RESUMEN
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.
Asunto(s)
Intercambio Genético/fisiología , Recombinación Homóloga/fisiología , Meiosis/fisiología , Animales , Segregación Cromosómica/genética , Intercambio Genético/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Recombinación Homóloga/genética , Masculino , Meiosis/genética , Ratones , Ratones Endogámicos DBA , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Ácidos Nucleicos Heterodúplex/genéticaRESUMEN
During meiosis, numerous DNA double-strand breaks (DSBs) are formed as part of the normal developmental program. This seemingly destructive behavior is necessary for successful meiosis, since repair of the DSBs through homologous recombination (HR) helps to produce physical links between the homologous chromosomes essential for correct chromosome segregation later in meiosis. However, DSB formation at such a massive scale also introduces opportunities to generate gross chromosomal rearrangements. In this review, we explore ways in which meiotic DSBs can result in such genomic alterations.
Asunto(s)
Inestabilidad Genómica , Células Germinativas/metabolismo , Animales , Cromotripsis , Roturas del ADN de Doble Cadena , Reordenamiento Génico/genética , Humanos , Meiosis/genéticaRESUMEN
DNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homology-directed repair (HDR) is initiated by resection of DSBs to generate 3' single-stranded DNA overhangs. How resection and HDR are activated during DDR is not known, nor are the roles of ATM and ATR in HDR. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Nonphosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection, leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/fisiología , Extractos Celulares/aislamiento & purificación , Cromatina/metabolismo , Secuencia Conservada , División del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Conejos , Proteínas Supresoras de Tumor/química , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/fisiología , Xenopus laevisRESUMEN
DNA double-strand break (DSB) resection, which results in RPA-bound single-stranded DNA (ssDNA), is activated in S phase by Cdk2. RPA-ssDNA activates the ATR-dependent checkpoint and homology-directed repair (HDR) via Rad51-dependent mechanisms. On the other hand, the fate of DSBs sustained during vertebrate M phase is largely unknown. We use cell-free Xenopus laevis egg extracts to examine the recruitment of proteins to chromatin after DSB formation. We find that S-phase extract recapitulates a two-step resection mechanism. M-phase chromosomes are also resected in cell-free extracts and cultured human cells. In contrast to the events in S phase, M-phase resection is solely dependent on MRN-CtIP. Despite generation of RPA-ssDNA, M-phase resection does not lead to ATR activation or Rad51 chromatin association. Remarkably, we find that Cdk1 permits resection by phosphorylation of CtIP but also prevents Rad51 binding to the resected ends. We have thus identified Cdk1 as a critical regulator of DSB repair in M phase. Cdk1 induces persistent ssDNA-RPA overhangs in M phase, thereby preventing both classical NHEJ and Rad51-dependent HDR.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , División Celular/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Antígenos Nucleares/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Sistema Libre de Células , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromatina/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Autoantígeno Ku , Proteína Homóloga de MRE11 , Meiosis/fisiología , Mitosis/fisiología , Membrana Nuclear/fisiología , Óvulo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , RecQ Helicasas/metabolismo , Proteína de Replicación A/metabolismo , Fase S/fisiología , Helicasa del Síndrome de Werner , Proteínas de Xenopus/antagonistas & inhibidores , Xenopus laevisRESUMEN
The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.