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Increased Activity and Fidelity of DNA Polymerase β on Single-nucleotide Gapped DNA

1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês

10.1074/jbc.272.44.27501

ISSN

1083-351X

Autores

Alexander M. Chagovetz, Joann B. Sweasy, Bradley D. Preston,

Tópico(s)

DNA and Nucleic Acid Chemistry

Resumo

DNA polymerase β (pol β) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol β activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol β (k cat/K m,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5′-margin of the gap. pol β exhibited the highest catalytic efficiency on 5′-phosphorylated 1-nucleotide gapped DNA. This efficiency was ≥500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol β, as judged by its ability to form G-N mispairs, was also higher (10–100 times) on 5′-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol β is to fill 5′-phosphorylated 1-nucleotide gaps. DNA polymerase β (pol β) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol β activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol β (k cat/K m,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5′-margin of the gap. pol β exhibited the highest catalytic efficiency on 5′-phosphorylated 1-nucleotide gapped DNA. This efficiency was ≥500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol β, as judged by its ability to form G-N mispairs, was also higher (10–100 times) on 5′-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol β is to fill 5′-phosphorylated 1-nucleotide gaps. DNA polymerase β (pol β) 1The abbreviations used are: pol β, DNA polymerase β; BER, base excision repair; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; n-mer, single-stranded oligodeoxyribonucleotide n residues in length; gap-n, double-stranded DNA substrate with a single-stranded gap n nucleotides in length; P-gap-n, same as gap-n but with a phosphate at the 5′-margin of the gap. plays a central role in mammalian base excision repair (BER (1Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 2Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (784) Google Scholar, 3Nealon K. Nicholl I.D. Kenny M.K. Nucleic Acids Res. 1996; 24: 3763-3770Crossref PubMed Scopus (59) Google Scholar, 4Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (662) Google Scholar, 5Friedberg E.C. Wood R.D. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 249-269Google Scholar)). pol β is a monomeric 39-kDa enzyme organized into a carboxyl-terminal 31-kDa domain that includes the polymerase active site and an amino-terminal 8-kDa domain that participates in DNA binding and harbors 5′-deoxyribose phosphodiesterase (lyase) activity (6Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (650) Google Scholar, 7Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12742-12761Crossref PubMed Scopus (273) Google Scholar). The presence of both polymerase and lyase activities suggests that pol β catalyzes two steps in the "short-patch" BER pathway: removal of a 5′-deoxyribose phosphate intermediate and subsequent filling of the resultant 1-nt gap (4Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (662) Google Scholar, 6Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (650) Google Scholar). pol β has also been implicated in "long-patch" BER (4Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (662) Google Scholar) and may function in meiosis (8Plug A.W. Clairmont C.A. Sapi E. Ashley T. Sweasy J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1327-1331Crossref PubMed Scopus (97) Google Scholar) and nucleotide excision repair (9Horton J.K. Srivastava D.K. Zmudzka B.Z. Wilson S.H. Nucleic Acids Res. 1995; 23: 3810-3815Crossref PubMed Scopus (83) Google Scholar,10Oda N. Saxena J.K. Jenkins T.M. Prasad R. Wilson S.H. Ackerman E.J. J. Biol. Chem. 1996; 271: 13816-13820Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The biochemical activities of purified pol β are consistent with a role in gap-filling DNA synthesis. Early studies showed that pol β is non-processive on single-stranded DNA templates, prefers short-gapped DNA substrates, and is capable of filling gaps to completion (11Chang L.M.S. J. Mol. Biol. 1975; 93: 219-235Crossref PubMed Scopus (48) Google Scholar, 12Bambara R.A. Uyemura D. Choi T. J. Biol. Chem. 1978; 253: 413-423Abstract Full Text PDF PubMed Google Scholar, 13Matsukage A. Nishizawa M. Tkahashi T. J. Biochem. (Tokyo). 1979; 85: 1551-1554Crossref PubMed Scopus (12) Google Scholar, 14Wang T.S.-F. Korn D. Biochemistry. 