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Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization
1Department of Molecular Cell Pharmacology, National Children's Medical Research Center, Taishido 3-35-31, Setagaya-ku, Tokyo 154, Japan. ytakei@nch.go.jp
Abstract:
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.
Insights
A novel protein, Map80, interacts with human MCM3 (minichromosome maintenance complex component 3), a key protein in DNA replication. Map80 appears to facilitate MCM3
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Minichromosome maintenance complex component 3 (MCM3) is crucial for DNA replication initiation and cell cycle control.
- Ensuring DNA replication occurs only once per cell cycle requires precise regulation of MCM3 function.
Purpose of the Study:
- To identify and characterize novel proteins interacting with human MCM3.
- To investigate the functional relationship between MCM3 and its interacting partners, particularly concerning nuclear localization.
Main Methods:
- Yeast two-hybrid screening to identify MCM3-interacting proteins.
- Antibody generation and Western blotting to confirm protein expression and interaction via immunoprecipitation.
- Site-directed mutagenesis to analyze the nuclear localization signal (NLS) of MCM3.
- Recombinant protein expression and purification (His6-tagged Map80) for in vitro assays.
Main Results:
- A novel interacting protein, designated Map80 (MCM3-associated protein 80 kDa), was identified and confirmed to interact with human MCM3.
- Mutagenesis of the MCM3 nuclear localization signal disrupted the binding of Map80.
- Recombinant Map80 enhanced the nuclear localization of MCM3 in vitro.
Conclusions:
- Map80 is a novel MCM3-binding protein involved in the nuclear import pathway of MCM3.
- The interaction between Map80 and MCM3 is dependent on the MCM3 nuclear localization signal.
- Map80 plays a role in regulating MCM3's presence in the nucleus, potentially impacting DNA replication control.