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Genetic interactions between CDC7 and CDC28: growth inhibition of cdc28-1N by Cdc7 point mutants
1Department of Molecular and Developmental Biology, Institute of Medical Science, The University of Tokyo, Japan.
Background:
Cdc7 kinase of Saccharomyces cerevisiae, a nuclear phosphoprotein, regulates initiation of chromosomal DNA replication. Overexpression of kinase-negative Cdc7 point mutants (T281E, D182N and D163N) arrests the cell cycle of the wild-type Saccharomyces cerevisiae cells at the G1/S boundary. This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity.
Results:
We report here that kinase-negative Cdc7 mutants, when overexpressed in cdc28-1N(ts) at a permissive temperature, not only inhibit DNA replication by inactivating the wild-type Cdc7 but may also disturb coordination between DNA replication and cell division. Suppression of growth inhibition under this condition requires co-expression of both Dbf4 and Cdc28, whereas Dbf4 alone can counteract the growth inhibition in the wild-type cells. In cdc28-1N(ts), co-expression of the wild-type Dbf4 rescues only the G1/S defect and results in accumulation of those cells with less than 1C DNA as well as 2C DNA. On the other hand, co-expression of Cdc28 alone leads to increase of those cells arrested at the G1/S boundary, as found typically in the wild-type. We also report that overexpression of T281A, a 'weak' allele of Cdc7, causes growth arrest in cdc28-1N(ts) cells, but not in the CDC28 wild-type cells. This suggests that T281A is inactive in cdc28-1N(ts) and is consistent with the idea that Cdc28 activates Cdc7 by phosphorylation.
Conclusion:
We conclude that two essential serine-threonine kinases, Cdc28 and Cdc7, genetically interact for initiation of the S phase and possibly for G2/M progression and/or S phase checkpoint control.
Insights
Overexpressing kinase-inactive Cdc7 mutants in yeast halts DNA replication and cell division. Co-expressing Cdc28 and Dbf4 rescues these defects, revealing a genetic interaction between Cdc28 and Cdc7 kinases.
Area of Science:
- Cell cycle regulation in Saccharomyces cerevisiae
- Molecular mechanisms of DNA replication initiation
Background:
- Cdc7 kinase is crucial for initiating chromosomal DNA replication in yeast.
- Overexpression of kinase-inactive Cdc7 mutants arrests the cell cycle at the G1/S boundary by sequestering Dbf4.
Purpose of the Study:
- Investigate the role of Cdc7 kinase mutants in cell cycle progression.
- Determine the interaction between Cdc7 and Cdc28 in DNA replication and cell division.
Main Methods:
- Overexpression of kinase-negative Cdc7 point mutants in wild-type and cdc28-1N(ts) yeast strains.
- Analysis of cell cycle progression and DNA replication.
- Genetic suppression studies involving co-expression of Dbf4 and Cdc28.
Main Results:
- Kinase-inactive Cdc7 mutants inhibit DNA replication and disrupt cell division coordination in cdc28-1N(ts) cells.
- Co-expression of Dbf4 and Cdc28 is required to suppress growth inhibition in cdc28-1N(ts) cells.
- Cdc28 appears to activate Cdc7 by phosphorylation, as indicated by the T281A mutant phenotype.
Conclusions:
- Cdc28 and Cdc7 kinases genetically interact for S phase initiation.
- This interaction may also be involved in G2/M progression and S phase checkpoint control.