title: The role of Dbf4-dependent protein kinase in DNA polymerase ζ-dependent mutagenesis in Saccharomyces cerevisiae.
abstract: The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK's essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate "complex" spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication "gap filling" rather than during "polymerase switching" by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Luis N
authors.initials: LN
authors.lastname: Brandão
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Ferguson
authors.affiliation: Department of Biology, Emory University, Atlanta, Georgia 30322.
authors.forename: Irma
authors.initials: I
authors.lastname: Santoro
authors.affiliation: Department of Biology, Emory University, Atlanta, Georgia 30322 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Sue
authors.initials: S
authors.lastname: Jinks-Robertson
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Sclafani
mesh_terms: D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; C088096:Dbf4 protein, S cerevisiae; C069200:MCM5 protein, S cerevisiae; C092219:REV7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C425929:DNA polymerase zeta; D004259:DNA-Directed DNA Polymerase
keywords: DNA damage; DNA repair; mutations; translesion synthesis; yeast