Overexpression of RNR1 efficiently elevates dNTP concentration in yeast strains lacking TLS polymerases. (a) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were incubated in liquid YP media with 2% galactose and treated with 0.2 mg/L 4-NQO as shown in the diagram. (b) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for determination of dNTP pools. The numbers above the bars indicate the amount of the individual dNTP expressed in pmols/10⁸ cells. Four overlaid HPLC chromatograms (raw data, not normalized by the number of cells) are shown on the inset. (c) Samples (indicated by numbers 1–4) treated as outlined in (a) were used for analysis of Rnr1 protein levels by 6% SDS–PAGE. M indicates protein marker lane. (d) The cell cycle progression is not altered in the strains lacking TLS polymerases. wild-type, pGAL-RNR1, rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were inoculated in liquid YPD and incubated overnight at 30°C. Next morning cultures were diluted in fresh YPD to an OD₆₀₀ of 0.1 and grown at 30°C. Samples were collected after 4.5 h and prepared for flow-cytometric analysis.

4-NQO increases mutation frequency in the rev1Δ rad30Δ rev3Δ pol4Δ strain. (a) wild-type and rev1Δ rad30Δ rev3Δ pol4Δ logarithmically growing in YPD were treated with increasing amounts of 4-NQO for 2 h and were after appropriate dilutions spread on YPD plates in triplicates to determine survival. (b) Yeast cells treated as in (a) were after appropriate dilutions spread on synthetic complete medium −arginine +L-canavanine to determine mutation frequencies in CAN1 gene by dividing the number of Can1^r mutants by the average number of surviving cells (c) rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strain grown in YPRaf was divided into two cultures, one of which was induced by 2% galactose and mutation frequencies were determined after 2 and 4 h induction. Hatched bars: uninduced cells; open bars: galactose-induced cells.

Increased dNTP concentration improves DNA damage tolerance in the absence of TLS polymerases. (a) Stationary phase cultures grown in YPD were spotted at 10-fold serial dilutions on YPGal (control) and YPGal with 0.24 mg/l 4-NQO plates, and incubated for 4 days at 30°C. (b) rev1Δ rad30Δ rev3Δ pol4Δ and rev1Δ rad30Δ rev3Δ pol4Δ pGAL-RNR1 strains were grown overnight in YPD; appropriate dilutions were plated on YPGal plates containing indicated amounts of 4-NQO, and on YPGal plates to calculate the number of viable cells. Colonies were counted after 4 days of incubation at 30°C.

Polε bypasses an 8-oxoG and MeG lesions at DNA-damage-state, but not at normal S-phase-state, dNTP concentration. (a) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The lesion bypass was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater. The sequence of the template and the positions of the nucleotides are indicated on the right. (b) Assays under single-hit conditions were performed with 0.17 nM Polε and 2 nM wild-type or 8-oxoG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 2 min at 30°C. The bypass probability was calculated by dividing the sum of the products at position 5 (position after G/8-oxoG) or greater by the sum of the products at position 3 or greater as previously described (30). The amount of extended primer is the intensity of all products greater than the primer divided by the intensity of the primer and all products greater than the primer. The total amount of primer extended in all reactions was far <20%, demonstrating that the conditions for single completed hits were reached (40). The sequence of the template and the positions of the nucleotides are indicated on the right. (c) Primer extension assays were performed with 4 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations (see Table 1 for details) for 10 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right. (d) Assays under single-hit conditions were performed with 0.06 nM Polε and 2 nM wild-type or MeG templates at low (L), normal S-phase (N) and DNA-damage-state (H) dNTP concentrations for 2 min at 30°C. The sequence of the template and the positions of the nucleotides are indicated on the right.