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 G 1 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 G 1 cells. This G 1 -phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G 1 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.

The initiation of DNA replication in Saccharomyces cerevisiae requires the protein product of the CDC45 gene. We report that although Cdc45p is present at essentially constant levels throughout the cell cycle, it completes its initiation function in late G 1 , after START and prior to DNA synthesis. Shortly after mitosis, cells prepare for initiation by assembling prereplicative complexes at their replication origins. These complexes are then triggered at the onset of S phase to commence DNA replication. Cells defective for CDC45 are incapable of activating the complexes to initiate DNA replication. In addition, Cdc45p and Cdc7p͞Dbf4p, a kinase implicated in the G 1 ͞S phase transition, are dependent on one another for function. These data indicate that CDC45 functions in late G 1 phase in concert with CDC7͞DBF4 to trigger initiation at replication origins after the assembly of the prereplicative complexes.

Both randomized oligonucleotide cassette mutagenesis and site-directed mutagenesis have been used in combination with a yeast genetic screen to identify critical residues in the DNA-binding domain of heat shock transcription factor from Saccharomyces cerevisiae. Most of the surface residues in this highly conserved domain can be changed to alanine with no observable effect on function. Of nine critical residues identified in this screen, five are within helix alpha 3, previously designated as the probable DNA recognition helix in the crystal structure of the Kluyveromyces lactis protein. The other four residues may be involved in DNA-binding or protein-protein interactions.

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