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Negative regulation of Cdc18 DNA replication protein by Cdc2
A Lopez-Girona1, O Mondesert, J Leatherwood
1Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Molecular Biology of the Cell
|March 14, 1998
Summary
Fission yeast Cdc18 protein
Area of Science:
- Cell cycle regulation
- DNA replication
- Molecular biology
Background:
- Fission yeast Cdc18 is essential for DNA replication origin activation.
- Cdc18 overexpression or loss of Cdc2-Cdc13 kinase causes DNA rereplication.
- This suggests Cdc2 kinase inhibits Cdc18 to prevent re-replication.
Purpose of the Study:
- To investigate the inhibitory mechanism of Cdc18 by Cdc2 kinase.
- To identify the role of B-type cyclins (Cdc13 and Cig2) in regulating Cdc18 activity.
- To understand how DNA replication is limited to once per cell cycle.
Main Methods:
- In vivo interaction studies between Cdc18, Cdc2, Cdc13, and Cig2.
- In vitro phosphorylation assays of Cdc18 by Cdc2.
- Site-directed mutagenesis of Cdc18 phosphorylation sites (T104A).
- Genetic analysis using cdc18 and cig2 mutations.
- Rereplication assays in fission yeast.
Main Results:
- Cdc18 interacts with Cdc2 in complex with Cdc13 and Cig2 cyclins.
- Cdc2 phosphorylates Cdc18 in vitro, with T104 being a key phosphorylation site.
- Mutation of T104A renders Cdc18 constitutively active, leading to rereplication.
- Genetic data confirms Cdc2-Cig2 kinase inhibits Cdc18.
- Constitutive Cig2 expression prevents rereplication in cells lacking Cdc13.
Conclusions:
- Cdc18 is a direct target of Cdc2-Cdc13 and Cdc2-Cig2 kinases.
- Phosphorylation of Cdc18 by these kinases is crucial for preventing DNA rereplication.
- This regulatory mechanism ensures DNA replication occurs only once per cell cycle.
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