DNA synthesis past a 5-methylc-containing cis-syn-cyclobutane pyrimidine dimer by yeast Pol η is highly nonmutagenic

Cyclobutane pyrimidine dimers (CPDs) are responsible for a considerable fraction of sunlight-induced C to T and 5-methycytosine (^mC) to T mutations in mammalian cells, though the precise mechanism is unknown. One possibility is that the C or ^mC of a CPD is not mutagenic and must first deaminate to U or T, respectively, for A to be inserted by a DNA polymerase. Alternatively, A might be directly inserted opposite the C or ^mC prior to deamination via an E-imino tautomer of the C or ^mC or by a nontemplated mechanism in which the photoproduct is sterically excluded from the active site. We have taken advantage of the retarding effect of C5 methylation on the deamination rate of cis-syn-cyclobutane dimers to prepare a template containing the cis-syn-cyclobutane dimer of ^mCT. Through the use of single-hit and multiple-hit competition assays, the catalytic core of pol η was found to insert dGMP opposite the ^mC of the CPD with about a 120:1 selectivity relative to dAMP. No significant insertion of dTTP or dCMP was detected. The high fidelity of nonmutagenic insertion opposite the ^mC of the CPD provides strong support for the deamination-bypass mechanism for the origin of sunlight induced C → T mutations.