Abstract
DNA precursors, i.e., dNTPs in the nucleotide pool, are oxidized during aerobic metabolism as in the case of DNA. Oxidized dNTPs could be incorporated into the genome DNA by DNA polymerases. We have shown possible involvement of Y-family DNA polymerases in the incorporation of oxidized dNTPs. Interestingly, the DNA polymerases incorporate 8-oxo-dGTPs opposite template dA much more frequently than dC. In this study, we assessed the amino acids involved in the preferential incorporation of 8-oxo-dGTP by human DNA polymerase κ (hPolκ) and η (hPolη). In hPolκ, the substitution of Y112 with alanine resulted in the ratio of incorporation of 8-oxo-dGTP opposite template dA:dC from 11:1 to 1:1. It suggests that Y112 may stabilize the 8-oxo-dGTP in the syn formation by aligning the sugar, because Y112 distinguishes rNTPs from dNTPs by recognizing 2'-OH of rNTPs. In hPolη, the substitution of F18, which corresponds to Y112 in hPolκ, did not change the preference. However, substitution of R61, which is not conserved among other Y-family polymerases, with alanine or lysine substantially changed the preference for incorporation of 8-oxo-dGTP opposite dA and dC; the ratio (dA:dC) of 660:1 in the wild-type hPolη was changed to 63:1 in R61A and 7:1 in R61K. The structural analysis of the incoming 8-oxo-dGTP in the active site of yeast Polη suggest that C8 oxygen of 8-oxo-dGTP in the anti formation may cause a sterical clash with R61, which lead to destabilization of 8-oxo-dGTP in the active site, although 8-oxo-dGTP in the syn formation could not be affected because of the lack of the structural interference. These results suggest that the amino acids affecting the specificity for incorporation of 8-oxo-dGTP are different between hPolκ and hPolη.
