Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Line
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Cell Proliferation
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Centromere / genetics
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Centromere / metabolism*
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DNA Replication / genetics
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DNA Replication / physiology*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Fluorescent Antibody Technique
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Green Fluorescent Proteins
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Mice
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Minichromosome Maintenance Complex Component 3
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Mitosis / genetics
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Mitosis / physiology
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Origin Recognition Complex / genetics
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Origin Recognition Complex / metabolism*
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Polo-Like Kinase 1
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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RNA, Small Interfering / genetics
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RNA, Small Interfering / metabolism
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Tubulin / genetics
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Tubulin / metabolism
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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Mcm3 protein, mouse
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Nuclear Proteins
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Origin Recognition Complex
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Proto-Oncogene Proteins
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RNA, Small Interfering
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Tubulin
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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Protein Serine-Threonine Kinases
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Minichromosome Maintenance Complex Component 3