Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization.
MCM3 is essential for the initiation of DNA replication and also participates in controls that ensure DNA replication is initiated once per cell cycle. In a two-hybrid screen for proteins that interact with human MCM3, we identified and cloned a novel protein of which the calculated molecular weight is 80,291. A specific antibody against the protein identified a 80-kDa protein in HeLa cell extract, indicating the protein actually expressed in cells. The interaction of these proteins was confirmed by immunoprecipitation assay. Moreover, we clarified a nuclear localization signal of human MCM3, and we find that mutagenesis on the nuclear localization signal of MCM3 affected the binding of newly isolated MCM3-assosiated protein, Map80. Map80 was expressed in Escherichia coli as a fusion with His6 tag and purified with sequential column chromatographies. The addition of recombinant Map80 stimulated the amount of nuclear localized MCM3. These results suggest that Map80 is involved in the nuclear localization pathway of MCM3.
Department of Molecular Cell Pharmacology, National Children's Medical Research Center, Taishido 3-35-31, Setagaya-ku, Tokyo 154, Japan. ytakei@nch.go.jp
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Takei
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Tsujimoto
D000123:Acetyltransferases; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000378:metabolism; D064126:Minichromosome Maintenance Complex Component 3; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C066470:MCM3 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010750:Phosphoproteins; D000123:Acetyltransferases; C406825:MCM3AP protein, human; D064126:Minichromosome Maintenance Complex Component 3