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J. Biol. Chem., Vol. 282, Issue 2, 1456-1467, January 12, 2007

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Sulfolobus solfataricus DNA Polymerase Dpo4 Is Partially Inhibited by "Wobble" Pairing between O⁶-Methylguanine and Cytosine, but Accurate Bypass Is Preferred^*

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

We examined the effect of a single O⁶-methylguanine (O⁶-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O⁶-MeG, with C being the major product (70%) and T or A being the minor species (20% or 10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that k_pol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O⁶-MeG was 6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O⁶-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O⁶-MeG. A structure containing T opposite O⁶-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher K_m,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O⁶-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.

Received for publication, October 13, 2006 , and in revised form, November 10, 2006.

The atomic coordinates and structure factors (code 2j6s (r2j6ssf), 2j6u (r2j6usf), and 2j6t (r2j6tsf)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Bruns-wick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants R01 ES010375 (to F. P. G.), F32 CA119776 (to R. L. E.), P30 ES000267 (to F. P. G. and M. E.), and P01 ES05355 (to M. E.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S10 and Tables S1-S3.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Avenues, Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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