Translesion synthesis by yeast DNA polymerase zeta from templates containing lesions of ultraviolet radiation and acetylaminofluorene

Abstract

In the yeast Saccharomyces cerevisiae, DNA polymerase zeta (Polzeta) is required in a major lesion bypass pathway. To help understand the role of Polzeta in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polzeta performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3' T, and predominantly A opposite the 5' T. Purified yeast Polzeta predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polzeta catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3' T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3' T of the TT (6-4) photoproduct. In contrast, the 3' T of a cis-syn TT dimer completely blocked purified yeast Polzeta, whereas the 5' T was readily bypassed. These results support the following dual-function model of Polzeta. First, Polzeta catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polzeta.

Figure 1

Analyses of purified yeast Polζ. (A) Purified yeast Polζ (300 ng) was analyzed by electrophoresis on a 10% SDS–polyacrylamide gel and visualized by silver staining. Protein size markers (lane M) are indicated on the left. (B) Purified yeast Polζ (300 ng) was analyzed by a western blot using a mouse monoclonal antibody against the His₆ tag. (C) A DNA polymerase assay was performed with purified yeast Polζ (26 ng, 129 fmol) using the 30mer DNA template 5′-CCTTCTTCATTCGAACATACTTCTTCTTCC-3′ annealed with the 5′-³²P-labeled 17mer primer 5′-GGAAGAAGAAGTATGTT-3′. DNA size markers in nucleotides are indicated on the left.

Figure 2

Bypass of a template TT (6-4) photoproduct by yeast Polζ. A 15mer primer was labeled with ³²P at its 5′ end and annealed to a DNA template containing a TT (6-4) photoproduct, right before the lesion. DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs (lane 1), or a single deoxyribonucleoside triphosphate, dATP (lane 2), dCTP (lane 3), dGTP (lane 4) or dTTP (lane 5). DNA size markers in nucleotides are indicated on the left.

Figure 3

Nucleotide incorporation by yeast Polζ opposite the 5′ T of the TT (6-4) photoproduct. (A) Three 16mer primers that differed by 1 nt at the 3′ end were labeled with ³²P at their 5′ ends and separately annealed to the indicated DNA template containing a TT (6-4) photoproduct, right before the 5′ T of the lesion. (B) DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs (N₄), or a single deoxyribonucleoside triphosphate, dATP (A), dCTP (C), dGTP (G) or dTTP (T), as indicated. DNA size markers in nucleotides are indicated on the left.

Figure 4

Response of yeast Polζ to a cis-syn TT dimer in template DNA. (A) A 17mer primer was labeled with ³²P at its 5′ end and annealed 8 nt before a template TT dimer as indicated. A DNA polymerase assay was performed with 150 ng (743 fmol) of purified yeast Polζ in the presence of all four dNTPs. (B) A 15mer primer was labeled with ³²P at its 5′ end and annealed right before the TT dimer as indicated. DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs (lane 1), or a single deoxyribonucleoside triphosphate, dATP (lane 2), dCTP (lane 3), dGTP (lane 4) or dTTP (lane 5). DNA size markers in nucleotides are indicated on the left.

Figure 5

Efficient bypass of the 5′ T of the cis–syn TT dimer by yeast Polζ. A 16mer primer was labeled with ³²P at its 5′ end and annealed right before the 5′ T of the TT dimer as indicated. DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs (lane 1), or a single deoxyribonucleoside triphosphate, dATP (lane 2), dCTP (lane 3), dGTP (lane 4) or dTTP (lane 5). DNA size markers in nucleotides are indicated on the left.

Figure 6

Translesion synthesis by yeast Polζ opposite an AAF-G. (A) A 13mer primer was labeled with ³²P at its 5′ end and annealed 4 nt before the template G without (lane 1) or with (lane 2) an AAF adduct. DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs. Nucleotide incorporation opposite the template AAF-G would form an 18mer DNA band. (B) A 17mer primer was labeled with ³²P at its 5′ end and annealed right before the template AAF-G as indicated. DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs (lane 1), or a single deoxyribonucleoside triphosphate, dATP (lane 2), dCTP (lane 3), dGTP (lane 4) or dTTP (lane 5). DNA size markers in nucleotides are indicated on the left.

Figure 7

Primer extension from opposite the AAF-G by yeast Polζ. (A) Four 18mer primers that differed by 1 nt at the 3′ end were labeled with ³²P at their 5′ ends and separately annealed to the indicated DNA template, with the primer 3′ end opposite the template AAF-G. (B) DNA polymerase assays were performed with 39 ng (193 fmol) of purified yeast Polζ in the presence of all four dNTPs (N₄), or a single deoxyribonucleoside triphosphate, dATP (A), dCTP (C), dGTP (G) or dTTP (T) as indicated. DNA size markers in nucleotides are indicated on the left.