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J. Biol. Chem., Vol. 281, Issue 4, 2358-2372, January 27, 2006

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Efficient and High Fidelity Incorporation of dCTP Opposite 7,8-Dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA Polymerase Dpo4^*

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

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed 95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K_d,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E_a for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca²⁺. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.

Received for publication, October 5, 2005 , and in revised form, November 16, 2005.

The atomic coordinates and structure factors (codes 2C22, 2C2D, 2C2E, 2C28 and 2C2R) 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 in part by United States Public Health Service Grants R01 ES010375 and P30 ES000267. The use of the advanced Photon Source was supported by the United States Department of Energy, Basic Energy Sciences, Office of Science under contract W-31-109-Eng-38. The DuPont-Northwestern-Dow Collaborative Access Team Synchrotron Research Center at the Advanced Photon Source (Sector 5) was supported by E. I. DuPont de Nemours & Co., the Dow Chemical Co., the National Science Foundation, and the State of Illinois. 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 Figs. S1–S2 and Tables S1–S3.

¹ To whom correspondence should be addressed: 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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