Role of DNA Polymerase η in the Bypass of a (6-4) TT Photoproduct

Abstract

UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase η (Polη), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Polη result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Polη for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than acis-syn TT dimer. Opposite the 3′ T of a (6-4) TT photoproduct, both yeast and human Polη preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polζ, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3′ T of the (6-4) TT lesion by Polη, and Polζ inserts the correct nucleotide A opposite the 5′ T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Polη and Polζ, wherein Polη inserts a nucleotide opposite the 3′ T of the lesion and Polζ extends from it. These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3′ T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3′ T→C change that would result from the insertion of a G opposite the 3′ T of the (6-4) TT lesion.

ACKNOWLEDGMENTS

We thank M. T. Washington for helpful discussions.

This work was supported by National Institutes of Health grant GM19261. The (6-4) TT photoproduct containing DNA was constructed in the Synthetic Organic Chemistry Core Laboratory, supported by NIEHS Center grant P30-ESO6676, and we are grateful to Richard Hodge for providing this DNA.