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 15-and 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 gapfilling synthesis is discussed.In mammalian cells, single base lesions are the most common form of DNA damage that arises either from exogenous DNAdamaging agents (1) or from endogenous biological processes resulting in base alkylation (2, 3), base oxidation (4), spontaneous cytosine deamination (3), and hydrolytic base loss (3,5,6). Base excision repair (BER) 3 is the major pathway to repair single base lesions (7). Short-patch and long-patch BER are the two subpathways of BER that remove and replace 1 (8 -10) and 2-11 nucleotides (11-13), respectively. Short-patch BER starts with the excision of a modified base by a DNA glycosylase, leaving a noncoding apurinic or apyrimidinic site in DNA. This lesion is further processed and repaired by a 5Ј-acting apurinic or apyrimidinic endonuclease, a DNA polymerase, a 5Ј-2-deoxyribose-5-phosphate lyase (dRPase), and a DNA ligase (12, 14 -16). It has been established that in mammalian systems DNA polymerase ␤ (Fig. 1, Pol␤), an X-family DNA polymerase, plays a critical role in short-patch BER (8, 9). The polymerase activity of Pol␤ catalyzes single nucleotide gap-filling synthesis (17), while its dRPase activity removes the 5Ј-terminal 2-deoxyribose-5-phosphate moiety (dRP) of a downstream strand (18). The uraci...

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