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. 2003 Feb;23(4):1453-9.
doi: 10.1128/MCB.23.4.1453-1459.2003.

Yeast DNA polymerase zeta is an efficient extender of primer ends opposite from 7,8-dihydro-8-Oxoguanine and O6-methylguanine

Lajos Haracska  1 , Satya Prakash, Louise Prakash
Affiliations

Yeast DNA polymerase zeta is an efficient extender of primer ends opposite from 7,8-dihydro-8-Oxoguanine and O6-methylguanine

Lajos Haracska et al. Mol Cell Biol. 2003 Feb.

Abstract

Genetic studies in Saccharomyces cerevisiae have indicated the requirement of DNA polymerase (Pol) zeta for mutagenesis induced by UV light and by other DNA damaging agents. However, on its own, Pol zeta is highly inefficient at replicating through DNA lesions; rather, it promotes their mutagenic bypass by extending from the nucleotide inserted opposite the lesion by another DNA polymerase. So far, such a role for Pol zeta has been established for cyclobutane pyrimidine dimers, (6-4) dipyrimidine photoproducts, and abasic sites. Here, we examine whether Pol zeta can replicate through the 7,8-dihydro-8-oxoguanine (8-oxoG) and O(6)-methylguanine (m6G) lesions. We chose these two lesions for this study because the replicative polymerase, Pol delta, can replicate through them, albeit weakly. We found that Pol zeta is very inefficient at inserting nucleotides opposite both these lesions, but it can efficiently extend from the nucleotides inserted opposite them by Pol delta. Also, the most efficient bypass of 8-oxoG and m6G lesions occurs when Pol delta is combined with Pol zeta, indicating a role for Polzeta in extending from the nucleotides inserted opposite these lesions by Pol delta. Thus, Pol zeta is a highly specialized polymerase that can proficiently extend from the primer ends opposite DNA lesions, irrespective of their degree of geometric distortion. Pol zeta, however, is unusually sensitive to geometric distortion of the templating residue, as it is highly inefficient at incorporating nucleotides even opposite the moderately distorting 8-oxoG and m6G lesions.

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Figures

FIG. 1.
FIG. 1.
Bypass of 8-oxoG and m6G lesions by the combined action of Polδ and Polζ. (A) DNA synthesis on an 8-oxoG-containing DNA template. Lanes 1 to 7, undamaged DNA; lanes 8 to 14, 8-oxoG-containing DNA. Sequences adjacent to the primer-template junction are shown for the 40-nt, 5′-32P-labeled primer and 75-nt template. The position corresponding to the undamaged G or the 8-oxoG site on the template is indicated by ∗G. Yeast Polζ (10 nM), yeast Polδ (10 nM), or a combination of these two enzymes were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (5 or 100 μM) at 30°C for 10 min. The reaction products were resolved on a 15% denaturing polyacrylamide gel and visualized by autoradiography. The gel was analyzed by using a PhosphorImager, and the concentrations of the products of the synthesis past the undamaged G or 8-oxoG are indicated. (B) DNA synthesis on an m6G-containing DNA template. Lanes 1 to 7, undamaged DNA; lanes 8 to 14, m6G-containing DNA. Reactions were carried out as indicated in the legend for panel A, except that the incubation time of the reactions was 15 min.
FIG. 2.
FIG. 2.
Extension of primers with various 3′ ends opposite 8-oxoG and m6G lesions by Polζ. Each of the four different primers, differing only in the 3′-terminal nucleotide, was annealed to the template oligonucleotide containing an undamaged G, an 8-oxoG, or an m6G residue at the same position. In the primer-template pair shown, N designates the position of the variable terminal primer nucleotide and ∗G designates the position of a G, an 8-oxoG, or an m6G residue. Polζ (10 nM) was incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM) at 30°C for 15 min. The gel was analyzed by using a PhosphorImager, and the amount of the product of primer extension opposite from the undamaged G, 8-oxoG, or m6G is indicated.
FIG. 3.
FIG. 3.
Steady-state kinetic analysis of insertion and extension reactions catalyzed by Polζ on 8-oxoG- and m6G-containing DNA templates. A portion of the DNA substrate used for insertion reactions (top left) or for extension reactions (top right) is shown, and the position of an 8-oxoG, m6G, or undamaged G residue in the template oligonucleotide is indicated by an asterisk (top). Polζ (2 nM) was incubated with the primer-template DNA substrate (30 nM) and increasing concentrations of a single deoxynucleotide for 10 min at 30°C. The quenched samples were analyzed by 15% denaturing polyacrylamide gel electrophoresis, and the steady-state kinetic parameters kcat and Km were determined.
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