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

Abstract

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...

Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

“…Similar single-nucleotide incorporation assays were performed for each enzyme, incorporating dATP, PMPA-DP, dCTP, L-3TC-TP, L-FTC-TP, dTTP, or AZT-TP opposite the complementary template base. Please note, a single-nucleotide-gap DNA substrate was used in the assays for the gap-filling Pols ␤ and , since the catalytic efficiencies of these enzymes are enhanced by a 5Ј-phosphorylated downstream strand (1,10) and the gap DNA substrate models a more physiologically relevant DNA substrate. Meanwhile, Pols , , , and Rev1 were assayed with a primer-template DNA substrate.…”

Single-turnover Kinetic Analysis of the Mutagenic Potential of 8-Oxo-7,8-dihydro-2′-deoxyguanosine during Gap-filling Synthesis Catalyzed by Human DNA Polymerases λ and β

“…[37][38][39] Thus, the experiments presented here were performed with human fPolλ in molar excess over DNA to allow the direct observation of nucleotide incorporation in a single pass of the reactants through the enzymatic pathway without complications resulting from the steady-state formation of products. 40 Single-turnover kinetic analysis of 8-oxodGTP incorporation Nucleotide pools are susceptible to oxidative DNA damage induced by reactive oxygen species, therefore, it is possible that a DNA polymerase may encounter and subsequently incorporate 8-oxodGTP in vivo, particularly under conditions of severe oxidative stress and/or a disfunctional 8-oxodGTPase.…”

Creative template-dependent synthesis by human polymerase mu

“…S2). Although Pol λ and Pol β are more active and more processive on gapped DNA substrates containing a 5ʹ-phosphate on the downstream end of the gap (22)(23)(24), Pol μ is the first gap-filling polymerase, to our knowledge, to engage single-and doublestrand break intermediate substrates such that the template base at the 5ʹ end of the gap directs incoming nucleotide incorporation. Given the strong dependence of Pol μ polymerase activity on 5ʹ-phosphate-containing gapped DNA (11,25), we postulate that interaction between the 8-kDa domain and the 5ʹ-phosphate on the downstream end of the gap may represent a major driving force for substrate binding.…”