Cell cycle and checkpoint regulation of the budding yeast S-phase promoting factor Dbf4p

The Dbf4/Cdc7 protein kinase is essential for origin firing during S phase in the budding yeast Saccharomyces cerevisiae. The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle despite its kinase activity being periodic. Cdc7p kinase requires association with Dbf4p, the regulatory subunit, for its activation. This study shows that Dbf4 protein levels are periodic during the cell cycle. Dbf4p is absent from early G1 cells, appears in late G1 and accumulates during S and G2 phases. This pattern of expression resembles the periodicity of the Cdc7p kinase activity. Dbf4p is rapidly degraded at the onset of anaphase in mitosis and remains highly unstable in pre-START G1 phase. The rapid degradation of Dbf4p requires the function of the Anaphase Promoting Complex (APC). Dbf4p contains a destruction box sequence in its N-terminus, that when mutated, eliminates this APC dependent degradation. Conditions that block the elongation step of DNA replication prevent the activation of late origins of replication in a MEC1/RAD53 dependent manner. During such a block, CDC7 cannot execute its function at late replicating origins. In this study, it was observed that Dbf4p is phosphorylated in response to blocks to S phase progression. Phosphorylation of Dbf4p is dependent upon Rad53p and Cdc7 protein kinases both in vivo and in vitro. Although phosphorylated Dbf4/Cdc7p retains its kinase activity during the S phase block, its ability to associate with chromatin is inhibited. A model is suggested whereby in response to blocks to S phase progression, activation of the Mec1p/Rad53p checkpoint pathway results in the phosphorylation of Dbf4p. This phosphorylated form would no longer be able to associate with origins of replication, thereby preventing Cdc7p from activating late origins of replication