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.

Keywords: DNA damage; DNA repair; mutations; translesion synthesis; yeast.

Figure 1

Induced mutagenesis requires CDC7. CDC7 (RSY466) and mcm5-bob1 cdc7Δ (P211) congenic strains (A364a background) were treated with different doses of UV light and plated for viability and survival on canavanine-containing medium. Values shown are mean of two independent experiments.

Figure 2

CDC7 is required for the production of complex mutations (CINS) in the lys2ΔA746 frameshift reversion assay. The spectra of mutations compiled from independent Lys⁺ revertants isolated from (A) RSY1183, (B) RSY1190, and (C) RSY1224 are shown. N corresponds to the number or revertants sequenced in each strain background.

Figure 3

Co-IP of Rev7 with Dbf4 or Cdc7. Cell extracts were incubated with anti-HA (A and C) or anti-Myc (B) antibody, and precipitated proteins were separated by SDS–PAGE. Western blots were probed individually with an anti-HA or anti-Myc antibody to detect tagged proteins in the immunoprecipitate. Strain YSS13 (Rev7-Myc) with plasmid pYQ118 (A, HA-Dbf4), pCH766 (B and C, HA-Cdc7), or pCH777 (HA-Cdc7-KD). In B and C, extracts were from cells containing a tagged wild-type (WT) or kinase-dead (KD) Cdc7, as indicated. Lane 1, input extract only; lane 2, supernatant of IP; lane 3, supernatant of mock IP with no antibody; lane 4, pellet of mock IP; lane 5, pellet of IP. Twenty percent of the IP was used for SDS–PAGE as compared to 5% of the supernatants or the input extract.

Figure 4

Control co-immunoprecipitation for nonspecific interactions between Rev7-myc or HA-Cdc7 with Pcf11 or Adh1. Cell extracts were directly loaded (input in lane 1) or incubated and immunoprecipitated with anti-Myc antibody (lanes 2 and 3), anti-HA (lanes 4 and 5), or no antibody (lines 6 and 7) and then proteins were separated by SDS–PAGE. Western blots were probed individually with an anti-Pcf11 (top) or anti-Adh1 antibody (bottom) to detect any tagged proteins in the immunoprecipitate.

Figure 5

Phosphorylation of yeast GST-Rev7 by yeast and Xenopus DDK. A total of 2–4 μg of the indicated GST-tagged yeast protein were incubated with (A) Cdc7-Myc immunopreciptates from strain YJO235 or (B) 10 μg of Xenopus DDK (xCdc7-xDbf4) in the presence of γ³²P-ATP. Reactions were subjected to SDS–PAGE, proteins were transferred to nitrocellulose, and protein phosphorylation was visualized by autoradiography. Lanes in A were from different gels and aligned by the molecular weight standards. Anti-GST (UBI) blots of the filters showed the position and confirmed equivalent amounts of the indicated GST fusion proteins (not shown). (A) Lane 1, Cdc7-Myc IP plus GST-Mcm2 (103 kDA); lane 2, Cdc7-Myc IP plus GST-Rev7 (55 kDA); lane 3, Cdc7-Myc IP plus GST (22 kDA); lane 4, Cdc7-Myc only. Note autophosphorylation of Dbf4 by the Cdc7-Myc IP in lanes 2–4. (B) Lane 1, Xenopus DDK only; lane 2, GST only; lane 3, GST-Mcm2 only; lane 4, GST-Rev7 only; lane 5, GST plus DDK; lane 6, GST-Mcm2 plus DDK; lane 7, GST-Rev7 plus DDK. Note DDK autophosphorylation of both xDbf4 and xCdc7 subunits.

Figure 6

Analysis of the co-IP of Rev7 by each subunit of DDK. Cell extracts were incubated with anti-HA antibody, and precipitated proteins were separated by SDS–PAGE. Western blots were probed individually with an anti-HA or anti-Myc antibody to detect tagged proteins in the immunoprecipitate. (A) WT strain YSS13 (lanes 1–5) or dbf4Δ strain RSY1445 (lanes 6–10) with HA-Cdc7 plasmid pCH766 was used. (B) WT strain YSS13 (lanes 1–5) or cdc7Δ strain yLPB163 (lanes 6–10) with HA-Dbf4 plasmid pYQ118 was used. Lanes are labeled the same in A and B. Lane 1, input extract only; lane 2, supernatant of mock IP with no antibody; lane 3, supernatant of IP; lane 4, pellet of mock IP; lane 5, pellet of IP; lane 6, input extract only; lane 7, supernatant of mock IP with no antibody; lane 8, supernatant of IP; lane 9, pellet of mock IP; lane 10, pellet of IP; 20% of the IP was used for SDS–PAGE as compared to 5% of the supernatants or the input extract.

Figure 7

Survival of single- and double-mutant strains following UV irradiation. (A) UV sensitivity of cdc7∆ mcm5-bob1 with pol30-K127,K164R. (B) UV sensitivity of cdc7∆ mcm5-bob1 with pol32Δ. Viability of relevant mutant strains was determined following exposure of log-phase cultures to UV. Error bars represent the standard deviation of the mean value of three independent experiments.