1980; 19: 1782-1790Crossref PubMed Scopus (76) Google Scholar, 15Siedlecki J.A. Szysko J. Pietrzykowska I. Zmudzka B. Nucleic Acids Res. 1980; 8: 361-375Crossref PubMed Scopus (14) Google Scholar, 16Mosbaugh D.W. Linn S. J. Biol. Chem. 1983; 258: 108-118Abstract Full Text PDF PubMed Google Scholar). More recently, Wilson and colleagues (17Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar) observed that pol β fills short gaps (2–6 nt) by a processive mechanism that requires a PO4 group at the 5′-margin of the gap. Binding of pol β to these short-gapped substrates is also strongly enhanced by the presence of a 5′-PO4 (18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar). These experiments, together with recent structural data, suggest a model in which pol β binding to gapped DNA is mediated by interactions between the 8-kDa domain of pol β and the 5′-PO4 at the downstream margin of the gap (7Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12742-12761Crossref PubMed Scopus (273) Google Scholar,18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar). Processive DNA synthesis on short (2–6-nt) gaps is consistent with roles for pol β in long-patch BER (4Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (662) Google Scholar) and in the completion of gap-filling synthesis initiated by other cellular DNA polymerases (9Horton J.K. Srivastava D.K. Zmudzka B.Z. Wilson S.H. Nucleic Acids Res. 1995; 23: 3810-3815Crossref PubMed Scopus (83) Google Scholar, 10Oda N. Saxena J.K. Jenkins T.M. Prasad R. Wilson S.H. Ackerman E.J. J. Biol. Chem. 1996; 271: 13816-13820Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Wang T.S.-F. Korn D. Biochemistry. 1980; 19: 1782-1790Crossref PubMed Scopus (76) Google Scholar, 15Siedlecki J.A. Szysko J. Pietrzykowska I. Zmudzka B. Nucleic Acids Res. 1980; 8: 361-375Crossref PubMed Scopus (14) Google Scholar, 16Mosbaugh D.W. Linn S. J. Biol. Chem. 1983; 258: 108-118Abstract Full Text PDF PubMed Google Scholar). Although DNA polymerization by pol β on single-stranded and short-gapped DNAs is understood in some detail, much less is known about pol β activity on its short-patch BER substrate, 1-nt gapped DNA. The model of pol β binding through its 8-kDa domain to the 5′-PO4 in short-gapped DNA does not appear to apply to 1-nt gaps; reducing the gap size from 5 to 1 nt decreases binding slightly, and the 5′-phosphorylation requirement is lost (18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar). This suggests that pol β may interact with 1-nt gapped DNA by a distinct mechanism. To better characterize the parameters governing pol β activity on 1-nt gapped DNA, we examined the steady-state kinetics of DNA polymerization on synthetic primer-templates of different structure. We show that the catalytic efficiency (k cat/ K m,dNTPapp) and nucleotide insertion fidelity of pol β are strongly influenced by gap size and that the 5′-phosphorylation requirement is retained for these activities even on 1-nt gapped DNA. These data have important implications for models of pol β DNA binding and provide biochemical evidence that 5′-phosphorylated 1-nt gapped DNA is the preferred substrate for pol β. Recombinant rat DNA polymerase β was purified as described previously (19Washington S.L. Yoon M.S. Chagovetz A.M. Li S.X. Clairmont C.A. Preston B.D. Eckert K.A. Sweasy J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1321-1326Crossref PubMed Scopus (55) Google Scholar). All oligonucleotides were synthesized and high pressure liquid chromatography-purified by Operon Technologies. 5′-32P Labeling of the primers was performed with [γ-32P]ATP (3,000 Ci/mmol; Amersham Corp.) using T4 polynucleotide kinase (U. S. Biochemical Corp.) according to the manufacturer's protocol. Labeled primers were separated from excess [γ-32P]ATP after labeling by gel filtration through 0.5-ml Sephadex G-50 (Pharmacia Biotech Inc., DNA grade) spin columns. 2′-Deoxyribonucleoside 5′-triphosphates (dNTPs) were from Calbiochem or Pharmacia. Concentrations of individual dNTPs were determined by UV spectroscopy (Beckman DU65). Protein concentrations were determined by the method of Bradford (Bio-Rad) according to the manufacturer's protocol. All other reagents were of the highest grade available from Fisher Scientific or Sigma. Primer-templates of different structure were constructed from synthetic oligodeoxyribonucleotides (see Fig. 1). Hybridizations were done by mixing equimolar amounts of the required oligonucleotides in 250 mm KCl, 50 mm Tris-HCl, pH 8.0 (22 °C) and incubating sequentially at 65 °C (10 min), 37 °C (10 min), 22 °C (10 min), and 0 °C (10 min). Annealing efficiencies were >95%, as evidenced by mobility shifts on non-denaturing polyacrylamide gel electrophoresis (PAGE) and by the proportion of 5′-32P-primers extended in prolonged incubations with excess pol β and saturating concentrations of dNTPs (data not shown). The kinetics of dNMP incorporation opposite template