Characterization of the Yeast Cdc7p/Dbf4p Complex Purified from Insect Cells

Abstract

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, 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 Cdc7-1p or Dbf4-1ϳ5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of various dbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequencebinding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.Initiation of chromosomal DNA replication and cell cycle progression are tightly regulated in eukaryotes. In the yeast Saccharomyces cerevisiae, several cdc (cell division cycle) mutants that block initiation of chromosomal DNA replication have been isolated and characterized (1, 2), for example, cdc28, cdc4, cdc6, and cdc7. The stepwise assembly of proteins at origins of DNA replication is a crucial part of regulating entry into S phase. Two key factors that mediate such cell cycle regulation are Cdc6 protein level and availability and the presence of an active cyclin-dependent kinase (Cdk) (see Ref. 3 for review). The origin recognition complex is bound to origins of DNA replication at all stages of the cell cycle in S. cerevisiae (4 -6). However, Cdc6 is not recruited to origins until late in M phase and is required for the association of the Mcm2-7 family proteins at origins to form a prereplicative complex (6 -8).The Cdc6 protein-dependent stage of the assembly reaction is inhibited by active Clb-Cdks (6, 9, 10). Because Cdc6 protein is synthesized only from late M phase until late G1 (11), prereplicative complexes can only be assembled during this period of the cell cycle. S phase cyclin-Cdk (Cdc28p/Clb5p or Cdc28p/ Clb6p) activity is required for the chromatin association of Cdc45p just before the initiation of chromosomal DNA replication (12). The previous results demonstrated that Cdc28 protein-Clb kinase is required throughout S phase to activate origins when they are scheduled to fire (13).The Cdc7/Dbf4 complex is a Cdk-like protein kinase (see Refs. 14 and 15 for review) that is also required for entry into S phase at a very late stage. CDC7 transcript levels are constant throughout the cell cycle, whereas DBF4 transcription is cell cycle regulated a...

Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

“…The binding of MCM2 to Cdc7 is consistent with the published data that MCM2 protein is the major physiological substrate of Cdc7 (16,35,36) (Fig. 1B, MCM2).…”

Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication inSaccharomyces cerevisiae

“…Our original hypothesis was that this lethality results from reduced protein levels of Dbf4, the regulatory subunit of DDK. Consistent with this hypothesis, Rad53 binds (Dohrmann et al 1999;Duncker et al 2002) and phosphorylates Dbf4 (Weinreich and Stillman 1999;Kihara et al 2000). However, we show in this report that the original hypothesis is incorrect and propose a new role for Rad53 in the initiation of DNA replication that is independent of Rad53's known roles in checkpoint or deoxynucleotide regulation.…”

Rad53 Kinase Activation-independent Replication Checkpoint Function of the N-terminal Forkhead-associated (FHA1) Domain

“…In vivo, Dbf4 is phosphorylated and displaced from chromatin in a Rad53-dependent manner in response to HU to prevent Cdc45 and polymerase ␣ loading onto origins and new origin activation (60,61). Mcm2-7 are suggested to be direct targets of the Cdc7-Dbf4 kinase from in vitro studies (58,59,62), and it is speculated that their phosphorylation by Cdc7-Dbf4 may allow Mcm2-7 to bind Cdc45 required for replication initiation (42). The interaction of Dbf4 with the FHA1 may therefore be required to bring the Rad53 kinase close to Cdc7-Dbf4 for its inhibitory phosphorylation in the intra-S checkpoint (27).…”

“…The most plausible candidate would be the Cdc7 kinase regulatory subunit Dbf4 that can directly bind to the FHA1 domain in co-immunoprecipitation and yeast two-hybrid assays (27). Cdc7-Dbf4 is required for initiation of DNA replication (56, 57) and a likely Rad53 target to inhibit late origin firing, as activated Rad53 can phosphorylate Cdc7-Dbf4 and inhibit its kinase activity in vitro (58,59). In vivo, Dbf4 is phosphorylated and displaced from chromatin in a Rad53-dependent manner in response to HU to prevent Cdc45 and polymerase ␣ loading onto origins and new origin activation (60,61).…”