RESUMO
Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/química , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células Cultivadas , Proteínas Cromossômicas não Histona/análise , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/análise , Dimerização , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosfotreonina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Treonina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.
Assuntos
Cromatina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA , Transdução de Sinais/genética , Autoantígenos/genética , Autoantígenos/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Cromatina/genética , Cromossomos/metabolismo , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Genes cdc , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Radiação Ionizante , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
The MRE11-RAD50-NBS1 (MRN) complex accumulates at sites of DNA double-strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1-dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation.
