Preparation of site-specific T=mCG cis-syn cyclobutane dimer-containing template and its error-free bypass by yeast and human polymerase η

Abstract

C-to-T mutations are a hallmark of UV light and, in humans, occur preferentially at methylated Py(m)CG sites, which are also sites of preferential cyclobutane pyrimidine dimer (CPD) formation. In response, cells have evolved DNA damage bypass polymerases, of which polymerase η (pol η) appears to be specifically adapted to synthesize past cis-syn CPDs. Although T=T CPDs are stable, CPDs containing C or 5-methylcytosine ((m)C) are not and spontaneously deaminate to U or T at pH 7 and 37 °C over a period of hours or days, making their preparation and study difficult. Furthermore, there is evidence to suggest that, depending on solvent polarity, a C or an (m)C in a CPD can adopt three tautomeric forms, one of which could code as T. Although many in vitro studies have established that synthesis past T or U in a CPD by pol η occurs in a highly error-free manner, the only in vitro evidence that synthesis past C or (m)C in a CPD also occurs in an error-free manner is for an (m)C in the 5'-position of an (m)C=T CPD. Herein, we describe the preparation and characterization of an oligodeoxynucleotide containing a CPD of a T(m)CG site, one of the major sites of C methylation and C-to-T mutations found in the p53 gene of basal and squamous cell cancers. We also demonstrate that both yeast and human pol η synthesize past the 3'-(m)C CPD in a >99% error-free manner, consistent with the highly water-exposed nature of the active site.

FIGURE 1.

Mutagenic properties of ^mC and its deamination product, T, in cis-syn dimer. The major tautomers of ^mC in a cis-syn dimer and their base pairing properties are compared with those of the deamination product, T.

FIGURE 2.

Single-hit primer extension competition experiment opposite 14-mer templates. An equimolar mixture of all dNTPs (200 μm each) together with sonicated/denatured salmon sperm DNA was added to a preincubated mixture of the indicated 9-mer primer-14-mer template and pol η. The reactions were terminated after 10 s with EDTA and unlabeled primer, and the products were electrophoresed on a citrate (pH 3.5)-10% polyacrylamide gel. The standards corresponding to extension of the 9-mer by A or G were prepared by automated synthesis.

FIGURE 3.

Multiple-hit full-length primer extension experiment. The indicated 9-mer primer-14-mer templates were incubated with pol η and 200 μm each dNTP for 2 min, and the products were electrophoresed on a citrate (pH 3.5)-20% polyacrylamide denaturing gel in comparison with standards.

FIGURE 4.

Single-hit primer extension biased nucleotide pool competition experiment opposite 14-mer templates. A 32:1 mixture of the indicated dNTP/dGTP (100 μm in the dNTP) together with sonicated/denatured salmon sperm DNA was added to a preincubated mixture of the indicated 9-mer primer-14-mer template and pol η. The reaction was terminated after 10 s with EDTA and unlabeled primer, and the products were electrophoresed on a citrate (pH 3.5)-10% polyacrylamide gel. The standards corresponding to extension of the 9-mer by A or G were prepared by automated synthesis.

FIGURE 5.

Selectivity of dGMP versus dAMP insertion opposite ^mC of T=^mC CPD via single-hit assay. Varying ratios of dATP to dGTP (with dATP fixed at 100 μm) together with sonicated/denatured salmon sperm DNA were added to a preincubated mixture of the 9-mer primer-14-mer template and pol η. The reaction was terminated after 10 s with EDTA and unlabeled primer, and the products were electrophoresed on a citrate (pH 3.5)-10% polyacrylamide gel. The standards corresponding to extension of the 9-mer by A or G were prepared by automated synthesis.

FIGURE 6.

Visual comparison of water accessibility of O6 of template guanine (yellow) in active site of yeast pol δ (Protein Data Bank code 3IAY) (A) and of O4 of template 3′-T (B) and template 5′-T (C) of cis-syn TT CPD (yellow) in active site of human pol η (code 3MR4).