Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage and mitochondrial family members to very low for certain eukaryotic homologues. The latter include Pol ν which, among all A-family polymerases, is uniquely prone to misincorporate dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate Family A homologue, the Klenow fragment of E. coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, kpol. In the sequence context where fidelity is highest, kpol for correct G-dCTP incorporation by Pol ν is ~ 15-fold faster than kpol for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, kpol is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous, and that the relaxed active-site specificity towards the G-dTTP mispair may be associated with its cellular function(s).