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1.
Cell Rep ; 17(12): 3099-3106, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-28009280

RESUMO

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.


Assuntos
Proteína BRCA1/química , Proteínas de Transporte/química , Enzimas Desubiquitinantes/química , Histonas/química , Complexos Multiproteicos/química , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/genética , Proteína BRCA1/ultraestrutura , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Cromatina/química , Cromatina/genética , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/ultraestrutura , Instabilidade Genômica , Histonas/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Reparo de DNA por Recombinação/genética , Ubiquitina/genética
2.
Mol Cell ; 45(3): 371-83, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22325354

RESUMO

Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.


Assuntos
Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Sequência de Aminoácidos , Proteína BRCA2/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Sequência Conservada , Instabilidade Genômica , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/química , Quinase 1 Polo-Like
3.
Nucleic Acids Res ; 40(9): 3913-28, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22234878

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 p53
4.
Cell ; 139(1): 100-11, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19804756

RESUMO

The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Reparo do DNA , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência
5.
J Biol Chem ; 283(51): 36019-30, 2008 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-18948271

RESUMO

Chk2/CHEK2/hCds1 is a modular serine-threonine kinase involved in transducing DNA damage signals. Phosphorylation by ataxia telangiectasia-mutated kinase (ATM) promotes Chk2 self-association, autophosphorylation, and activation. Here we use expressed protein ligation to generate a Chk2 N-terminal regulatory region encompassing a fork-head-associated (FHA) domain, a stoichiometrically phosphorylated Thr-68 motif and intervening linker. Hydrodynamic analysis reveals that Thr-68 phosphorylation stabilizes weak FHA-FHA interactions that occur in the unphosphorylated species to form a high affinity dimer. Although clearly a prerequisite for Chk2 activation in vivo, we show that dimerization modulates potential phosphodependent interactions with effector proteins and substrates through either the pThr-68 site, or the canonical FHA phosphobinding surface with which it is tightly associated. We further show that the dimer-occluded pThr-68 motif is released by intra-dimer autophosphorylation of the FHA domain at the highly conserved Ser-140 position, a major pThr contact in all FHA-phosphopeptide complex structures, revealing a mechanism of Chk2 dimer dissociation following kinase domain activation.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Ativação Enzimática/fisiologia , Humanos , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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