RESUMEN
BRCA1 promotes error-free, homologous recombination-mediated repair (HRR) of DNA double-stranded breaks (DSBs). When excessive and uncontrolled, BRCA1 HRR activity promotes illegitimate recombination and genome disorder. We and others have observed that the BRCA1-associated protein RAP80 recruits BRCA1 to postdamage nuclear foci, and these chromatin structures then restrict the amplitude of BRCA1-driven HRR. What remains unclear is how this process is regulated. Here we report that both BRCA1 poly-ADP ribosylation (PARsylation) and the presence of BRCA1-bound RAP80 are critical for the normal interaction of BRCA1 with some of its partners (e.g., CtIP and BACH1) that are also known components of the aforementioned focal structures. Surprisingly, the simultaneous loss of RAP80 and failure therein of BRCA1 PARsylation results in the dysregulated accumulation in these foci of BRCA1 complexes. This in turn is associated with the intracellular development of a state of hyper-recombination and gross chromosomal disorder. Thus, physiological RAP80-BRCA1 complex formation and BRCA1 PARsylation contribute to the kinetics by which BRCA1 HRR-sustaining complexes normally concentrate in nuclear foci. These events likely contribute to aneuploidy suppression.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/metabolismo , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cromosomas/genética , Cromosomas/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Chaperonas de Histonas/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli ADP Ribosilación , Unión ProteicaRESUMEN
UNLABELLED: BRCA1 promotes homologous recombination-mediated DNA repair (HRR). However, HRR must be tightly regulated to prevent illegitimate recombination. We previously found that BRCA1 HRR function is regulated by the RAP80 complex, but the mechanism was unclear. We have now observed that PARP1 interacts with and poly-ADP-ribosylates (aka PARsylates) BRCA1. PARsylation is directed at the BRCA1 DNA binding domain and downmodulates its function. Moreover, RAP80 contains a poly-ADP-ribose-interacting domain that binds PARsylated BRCA1 and helps to maintain the stability of PARP1-BRCA1-RAP80 complexes. BRCA1 PARsylation is a key step in BRCA1 HRR control. When BRCA1 PARsylation is defective, it gives rise to excessive HRR and manifestations of genome instability. BRCA1 PARsylation and/or RAP80 expression is defective in a subset of sporadic breast cancer cell lines and patient-derived tumor xenograft models. These observations are consistent with the possibility that such defects, when chronic, contribute to tumor development in BRCA1+/+ individuals. SIGNIFICANCE: We propose a model that describes how BRCA1 functions to both support and restrict HRR. BRCA1 PARsylation is a key event in this process, failure of which triggers hyper-recombination and chromosome instability. Thus, hyperfunctioning BRCA1 can elicit genomic abnormalities similar to those observed in the absence of certain BRCA1 functions.
Asunto(s)
Proteína BRCA1/metabolismo , Reparación del ADN , Recombinación Homóloga , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/química , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromosomas Humanos , Daño del ADN , Proteínas de Unión al ADN , Femenino , Expresión Génica , Inestabilidad Genómica , Células HeLa , Chaperonas de Histonas , Humanos , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos , Poli(ADP-Ribosa) Polimerasa-1 , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.