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

Human DNA polymerase iota (Pol iota) 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 iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota 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 N-6 of an A or the 06 of a G, here we provide evidence that whereas hydrogen bonding at N-6 is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O-6 is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O-6 and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the O-6-methyl guanine and 8-oxoguanine lesions, which affect the O-6 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 requiired, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and 0 6 is an imperative. The dispensability of N-6 hydrogen bonding for proficient T incorporation opposite template A, has important biological implications, as that would endow Pol iota with the ability to replicate through lesons which impair the Watson-Crick hydrogen bonding potential at both the N1 and N-6 positions of templating A.