Role of DNA polymerase eta 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 eta (Poleta), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Poleta result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Poleta for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer. Opposite the 3' T of a (6-4) TT photoproduct, both yeast and human Poleta preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polzeta, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3' T of the (6-4) TT lesion by Poleta, and Polzeta 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 Poleta and Polzeta, wherein Poleta inserts a nucleotide opposite the 3' T of the lesion and Polzeta 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.

FIG. 1

Bypass of the (6-4) TT photoproduct by the combined action of Polη and Polζ. Lanes 1 to 3, undamaged DNA; lanes 4 to 9, (6-4) TT photoproduct-containing DNA. Positions of the two T's in the undamaged or the (6-4) TT photoproduct-containing template are indicated on the right. hPolη (1 nM), yPolη (1 nM), yPolζ (1.8 nM), or either yeast or human Polη combined with yPolζ was incubated with the DNA substrate for 5 min at 37°C in the presence of 100 μM each of the four dNTPs.

FIG. 2

Nucleotide incorporation by hPolη opposite the 3′ T residue in a nondamaged template or a (6-4) TT photoproduct-containing template. (A) Incorporation of nucleotides opposite the nondamaged T residue; (B) incorporation opposite the equivalent 3′ T residue of a (6-4) TT photoproduct. A portion of each primer:template substrate is shown at the top. hPolη (0.5 nM) was incubated with DNA substrate (10 nM) and the indicated concentrations of dNTPs for 5 min at 30°C.

FIG. 3

Nucleotide incorporation opposite the 5′ T of the (6-4) TT photoproduct by yPolζ. (A) Nucleotide incorporation following a G residue opposite the 3′ T of the (6-4) photoproduct; (B) nucleotide incorporation following an A residue opposite the 3′ T of a (6-4) photoproduct. yPolζ (5 nM) was incubated for 3 min at 30°C with the primer:template DNA substrate (20 nM) and with the indicated concentrations of dNTPs.