RESUMEN
Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.
Asunto(s)
Proteína BRCA2/metabolismo , Replicación del ADN , ADN/biosíntesis , Recombinasa Rad51/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Proteína BRCA2/genética , Sitios de Unión , ADN/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa I/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Femenino , Inestabilidad Genómica , Humanos , Proteína Homóloga de MRE11 , Masculino , Mutación , Unión Proteica , Recombinasa Rad51/genética , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
Asunto(s)
Replicación del ADN , Interferón Tipo I/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Citosol/metabolismo , ADN de Cadena Simple/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Interferón Tipo I/inmunología , Proteína Homóloga de MRE11/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , RecQ Helicasas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/deficienciaRESUMEN
Fanconi anemia (FA) is a genetic disorder characterized by a defect in DNA interstrand crosslink (ICL) repair, chromosomal instability, and a predisposition to cancer. Recently, two RAD51 mutations were reported to cause an FA-like phenotype. Despite the tight association of FA/HR proteins with replication fork (RF) stabilization during normal replication, it remains unknown how FA-associated RAD51 mutations affect replication beyond ICL lesions. Here, we report that these mutations fail to protect nascent DNA from MRE11-mediated degradation during RF stalling in Xenopus laevis egg extracts. Reconstitution of DNA protection in vitro revealed that the defect arises directly due to altered RAD51 properties. Both mutations induce pronounced structural changes and RAD51 filament destabilization that is not rescued by prevention of ATP hydrolysis due to aberrant ATP binding. Our results further interconnect the FA pathway with DNA replication and provide mechanistic insight into the role of RAD51 in recombination-independent mechanisms of genome maintenance.