Cdc7p-Dbf4p regulates mitotic exit by inhibiting Polo kinase.

Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.

A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast.

Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved approximately 40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called "motif N." BRCT motifs encode approximately 100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved approximately 100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p-Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest.

DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

Budding yeast Dbf4 sequences required for Cdc7 kinase activation and identification of a functional relationship between the Dbf4 and Rev1 BRCT domains.

Cdc7-Dbf4 is a two-subunit kinase required for initiating DNA replication. The Dbf4 regulatory subunit is required for Cdc7 kinase activity. Previous studies have shown that the C termini of Dbf4 orthologs encode a single (putative) C(2)H(2) zinc (Zn) finger, referred to as "motif C." By mutational analysis we show that the Zn finger is not required for the essential function of Dbf4. However, deletion and point mutants altering conserved Zn-finger residues exhibit a substantially slowed S-phase, DNA damage sensitivity, and a hypo-mutagenic phenotype following UV irradiation. Using two-hybrid and biochemical assays, we show that the Dbf4 Zn finger interacts with Cdc7 and stimulates its kinase activity. However, a separable Dbf4 region also mediates an interaction with Cdc7 such that only the loss of both Cdc7-interacting regions results in lethality. In contrast, an N-terminal BRCT-like domain is not required for induced mutagenesis nor does it interact with Cdc7. By making chimeric Dbf4 proteins that contain known BRCT domains in Saccharomyces cerevisiae, we show that the BRCT domain from Rev1, a translesion DNA polymerase, can uniquely substitute for the Dbf4 BRCT domain. Thus, we have mapped regions on budding yeast Dbf4 required for binding and activating Cdc7 kinase. Our data also suggest that the Dbf4 and Rev1 BRCT domains interact with a common protein or structure, although the precise function of both domains and their binding partners remains elusive.