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.
Harkins
Victoria
V
Laboratory of Chromosome Replication, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.
Gabrielse
Carrie
C
Haste
Louise
L
Weinreich
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D017124:Conserved Sequence; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000378:metabolism; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000528:radiation effects; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000378:metabolism; D013312:Stress, Physiological; D035582:Transcription Factor TFIIIA / Q000737:chemistry; D014466:Ultraviolet Rays / Q000009:adverse effects; D016335:Zinc Fingers
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D035582:Transcription Factor TFIIIA; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases