A Mutation in Dbf4 Motif M Impairs Interactions with DNA Replication Factors and Confers Increased Resistance to Genotoxic Agents

Abstract

Dbf4/Cdc7 is required for DNA replication in Saccharomyces cerevisiae and appears to be a target in the S-phase checkpoint. Previously, a 186-amino-acid Dbf4 region that mediates interactions with both the origin recognition complex and Rad53 was identified. We now show this domain also mediates the association between Dbf4 and Mcm2, a key Dbf4/Cdc7 phosphorylation target. Two conserved sequences, the N and M motifs, have been identified within this Dbf4 region. Removing motif M (Dbf4⌬M) impairs the ability of Dbf4 to support normal cell cycle progression and abrogates the Dbf4-Mcm2 association but has no effect on the Dbf4-Rad53 interaction. In contrast, deleting motif N (Dbf4⌬N) does not affect the essential function of Dbf4, disrupts the Dbf4-Rad53 interaction, largely preserves the Dbf4-Mcm2 association, and renders the cells hypersensitive to genotoxic agents. Surprisingly, Dbf4⌬M interacts strongly with Orc2, while Dbf4⌬N does not. The DBF4 allele dna52-1 was cloned and sequenced, revealing a single point mutation within the M motif. This mutant is unable to maintain interactions with either Mcm2 or Orc2 at the semipermissive temperature of 30°C, while the interaction with Rad53 is preserved. Furthermore, this mutation confers increased resistance to genotoxic agents, which we propose is more likely due to a role for Dbf4 in the resumption of fork progression following checkpoint-induced arrest than prevention of late origin firing. Thus, the alteration of the M motif may facilitate the role of Dbf4 as a checkpoint target.

Cdc7 Is an Active Kinase in Human Cancer Cells Undergoing Replication Stress

“…This observation is reminiscent of the fact that specific Cdc7 and Dbf4 mutations that alter sensitivity to HU and other genotoxic agents can be isolated in both budding (5,25,27) and fission yeast (28 -31).…”

“…In the presence of DNA damage caused by UV radiation or by the alkylating drug methyl methanesulfonate, Cdc7 was shown to be important for induced mutagenesis (5,25). Finally, deletion analysis has defined an N-terminal domain within the Dbf4 protein, which is dispensable for DNA replication, but is required for maintaining cell viability in HU, possibly by targeting the kinase to stalled forks (26,27). Similarly, a growing body of genetic evidence indicates that HSK1, the fission yeast Cdc7 homologue is important for HU and alkylating agent-induced checkpoint responses (28 -31).…”

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

“…The association of Rad53 and Dbf4 mediates the Rad53-dependent phosphorylation of Dbf4 and the consequent inhibition of Cdc7, thus preventing late origin firing (7)(8)(9)(10). This interaction involves the N-terminal region of Dbf4 that folds as a modified BRCA1 C-terminal (BRCT) domain (4,(11)(12)(13).…”

Extending the Interaction Repertoire of FHA and BRCT Domains

“…The structure revealed that this region of Dbf4 folds as a modified BRCT domain that requires an additional N-terminal α-helix to form a stable unit (Figure 7). A fragment of Dbf4 consisting of the canonical BRCT domain but missing the additional helix did not support binding to the FHA1 domain of the checkpoint effector kinase, Rad53 [9,10,31,88]. It was proposed that this additional helix (α0) defines, at least in part, the interaction interface [10].…”