Increased catalytic activity and altered fidelity of human DNA polymerase ι in the presence of manganese

All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg2+ is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn2+ in vitro. Here we report on the effects of Mn2+ and Mg2+ on the enzymatic properties of human DNA polymerase iota (pol iota). pol iota exhibited the greatest activity in the presence of low levels of Mn2+ (0.05-0.25 mM). Peak activity in the presence of Mg2+ was observed in the range of 0.1-0.5 mM and was significantly reduced at concentrations > 2 mM. Steady-state kinetic analyses revealed that Mn2+ increases the catalytic activity of pol iota by similar to 30-60,000-fold through a dramatic decrease in the Km value for nucleotide incorporation. Interestingly, whereas pol iota preferentially misinserts G opposite T by a factor of similar to 1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg2+, respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mM Mn2+. Low levels of Mn2+ also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol iota for Mn2+ even when Mg2+ is present in a > 10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn2+ rather than Mg2+, as tacitly assumed.