Role of Hoogsteen edge hydrogen bonding at template purines in nucleotide incorporation by human DNA polymerase ι

Human DNA polymerase ι (Pol ι) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol ι with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol ι active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N⁶ of an A or the O⁶ of a G, here we provide evidence that whereas hydrogen bonding at N⁶ is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O⁶ is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O⁶ and N7 hydrogen bonding to DNA synthesis by Pol ι, we have examined its proficiency for replicating through the ⁶O-methyl guanine and 8-oxoguanine lesions, which affect the O⁶ and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O⁶ is an imperative. The dispensability of N⁶ hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol ι with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N ⁶ positions of templating A.