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 (Dbf4DeltaM) 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 (Dbf4DeltaN) 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, Dbf4DeltaM interacts strongly with Orc2, while Dbf4DeltaN 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 degrees 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.