Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

Abstract

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G₁ phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G₁ cells. This G₁-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G₁ phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

ACKNOWLEDGMENTS

G.O. and J.C.O. contributed equally to the work reported in this paper.

We thank Bik Tye, Yasuo Kawasaki, and Akio Sugino for strains and plasmids and Tom Wang for help in plasmid constructions. University of Colorado Cancer Center Core facilities were used for DNA sequencing, flow cytometry, and monoclonal antibody production (supported by PHS grant P30 CA46934).

This work was supported by PHS grant GM35078 awarded to R.A.S. J.C.O. is supported by an Achievement Rewards for College Scientists Foundation graduate fellowship and by a National Institutes of Health training grant. J.J.L. is a Searle Scholar and a Rita Allen Foundation Scholar.