RESUMO
Initiation of eukaryotic DNA replication is achieved by the sequential binding of different proteins to origins of DNA replication. Using EGFP-tagged initiator proteins and immunofluorescence techniques we found that most of the ORC and the MCM subunits are localised at centrosomes and are colocalised with the polo-like protein kinase, Plk1. Yeast two-hybrid studies revealed interactions of Plk1 with the Mcm2 as well as the Orc2 protein. Co-immunoprecipitations showed an interaction of Plk1 with Mcm2 as well as interactions of gamma-tubulin with Mcm3 and Orc2, respectively. An in vitro phosphorylation assay showed that the Orc2 protein is a substrate of Plk1. Depletion of Orc2 and Mcm3 by siRNA leads to an inhibition of cell proliferation, an altered cell cycle distribution as well as to multinucleated cells with insufficiently organised microtubules. These results indicate an important role of the MCM and ORC proteins in mitosis besides their described role in the establishment of the pre-replicative complex.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Centrômero/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Imunofluorescência , Proteínas de Fluorescência Verde , Camundongos , Componente 3 do Complexo de Manutenção de Minicromossomo , Mitose/genética , Mitose/fisiologia , Proteínas Nucleares/genética , Complexo de Reconhecimento de Origem/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Quinase 1 Polo-LikeRESUMO
A replication fork barrier at the 3'-end of mouse ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. This barrier is an inherent property of a defined DNA-protein complex including transcription termination factor I, and specific protein-protein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNA-binding protein is essential for barrier activity. We have purified and functionally characterised the protein from HeLa cells. The final preparation contained two polypeptides with molecular masses of 70 and 86 kDa, respectively. Both polypeptides interact with a GC-stretch adjacent to the binding site of transcription termination factor I. The specificity of binding to the barrier DNA was demonstrated in an electrophoretic mobility shift assay. The biochemical properties of this protein resemble that of Ku antigen, a human nuclear DNA-binding heterodimer that is the target of autoimmune-antibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from co-infected insect cells, is able to partially rescue the barrier activity in Ku-depleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent head-on collision between the replication and the transcription machinery.