Effect of N2-guanyl modifications on early steps in catalysis of polymerization by Sulfolobus solfataricus P2 DNA polymerase Dpo4 T239W.

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N(2)-alkyl guanine (G) adducts with N(2)-isobutylG showing the largest effect, decreasing approximately 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N(2)-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2'-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N(2)-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N(2)-alkylG adducts. With the smaller N(2)-alkylG adducts, the conformational rate constants were not changed dramatically (<3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (>or=(2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.