Effect of rev3 mutation on the spontaneous mutator phenotype of DNA replication mutants. The diagrams show the rates of Can^r mutation in the REV3⁺ and rev3 variants of pol3-Y708A (A), pol3t (B), pol2-1 (C), dpb3 (D), and pol1-1 (E) strains. The DNA polymerases affected by the mutations are indicated on top of each diagram. All data are from Supplementary Table I and are medians and 95% confidence limits for at least 18 independent determinations. On each graph, the upper confidence limit is not shown for the strongest mutator strain.

Polζ-dependent spontaneous mutagenesis in DNA replication mutants is promoted by post-translational modification of PCNA. Diagrams (A) and (B) show the effects of pol30-K164R and siz1 mutations on the spontaneous mutator phenotype of pol3-Y708A and pol2-1 strains, respectively. The data are rates of Can^r mutation from Supplementary Table I and are medians and 95% confidence limits for at least 18 independent determinations. (C) Detection of monoubiquitinated PCNA in whole-cell extracts of the pol3-Y708A strain by Western blot analysis. The positions of unmodified and monoubiquitinated (U1) PCNA are indicated. The MMS treatment (last lane) was for 1 h. The low-intensity band at U1 position in the untreated wild-type strain likely represents cross-reacting protein species, since it is also seen in pol30-K164R and rad18 strains.

Pcna-113 shows a general trimerization defect and a specific Polζ interaction defect. (A) Gel filtration analysis of wild-type PCNA, pcna-52, and pcna-113 on a superose 6 column was carried out as described (Ayyagari et al, 1995). The data for pcna-52 were taken from that study. (B) Replication of SS mp18 DNA by Polδ with wild-type and mutant PCNA was as described in Materials and methods. (C) PCNA ubiquitination was carried in an assay containing ubiquitin, E1, Rad6/Rad18, multiple-primed SS DNA, RFC, and the indicated concentration of wild-type or mutant PCNA as described (Garg and Burgers, 2005). A Western analysis with antibodies to PCNA was used for detection. (D) Monoubiquitination of PCNA in vivo in the wild-type strain E134 and its pol30-K164R and pol30-113 mutants was analyzed by Western blot with whole-cell extracts and antibodies to PCNA (see legend to Figure 3C for more details). (E) TLS on UV-irradiated (1000 J/m²) template/primer V9/C12-4 by Polζ with the indicated concentrations of PCNA or pcna-113 was for 5 min at 30°C. Bypass products are defined as those proceeding past the irradiated dT₂₅ stretch. (F) Bypass of an abasic site on template/primer V9AP2/C12-4 by Polδ or Polζ with the indicated concentrations of PCNA or pcna-113 was for 5 min at 30°C. (G) Bypass of an abasic site on template/primer V9AP2/C12-4 (see panel F) by Polη with the indicated concentrations of PCNA or pcna-113 was for 1 or 3 min at 30°C. See Materials and methods for details on all TLS assays.

Effect of DNA replication mutations on UV-induced mutagenesis. The data are average frequencies of UV-induced Can^r mutants for three independent determinations. Standard errors are shown where the size of the error bar exceeds the size of the plot symbol.

The pol30-113 mutant is defective in Polζ-dependent spontaneous and UV-induced mutagenesis. (A) Location of the pol30-K164R, pol30-113, and pol30-52 mutations on the PCNA structure. The three monomers of PCNA trimer are shown in yellow, magenta, and blue ribbon. The amino-acid residues affected by the mutations are shown in space fill mode. (B) Effect of pol30-113 mutation on the spontaneous mutator phenotype of pol3-Y708A (top) and pol2-1 (bottom) strains. The data are from Supplementary Table I and are medians and 95% confidence limits for at least 18 independent determinations. (C) Effect of the pol30-113 mutation on the frequency of UV-induced Can^r mutants (top), Trp⁺ revertants (middle), and His⁺ revertants (bottom). The data are the average for at least three independent determinations.