand invasion with template switching in mouse. Our methodology provides a high-throughput approach for characterization of gene function in meiotic recombination at low animal cost.
RESUMO
Over the past decade, genomic analyses of single cells-the fundamental units of life-have become possible. Single-cell DNA sequencing has shed light on biological questions that were previously inaccessible across diverse fields of research, including somatic mutagenesis, organismal development, genome function, and microbiology. Single-cell DNA sequencing also promises significant future biomedical and clinical impact, spanning oncology, fertility, and beyond. While single-cell approaches that profile RNA and protein have greatly expanded our understanding of cellular diversity, many fundamental questions in biology and important biomedical applications require analysis of the DNA of single cells. Here, we review the applications and biological questions for which single-cell DNA sequencing is uniquely suited or required. We include a discussion of the fields that will be impacted by single-cell DNA sequencing as the technology continues to advance.
Assuntos
Genoma , Genômica , DNA , Humanos , RNA , Análise de Sequência de DNARESUMO
The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.
Assuntos
Especiação Genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Hibridização Genética/genética , Infertilidade/genética , Engenharia de Proteínas , Dedos de Zinco/genética , Alelos , Animais , Sítios de Ligação , Pareamento Cromossômico/genética , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Feminino , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Estrutura Terciária de Proteína/genética , Recombinação Genética/genéticaRESUMO
Meiotic recombination commences with hundreds of programmed DNA breaks; however, the degree to which they are accurately repaired remains poorly understood. We report that meiotic break repair is eightfold more mutagenic for single-base substitutions than was previously understood, leading to de novo mutation in one in four sperm and one in 12 eggs. Its impact on indels and structural variants is even higher, with 100- to 1300-fold increases in rates per break. We uncovered new mutational signatures and footprints relative to break sites, which implicate unexpected biochemical processes and error-prone DNA repair mechanisms, including translesion synthesis and end joining in meiotic break repair. We provide evidence that these mechanisms drive mutagenesis in human germ lines and lead to disruption of hundreds of genes genome wide.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Genoma Humano , Meiose , Mutagênese , Recombinação Genética , Humanos , Masculino , Meiose/genética , Mutação , Óvulo/metabolismo , Sêmen/metabolismo , Síntese de DNA Translesão , FemininoRESUMO
Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.