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Binding of human minichromosome maintenance proteins with histone H3
Y Ishimi1, S Ichinose, A Omori
1Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan.
Abstract:
Minichromosome maintenance (MCM) proteins play essential roles in eukaryotic DNA replication, but their biochemical properties remain to be determined. We detected in HeLa cell extracts six proteins, CDC47, CDC46/MCM5, Cdc21, P1/MCM3, Mis5, and BM28/MCM2, by their binding to a specific antibody and by partial sequencing. The human homologs of the MCM2 (BM28), Mis5, Cdc21, and CDC47 proteins were tightly bound to a histone-Sepharose column and purified to near homogeneity, whereas the P1/MCM3 and CDC46/MCM5 proteins passed through. Among the four core histones, the human BM28/MCM2, Mis5, Cdc21, and CDC47 proteins had high affinity for histone H3. Immunoprecipitation with anti-Cdc21 antibody revealed that these four MCM proteins form complexes. These results are consistent with the findings that MCM proteins bind with chromatin in vivo.
Insights
Six Minichromosome maintenance (MCM) proteins were identified in HeLa cells. Four MCM proteins (MCM2, Mis5, Cdc21, CDC47) bind tightly to histone H3, suggesting chromatin association during DNA replication.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Minichromosome maintenance (MCM) proteins are crucial for eukaryotic DNA replication.
- The specific biochemical properties and interactions of MCM proteins are not fully elucidated.
Purpose of the Study:
- To identify and characterize MCM proteins involved in DNA replication in HeLa cell extracts.
- To investigate the biochemical properties and chromatin binding of identified MCM proteins.
Main Methods:
- Antibody-based detection and partial protein sequencing were used to identify MCM proteins.
- Histone-Sepharose chromatography was employed for protein purification.
- Co-immunoprecipitation assays were performed to analyze protein complex formation.
Main Results:
- Six MCM proteins (CDC47, CDC46/MCM5, Cdc21, P1/MCM3, Mis5, BM28/MCM2) were detected in HeLa cell extracts.
- Human homologs of MCM2 (BM28), Mis5, Cdc21, and CDC47 bound tightly to histone-Sepharose, indicating chromatin association.
- These four MCM proteins exhibited high affinity for histone H3 and formed complexes, as shown by immunoprecipitation.
Conclusions:
- The study identified key MCM proteins involved in DNA replication.
- Evidence suggests that specific MCM proteins interact with histone H3 and associate with chromatin in vivo.