RESUMO
The ubiquitin ligase CRL4Cdt2 plays a vital role in preserving genomic integrity by regulating essential proteins during S phase and after DNA damage. Deregulation of CRL4Cdt2 during the cell cycle can cause DNA re-replication, which correlates with malignant transformation and tumor growth. CRL4Cdt2 regulates a broad spectrum of cell cycle substrates for ubiquitination and proteolysis, including Cdc10-dependent transcript 1 or Chromatin licensing and DNA replication factor 1 (Cdt1), histone H4K20 mono-methyltransferase (Set8) and cyclin-dependent kinase inhibitor 1 (p21), which regulate DNA replication. However, the mechanism it operates via its substrate receptor, Cdc10-dependent transcript 2 (Cdt2), is not fully understood. This review describes the essential features of the N-terminal and C-terminal parts of Cdt2 that regulate CRL4 ubiquitination activity, including the substrate recognition domain, intrinsically disordered region (IDR), phosphorylation sites, the PCNA-interacting protein-box (PIP) box motif and the DNA binding domain. Drugs targeting these specific domains of Cdt2 could have potential for the treatment of cancer.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Proteínas de Ciclo Celular/genética , DNA/genética , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA-damaging agents and during the S phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the S phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Fosforilação/fisiologia , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Espectrometria de Massas em Tandem/métodos , Ubiquitinação/fisiologiaRESUMO
Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polµ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polµ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polµ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polµ. We identified Ser12, Thr21 (located in the BRCT domain) and Ser372 (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser372 is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polµ could be regulated in vivo by phosphorylation of the BRCT domain (Ser12/Thr21) and of Ser372, affecting the function of loop1. Consequently, Polµ's most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.