Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells - Its protein kinase activity is regulated by Rad53p

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was p urified to near homogeneity from insect cells. The complex efficiently phos phorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or o ther replication proteins. Significantly, when pretreated with alkaline pho sphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc 7-1p or Dbf4-1 similar to 5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequ ence binding activity of various dbf4 mutants was also analyzed. These stud ies suggest that the autonomously replicating sequence-binding and Cdc7p pr otein kinase activation domains of Dbf4p collaborate to form an active Cdc7 p/Dbf4p complex and function during S phase in S. cerevisiae, It is shown t hat Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this ph osphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p, This res ult suggests that Rad53p controls the initiation of chromosomal DNA replica tion by regulating the protein kinase activity associated with the Cdc7p/Db f4p complex.