RAD53 regulates DBF4 independently of checkpoint function in Saccharomycescerevisiae

The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A gene tic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation group s. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53 whi ch was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlik e other RAD53 alleles, we demonstrate that the ran53-31 allele retains an i ntact checkpoint function. Thus, the checkpoint function and the DNA replic ation function of RAD53 can be functionally separated. The activation of DN A replication through RAD53 most likely occurs through DBF4. Two-hybrid ana lysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the st eady-state level of DBF4 message and Dbf4p protein is reduced in several ra d53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulat ed through the common intermediate RAD53.