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Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclin A/Cdk2
Y Ishimi1, Y Komamura-Kohno, Z You
1Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. yukio@libra.m-kagaku.co.jp
Abstract:
A strong body of evidence indicates that cyclin-dependent protein kinases are required not only for the initiation of DNA replication but also for preventing over-replication in eukaryotic cells. Mcm proteins are one of the components of the replication licensing system that permits only a single round of DNA replication per cell cycle. It has been reported that Mcm proteins are phosphorylated by the cyclin-dependent kinases in vivo, suggesting that these two factors are cooperatively involved in the regulation of DNA replication. Our group has reported that a 600-kDa Mcm4,6,7 complex has a DNA helicase activity that is probably necessary for the initiation of DNA replication. Here, we examined the in vitro phosphorylation of the Mcm complexes with cyclin A/Cdk2 to understand the interplay between Mcm proteins and cyclin-dependent kinases. The cyclin A/Cdk2 mainly phosphorylated the amino-terminal region of Mcm4 in the Mcm4,6,7 complex. The phosphorylation was associated with the inactivation of its DNA helicase activity. These results raise the possibility that the inactivation of Mcm4,6,7 helicase activity by Cdk2 is a part of the system for regulating DNA replication.
Insights
Cyclin-dependent kinases (CDKs) regulate DNA replication. Phosphorylation of Mcm4,6,7 complex by cyclin A/Cdk2 inactivates its DNA helicase activity, preventing DNA over-replication.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Replication
Background:
- Cyclin-dependent kinases (CDKs) are crucial for initiating DNA replication and preventing re-replication in eukaryotes.
- Mcm proteins are key components of the replication licensing system, ensuring one round of DNA replication per cell cycle.
- Previous studies suggest in vivo phosphorylation of Mcm proteins by CDKs indicates cooperative regulation of DNA replication.
Purpose of the Study:
- To investigate the in vitro interaction between Mcm proteins and cyclin-dependent kinases.
- To elucidate the role of Mcm protein phosphorylation by cyclin A/Cdk2 in DNA replication regulation.
Main Methods:
- In vitro phosphorylation assays using Mcm4,6,7 complex and cyclin A/Cdk2.
- Analysis of Mcm4 phosphorylation sites.
- Assay of DNA helicase activity of the Mcm complex before and after phosphorylation.
Main Results:
- Cyclin A/Cdk2 predominantly phosphorylated the N-terminal region of Mcm4 within the Mcm4,6,7 complex.
- This phosphorylation event was directly correlated with the inactivation of the Mcm4,6,7 complex's DNA helicase activity.
- The findings suggest a mechanism for regulating DNA replication initiation.
Conclusions:
- CDK-mediated phosphorylation of Mcm proteins plays a significant role in controlling DNA replication.
- Inactivation of Mcm4,6,7 helicase activity by Cdk2 is a potential regulatory step in preventing DNA over-replication.
- This study provides insights into the intricate interplay between CDKs and Mcm proteins in cell cycle control.