RESUMO
The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. Here, we show that Cdc6 localized to the centrosomes during S and G2 phases of the cell cycle. The centrosomal localization was mediated by Cdc6 amino acid residues 311-366, which are conserved within other Cdc6 homologues and contains a putative nuclear export signal. Deletions or substitutions of the amino acid residues did not allow the proteins to localize to centrosomes. In contrast, DsRed tag fused to the amino acid residues localized to centrosomes. These results indicated that a centrosome localization signal is contained within amino acid residues 311-366. The cell cycle-dependent centrosomal localization of Cdc6 in S and G2 phases suggest a novel function of Cdc6 in centrosomes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Fase G2 , Proteínas Nucleares/metabolismo , Fase S , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Deleção de SequênciaRESUMO
During the late M to the G(1) phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2-5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2-5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.
Assuntos
Cromatina/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Processamento de Proteína Pós-Traducional , Origem de Replicação , Sequência de Aminoácidos , Sequência Conservada , Células HeLa , Humanos , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem/química , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Fase SRESUMO
Human TopBP1 is involved in the DNA damage checkpoint response, chromosome replication, and other functions of cell cycle control. The C-terminal region of TopBP1 (TbpCtr: amino acid residues 1222-1522) is involved in the localization of TopBP1 to the centrosomes during mitosis. Here, we showed that the amino acid residues 741-885 of TopBP1, in addition to TbpCtr, are necessary for the centrosomal localization of TopBP1. Whereas oligomeric tags fused to TbpCtr localized to mitotic centrosomes, monomeric tags fused to TbpCtr did not. Insertion of the amino acid residues 741-885 into the monomeric tag fused to TbpCtr allowed the protein to localize to the mitotic centrosome. These results suggest that the amino acid residues 741-885 are necessary for oligomerization of TopBP1 for centrosomal localization.
Assuntos
Proteínas de Transporte/metabolismo , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Mitose , Proteínas Nucleares/metabolismo , Ciclo Celular , Centrossomo/ultraestrutura , Dano ao DNA , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fosforilação , Combinação Trimetoprima e Sulfametoxazol/químicaRESUMO
TopBP1 contains repeats of the BRCA1 C-terminal (BRCT) domain and plays important roles in DNA damage response, DNA replication, and other cellular regulatory functions during the interphase. In prometaphase, metaphase, and anaphase, TopBP1 localizes to the mitotic centrosomes, which function as spindle-poles for the bipolar separation of sister chromatids. The localization of TopBP1 to the mitotic centrosomes is mediated by amino acid residues 1259 to 1420 in the TopBP1 C-terminal region (TbpCtr). GST and DsRed2 tags fused to TbpCtr were localized in the mitotic centrosomes, thereby suggesting that TbpCtr functions as a mitosis-specific centrosome localization signal (CLS). Mutations of Ser 1273 and/or Lys 1317, which were predicted to interact with a putative phosphoprotein, inhibited CLS function. Ectopic expression of TbpCtr specifically eliminated endogenous TopBP1 from the mitotic centrosomes, whereas mutant TbpCtr derivatives, containing substitutions at Ser 1273 and/or Lys 1317, did not. The specific elimination of TopBP1 from the mitotic centrosomes prolonged the durations of prometaphase and metaphase and shortened the inter-kinetochore distances of metaphase sister chromatids while maintaining the spindle assembly checkpoint. These results suggest that the localization of TopBP1 to the mitotic centrosomes is necessary for proper mitotic progression.
Assuntos
Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cromátides/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Deleção de Genes , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
In model organisms, MCM10 is required for forming the pre-initiation complex for initiation of chromosome replication and is involved in the elongation step. To investigate the role of MCM10 in human chromosome replication, we used small interfering RNA (siRNA) in MCM10-knockdown experiments and found that knockdown accumulated S and G2 phase cells. The chromosome replication of MCM10-knockdown cells was slowed during early and mid S phases, although Cdc45, Polalpha, and PCNA proteins were loaded onto the chromatin, and was aberrant during late S phase. Our results indicate that MCM10 is essential for the efficient elongation step of chromosome replication.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Cromossomos Humanos/genética , Replicação do DNA/genética , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , DNA Polimerase I/análise , DNA Polimerase I/metabolismo , Replicação do DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Fase G2/genética , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo , Antígeno Nuclear de Célula em Proliferação/análise , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/farmacologia , Fase S/efeitos dos fármacos , Fase S/genéticaRESUMO
MCM10 has been shown to be a component for the elongation step of chromosome replication in model organisms such as yeast. In the accompanying manuscript [J.H. Park, S.W. Bang, Y. Jeon, S. Kang, D.S. Hwang, Knockdown of human MCM10 exhibits delayed and incomplete chromosome replication, Biochem. Biophys. Res. Commun. (2007) 365 (2008) 575-582.], we reported that knockdown of human MCM10 protein exhibits delayed and incomplete chromosomal DNA replication. In this report, we examined the consequences of the delayed and incomplete chromosome replication in the cell cycle. Defective and incomplete chromosome replication by MCM10 knockdown activated a checkpoint pathway, composed of Chk1 and Cdc25, that inhibited Cdk1. Chk2 appeared not to be involved in the Cdk1 inhibition. The function of Cdk1 is necessary for the transition from G2 to mitotic phase, thereby Cdk1 inhibition by checkpoint arrested MCM10-knockdown cells in G2 phase. The prolonged depletion of MCM10 resulted in DNA damage followed by cell death. These results indicate that MCM10 protein is essential for maintaining genome integrity as well as cell cycle progression.