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Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex
M Hendrickson1, M Madine, S Dalton
1Roche Institute of Molecular Biology, Nutley, NJ 07110, USA.
Abstract:
In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.
Insights
Cell cycle regulation prevents DNA replication more than once per cycle. Phosphorylation of Xenopus MCM4 protein by cdc2/cyclinB kinase inactivates the MCM complex, preventing DNA replication during S phase and mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Eukaryotic cell cycle progression is tightly regulated.
- Minichromosome maintenance (MCM) proteins are crucial for preventing re-replication of DNA.
- The MCM complex's regulation during the cell cycle is not fully understood.
Purpose of the Study:
- To investigate the cell cycle-dependent regulation of Xenopus MCM4.
- To determine the role of MCM4 phosphorylation in controlling DNA replication.
Main Methods:
- Studied Xenopus MCM4 protein in cleaving embryos and cell-free extracts.
- Analyzed MCM4 phosphorylation patterns during the cell cycle.
- Investigated the kinase responsible for MCM4 phosphorylation using in vitro assays.
- Assessed the effect of phosphorylation on MCM4's chromatin binding affinity.
Main Results:
- Xenopus MCM4 is part of a large complex with other MCM proteins.
- MCM4 undergoes cell cycle-dependent phosphorylation, coinciding with its release from chromatin in S phase.
- Cdc2/cyclinB protein kinase phosphorylates MCM4 at specific sites, both in vitro and in vivo.
- Phosphorylation significantly reduces MCM4's affinity for chromatin.
Conclusions:
- Cell cycle-dependent phosphorylation of MCM4 by cdc2/cyclinB kinase inactivates the MCM complex.
- This mechanism prevents DNA replication from late S phase through mitosis.
- Ensures the fidelity of DNA replication by preventing re-replication within a single cell cycle.