The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis

Abstract

The Rev1-Polzeta pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N(6)-ethenoadenine were severely deficient. Specifically, the predominant A-->G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N(6)-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.

Figure 1.

Translesion synthesis of 1,N⁶-ethenoadenine DNA adducts by human Polη, Polκ, and Polι. (A) The DNA template for translesion synthesis. A 20-mer primer was labeled with ³²P at its 5′-end (*) and annealed right before a template 1,N⁶-ethenoadenine. (B) Translesion synthesis reactions were performed with purified human Polη (lanes 1–5), Polκ (lanes 6–10), and Polι (lanes 11–15) as indicated in the presence of a single deoxyribonucleoside triphosphate dATP (A), dCTP (C), dTTP (T) or dGTP (G), or all four dNTPs (N₄). Reaction products were separated by electrophoresis on denaturing polyacrylamide gel and visualized by autoradiography. Quantitation of extended primers is shown at the bottom of the gel. DNA size markers in nucleotides are indicated on the left.

Figure 2.

Relative frequencies of translesion synthesis (TLS) in various yeast strains. Using the plasmid pELUf1-ethenoA containing a site-specific 1,N⁶-ethenoadenine DNA adduct, in vivo translesion synthesis assays were performed as described in ‘Materials and Methods’ section. Relative TLS was obtained by comparing translesion synthesis in various mutant strains to that in the wild-type cells. Slightly different transformation efficiencies as determined with the undamaged pELUf1 were taken into account in calculating the relative efficiencies. Standard deviations are shown as error bars. The genetic background of all strains was isogenic to the BY4741 strain. WT, wild-type; rad30Δ, lacking Polη; rev1Δ, lacking Rev1; rev3Δ, lacking Polζ.

Figure 3.

In vitro assays for the dCMP transferase of Rev1 and the Rev1D467A/E468A mutant protein. (A) Purified mutant Rev1D467A/E468A protein, which was visualized by staining the 10% polyacrylamide gel with Coomassie blue. The full-length mutant Rev1 is indicated by the arrowhead. (B) Standard translesion synthesis assays were performed with purified wild-type (lanes 1–6) or mutant Rev1D467A/E468A (lanes 7–12) protein using either a G template or an AP template as indicated. The AP site in the template sequence is indicated by the X. The 17-mer DNA band is indicative of the dCMP transferase activity.

Figure 4.

UV sensitivity of various yeast strains. Yeast cells were grown in minimum medium. After appropriate dilution, cells were plated onto minimum medium plates. The uncovered plates were irradiated with UV light at the indicated doses. Surviving colonies were counted after incubation at 30°C for 3–4 days. Survival rates are expressed relative to those of non-irradiated cells. Results are averages of triplicate experiments with the standard deviations shown as error bars. The genetic background of all strains was isogenic to the CL1265-7C strain. Strains rev1Δ/REV1 and rev1Δ/REV1mt are rev1 deletion mutant (rev1Δ) cells containing the wild-type REV1 gene and the mutant rev1D467A/E468A gene, respectively, on a plasmid under the ADH1 promoter control.

Figure 5.

Relative frequencies of translesion synthesis (TLS) in yeast strains expressing the mutant Rev1D467A/E468A protein. Using the plasmid pELUf1-ethenoA containing a site-specific 1,N⁶-ethenoadenine, in vivo translesion synthesis assays were performed as described in ‘Materials and Methods’ section. Relative TLS was obtained by comparing translesion synthesis in various mutant strains to that in the wild-type cells. Slightly different transformation efficiencies as determined with the undamaged pELUf1 were taken into account in calculating the relative efficiencies. Standard deviations are shown as error bars. The genetic background of all strains was isogenic to the CL1265-7C strain. WT, wild-type; rev1Δ, rev1 deletion mutant; rev1Δ/REV1 and rev1Δ/REV1mt, rev1 deletion mutant cells containing the wild-type REV1 gene and the mutant rev1D467A/E468A gene, respectively, on a plasmid under the ADH1 promoter control.

Figure 6.

A mechanistic model for translesion synthesis of the 1,N⁶-ethenoadenine DNA adduct in yeast cells. The replication complex (represented by the filled blue oval) is blocked by the lesion, signaling translesion synthesis. Translesion synthesis is mediated predominantly by C insertion opposite the lesion catalyzed by the Rev1 dCMP transferase. Extension synthesis by Polζ completes the lesion bypass. This major mechanism of translesion synthesis results in A→G transition mutations.