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J. Biol. Chem., Vol. 282, Issue 34, 24689-24696, August 24, 2007

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Increased Catalytic Activity and Altered Fidelity of Human DNA Polymerase in the Presence of Manganese^*

From the Laboratory of Genomic Integrity, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725

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

Received for publication, March 13, 2007 , and in revised form, July 2, 2007.

^* This work was funded by the Intramural Research Programs of the NICHD, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Bldg. 6, Rm. 1A13, NICHD, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-2725. Tel.: 301-496-6175; Fax: 301-594-1135; E-mail: woodgate{at}nih.gov.

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