Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase η

DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polν. Polη displayed "burst" kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (K_d) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (k_pol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.