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J. Biol. Chem., Vol. 282, Issue 27, 19831-19843, July 6, 2007

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Hydrogen Bonding of 7,8-Dihydro-8-oxodeoxyguanosine with a Charged Residue in the Little Finger Domain Determines Miscoding Events in Sulfolobus solfataricus DNA Polymerase Dpo4^*

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

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a highly efficient and relatively accurate manner. Crystal structures have revealed a potential role for Arg³³² in stabilizing the anti conformation of the 8-oxoG template base by means of a hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8-oxoG and dATP less favorable. Site-directed mutagenesis was used to replace Arg³³² with Ala, Glu, Leu, or His in order to probe the importance of Arg³³² in accurate and efficient bypass of 8-oxoG. The double mutant Ala³³¹Ala³³² was also prepared to address the contribution of Arg³³¹. Transientstate kinetic results suggest that Glu³³² retains fidelity against bypass of 8-oxoG that is similar to wild type Dpo4, a result that was confirmed by tandem mass spectrometric analysis of full-length extension products. A crystal structure of the Dpo4 Glu³³² mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu³³² and the O-8 atom of 8-oxoG. The space normally occupied by Arg³³² side chain is empty in the crystal structures of the Ala³³² mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu³³² and Ala³³² mutants. These results support the view that a bond between Arg³³² and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.

Received for publication, March 16, 2007 , and in revised form, April 12, 2007.

The atomic coordinates and structure factors (codes 2uvw, 2uvv, 2uvu, 2uvr) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, 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-S31 and Tables S1-S6 detailing a large portion of the LC-MS/MS results used to analyze the full-length extension by WT Dpo4 and the mutant enzymes, steady-state parameters for the Ala³³¹Ala³³² mutant, and x-ray density.

This article was selected as a Paper of the Week.

¹ These authors contributed equally to this work.

² 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 Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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