RESUMO
The six minichromosome maintenance proteins (Mcm2-7) are required for both the initiation and elongation of chromosomal DNA, ensuring that DNA replication takes place once, and only once, during the S phase. Here we report on the cloning of a new human Mcm gene (hMcm8) and on characterisation of its protein product. The hMcm8 gene contains the central Mcm domain conserved in the Mcm2-7 gene family, and is expressed in a range of cell lines and human tissues. hMcm8 mRNA accumulates during G(1)/S phase, while hMcm8 protein is detectable throughout the cell cycle. Immunoprecipitation-based studies did not reveal any participation of hMcm8 in the Mcm3/5 and Mcm2/4/6/7 subcomplexes. hMcm8 localises to the nucleus, although it is devoid of a nuclear localisation signal, suggesting that it binds to a nuclear protein. In the nucleus, the hMcm8 structure-bound fraction is detectable in S, but not in G(2)/M, phase, as for hMcm3. However, unlike hMcm3, the hMcm8 structure-bound fraction is not detectable in G(1) phase. Overall, our data identify a new Mcm protein, which does not form part of the Mcm2-7 complex and which is only structure-bound during S phase, thus suggesting its specific role in DNA replication.
Assuntos
Proteínas de Ciclo Celular/genética , Família Multigênica/genética , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Evolução Molecular , Fase G1 , Expressão Gênica , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Filogenia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
The human replication protein Cdc6p is translocated from its chromatin sites to the cytoplasm during the replication phase (S phase) of the cell cycle. However, the amounts of Cdc6p on chromatin remain high during S phase implying either that displaced Cdc6p can rebind to chromatin, or that Cdc6p is synthesized de novo. We have performed metabolic labeling experiments and determined that [35S]methionine is incorporated into Cdc6p at similar rates during the G1 phase and the S phase of the cell cycle. Newly synthesized Cdc6p associates with chromatin. Pulse-chase experiments show that chromatin-bound newly synthesized Cdc6p has a half life of 2-4 h. The results indicate that, once bound to chromatin, pulse-labeled new Cdc6p behaves just as old Cdc6p: it dissociates and eventually disappears from the nucleus. The data suggest a surprisingly dynamic behaviour of Cdc6p in the HeLa cell cycle.
