Importance of hydrogen bonding for efficiency and specificity of the human mitochondrial DNA polymerase

To assess the contribution to discrimination afforded by base pair hydrogen bonding during DNA replication by the human mitochondrial DNA polymerase, we examined nucleoside mimics lacking hydrogen bond forming capability but retaining the overall steric shape of the natural nucleotide. We employed oligonucleotide templates containing either a deoxyadenosine shape mimic (dQ) or a deoxythymidine shape mimic (dF). Additionally, the nucleoside triphosphate analogs difluorotoluene deoxynucleoside triphosphate, 9-methyl-1-H-imidazo[(4,5)-b]pyridine deoxyribose triphosphate, and 4-methylbenzimidazole deoxyribose triphosphate (dZTP; another dATP shape mimic) were assayed. We used pre-steady state methods to determine the kinetic parameters governing nucleotide incorporation, k(pol) and K(d). In general, the loss of hydrogen bonding potential led to 2-3 kcal/mol reduction in ground state binding free energy, whereas effects on the maximum rate of polymerization were quite variable, ranging from negligible (dATP:dF) to nearly 4 kcal/mol (dZTP:dT). Although we observed only a 46-fold reduction in discrimination when dF was present in the template, there was a complete elimination of discrimination when dQ was present in the template. Our data with dF indicate that hydrogen bonding contributes 2.2 kcal/mol toward the efficiency of incorporation, whereas data with dQ (which may overestimate the effect due to poor steric mimicry) suggest a contribution of up to 6.8 kcal/mol. Taken together, the data suggest that sterics are necessary but not sufficient to achieve optimal efficiency and fidelity for DNA polymerase. Base pair hydrogen bonding contributes at least a third of the energy underlying nucleoside incorporation efficiency and specificity.