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J. Biol. Chem., Vol. 281, Issue 47, 35649-35655, November 24, 2006

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Kinetic Effect of a Downstream Strand and Its 5'-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase ^*

Wade W. Duym, Kevin A. Fiala¹, Nikunj Bhatt, and Zucai Suo^¶||**2

From the Department of Biochemistry, Biochemistry Program, ^¶Biophysics Program, ^||Molecular, Cellular, and Developmental Biology Program, and ^**Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase or is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase . The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (k_p/K_d) by 15and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant k_p rather than the ground state nucleotide binding affinity K_d. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase , it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in k_p rather than K_d. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

Received for publication, August 7, 2006 , and in revised form, September 21, 2006.

^* This work was supported in part by startup funds from The Ohio State University (to Z. S.). 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 Supported by a Herta Camerer Gross graduate research fellowship and American Heart Association Predoctoral Fellowship Grant 0415129B.

² To whom correspondence should be addressed: 740 Biological Sciences, 484 W. 12th Ave., Columbus, OH 43210. Tel.: 614-688-3706; Fax: 614-292-6773; E-mail: suo.3{at}osu.edu.

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