RESUMEN
The accurate repair of DNA double-strand breaks (DSBs), highly toxic DNA lesions, is crucial for genome integrity and is tightly regulated during the cell cycle. In mitosis, cells inactivate DSB repair in favor of a tethering mechanism that stabilizes broken chromosomes until they are repaired in the subsequent cell cycle phases. How this is achieved mechanistically is not yet understood, but the adaptor protein TOPBP1 is critically implicated in this process. Here, we identify CIP2A as a TOPBP1-interacting protein that regulates TOPBP1 localization specifically in mitosis. Cells lacking CIP2A display increased radio-sensitivity, micronuclei formation and chromosomal instability. CIP2A is actively exported from the cell nucleus in interphase but, upon nuclear envelope breakdown at the onset of mitosis, gains access to chromatin where it forms a complex with MDC1 and TOPBP1 to promote TOPBP1 recruitment to sites of mitotic DSBs. Collectively, our data uncover CIP2A-TOPBP1 as a mitosis-specific genome maintenance complex.
Asunto(s)
Autoantígenos , Proteínas Portadoras , Reparación del ADN , Proteínas de Unión al ADN , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteínas Nucleares , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica , ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitosis/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Nucléolo Celular/genética , ADN Ribosómico/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Secuencias de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Roturas del ADN de Doble Cadena , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , ARN Ribosómico/genética , ARN Ribosómico/metabolismoRESUMEN
In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.
