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Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm
D Schulte1, A Richter, R Burkhart
1Division of Biology, Universität Konstanz, Germany.
Abstract:
Recently we identified a cDNA fragment encoding a conserved part of a new human minichromosome maintenance (Mcm) protein, provisionally termed P1.1Mcm3. Here, we report that the protein is most highly related to a yeast cell-division-cycle protein, Cdc47, encoded by the open reading frame YBR1441 on chromosome 11 of Saccharomyces cerevisiae. The human protein migrates on a polyacrylamide gel with an apparent molecular mass of 85 kDa and shares areas of significant similarity with the Mcm family of replication proteins. It is, therefore, designated as p85Mcm. Microscopic immuno-fluorescence studies revealed that protein p85Mcm is located in the nuclei of interphase cells, but is evenly distributed throughout the cell during mitosis. The amounts of p85Mcm do not significantly change during the cell cycle, but mRNA levels rise with the beginning of the S phase. However, in vitro differentiation of HL60 cells results in a striking decrease of both p85Mcm mRNA and protein levels, suggesting a role for p85Mcm in proliferating, but not in differentiated cells. Under physiological salt conditions, p85Mcm is a component of a high molecular-mass complex including other Mcm proteins. The complex dissociates at high ionic strength giving rise to stable subcomplexes, one of which contains protein p85Mcm together with Mcm proteins hCdc21 and p1O5Mcm.
Insights
We identified a new human protein, p85Mcm, related to yeast Cdc47. This minichromosome maintenance protein is crucial for proliferating cells and forms complexes with other Mcm proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Minichromosome maintenance (Mcm) proteins are essential for DNA replication.
- A novel human Mcm protein, provisionally named P1.1Mcm3, was previously identified.
- This protein shows homology to yeast cell-division-cycle protein Cdc47.
Purpose of the Study:
- To characterize the novel human Mcm protein, designated p85Mcm.
- To investigate the cellular localization, expression, and complex formation of p85Mcm.
- To determine the role of p85Mcm in cell proliferation and differentiation.
Main Methods:
- Protein identification and characterization (e.g., molecular mass determination).
- Immunofluorescence microscopy for cellular localization studies.
- Cell cycle analysis of protein and mRNA levels.
- In vitro differentiation assays (HL60 cells).
- Biochemical analysis of protein complex formation.
Main Results:
- p85Mcm has an apparent molecular mass of 85 kDa and is highly similar to Mcm replication proteins.
- p85Mcm localizes to the nucleus during interphase and is distributed throughout the cell during mitosis.
- p85Mcm protein levels are stable, but mRNA levels increase during S phase.
- HL60 cell differentiation leads to decreased p85Mcm mRNA and protein, suggesting a role in proliferation.
- p85Mcm forms a high molecular-mass complex with other Mcm proteins, dissociating into subcomplexes at high salt concentrations.
Conclusions:
- p85Mcm is a novel human Mcm protein involved in DNA replication.
- Its expression pattern suggests a role in proliferating cells.
- p85Mcm functions as part of a larger Mcm complex, potentially regulating DNA replication initiation or elongation.