MNNG-induced mutations at the CAN1 locus in rad30Δ and pol32Δ yeast strains. can1^r mutation frequency (A) and viability (B) in MNNG-treated yeast strains are shown. Cells grown overnight in YPD medium were treated with MNNG at the concentrations indicated for a 20-min period. Appropriate dilutions were spread onto synthetic complete medium lacking arginine and containing canavanine for the determination of CAN1^s to can1^r mutation frequencies and onto YPD plates for viability determinations. Each curve represents the average of two or more experiments. ▵, EMY74.7 (wild type) (RAD30 POL32); ○, YPO-69 (pol32Δ); □, YR30.2 (rad30Δ); ■, YR30.97 (rad30Δ pol32Δ).

Translesion DNA synthesis activity of hPolη on template containing m6G. (A) Running-start DNA synthesis past m6G by hPolη. The position of undamaged G or the corresponding m6G is indicated by G*. Five nanomolar (lanes 1 and 3) or 2.5 nM (lanes 2 and 4) hPolη was incubated with DNA substrate (20 nM) for 5 min at 30°C under standard reaction conditions. Undamaged DNA template, lanes 1 and 2; m6G template, lanes 3 and 4. (B) Deoxynucleotide incorporation opposite m6G by hPolη. The position of the m6G or the undamaged G residue in template DNA is indicated by G*. hPolη (10 nM) was incubated with 20 nM undamaged (lane 5) or m6G-containing (lane 6) DNA substrate under standard reaction conditions. Electrophoretic mobilities of 18-nt reaction products (lanes 5 and 6) were compared with those of 18-nt synthetic oligomer markers containing a G, T, A, or C residue at position 13 (lanes 1 to 4, respectively). Two of these 18-nt marker oligomers, containing a C (lower band) or T (upper band) at position 13, were mixed and run in lane 7.

Translesion DNA synthesis by yeast Polη and yeast Polδ on templates containing m6G. (A) Running-start and standing-start DNA synthesis past m6G by yeast Polη and yeast Polδ. Sequences adjacent to the primer:template junction are shown for 75-nt template and 32-nt (S-1 substrate, lanes 1, 2, 5, and 6) or 44-nt (S-2 substrate, lanes 3, 4, 7, and 8) primer. The primers were ³²P-labeled at their 5′ end. The position of undamaged G (lanes 1, 3, 5, and 7) or m6G (lanes 2, 4, 6, and 8) on the template is indicated by G*. Yeast Polδ (10 nM) (lanes 1 to 4) or yeast Polη (2.5 nM) (lanes 5 to 8) was incubated with the DNA substrate (20 nM) for 5 min at 30°C. Reaction products were resolved on a 10% denaturing polyacrylamide gel and were visualized by autoradiography. The amount of synthesis past the undamaged G or m6G is indicated. (B) Nucleotides incorporated opposite m6G by yeast Polη and yeast Polδ. Standing-start reactions were carried out on a G (lanes 5 and 7)- or m6G (lanes 6 and 8)-containing 18-nt template primed with a 5′ ³²P-labeled 12-nt oligomer. The position corresponding to the G or m6G residue in the template is indicated by G*. Reactions were carried out as described for panel A above, except that the following DNA polymerase concentrations were used: yeast Polδ, 40 nM (lanes 5 and 6); and yeast Polη, 10 nM (lanes 7 and 8). Reaction mixtures were resolved on 20% denaturing polyacrylamide gel, and electrophoretic mobilities of the 18-nt reaction products (lanes 5 to 8) were compared with those of 18-nt synthetic oligomers containing a G (lane 1), a T (lane 2), an A (lane 3), or a C (lane 4) residue at position 13.