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 λ, may preferentially utilize Mn2+ in vitro. Here we report on the effects of Mn2+ and Mg2+ on the enzymatic properties of human DNA polymerase ι (pol ι). pol ι 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 ι by ∼30–60,000-fold through a dramatic decrease in the Km value for nucleotide incorporation. Interestingly, whereas pol ι preferentially misinserts G opposite T by a factor of ∼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 ι to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol ι 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 ι in vivo may actually be Mn2+ rather than Mg2+, as tacitly assumed.

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