positions G21 (see Fig. 1) were determined in polymerization reactions (10 or 20 μl) containing 0.1–20 nm pol β, 20 nm primer-template, and 0–5,000 μm of a single dNTP in 50 mm Tris-HCl, pH 8.0 (22 °C), 10 mm MgCl2, 2 mm dithiothreitol, 20 mm NaCl, 20 mm KCl, 2.5% glycerol, 0.2 mg/ml bovine serum albumin. Primer-templates were first incubated with pol β for 5 min at 37 °C in the absence of dNTPs, and then polymerizations were initiated by the addition of a single dNTP. After continued incubation for 3–15 min at 37 °C, reactions were terminated by adding 0.1 volume of 0.5 m EDTA. 1–2-μl aliquots were removed and mixed with 5 μl of formamide loading dye (20Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (685) Google Scholar), boiled for 5 min, and immediately transferred into an ice slurry for 5 min. Products were resolved by PAGE (7 m urea, 16% acrylamide) and then visualized and quantified using a PhosphorImager and Imagequant software (Molecular Dynamics). Reaction times and enzyme concentrations were adjusted for each substrate to optimize product detection while ensuring that all reactions were conducted in the steady state. Only those reactions that fell within the linear range of substrate utilization (≤20% primer extension) were used for kinetic analyses. Steady-state kinetic analyses were based on the Michaelis-Menten equation. For correct dCMP incorporation, k catand K m,dNTPapp values were determined using a non-linear curve fitting program (SigmaPlot). For mispairs,k cat/ K m,dNTPappvalues were determined from the initial slopes of Michaelis-Menten plots (20Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (685) Google Scholar, 21Boosalis M.S. Mosbaugh D.W. Hamatake R. Sugino A. Kunkel T.A. Goodman M.F. J. Biol. Chem. 1989; 264: 11360-11366Abstract Full Text PDF PubMed Google Scholar), and the frequencies of misincorporation were calculated as described (22Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (403) Google Scholar). To detect extension products resulting from dNMP misincorporation, it was often necessary to increase pol β concentrations and/or incubation times. As expected in steady state (22Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (403) Google Scholar), V max values were directly proportional to enzyme concentration (data not shown). The processivity of pol β was determined on the same substrates used in the kinetic assays (see Fig. 1) and under similar conditions, except the reactions were started by adding all four dNTPs at saturating concentrations (1.25 mm each). Linear regions of product yield versus pol β concentration curves were used to quantify average (statistically weighted) processivities. Statistical weighting was performed by multiplying the lengths of the products by the relative intensities of corresponding bands on the gel. To examine pol β activity on DNA substrates of different structure, a series of primer-templates was constructed (Fig.1). These DNAs all contained the same 46-mer template sequence based on a region of bacteriophage φX174 DNA used in previous fidelity studies (Refs. 20Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (685) Google Scholar and 23Roberts J.D. Kunkel T.A. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 217-247Google Scholar, and references therein). All of the substrates also contained the same [5′-32P]20-mer primer hybridized to template residues 22–41. This places the primer 3′-OH terminus such that polymerization of the first dNTP occurs opposite template G21 (which corresponds to residue 587 in φX174 DNA). The simplest DNA substrate, comprised of [5′-32P]20-mer primer hybridized to 46-mer template, had a "recessed" primer with 21 nt of downstream single-stranded DNA template (Fig. 1 A). Two gapped substrates were also constructed (Fig. 1, B andC). The substrate designated gap-6 contained a second oligonucleotide (15-mer) hybridized to template residues 1–15, thereby creating a primer-template with a 6-nt gap immediately downstream from the 5′-32P-primer 3′-OH (Fig.1 B). A similar substrate with a 1-nt gap (designatedgap-1) was constructed by hybridizing a 20-mer oligonucleotide to template residues 1–20 (Fig. 1 C). Variants of gap-6 and gap-1 containing PO4 moieties at the 5′ margins of the gaps (designated P-gap-6 andP-gap-1, respectively) were also made by starting with 5′-phosphorylated oligonucleotides. Proper assembly of these oligonucleotides into the desired structures was confirmed in two ways. First, native PAGE showed that hybridization efficiencies were >95% as evidenced by different mobilities of the [5′-32P]20-mer primer before and after hybridization to the 46-mer template alone and in combination with the downstream oligonucleotides (data not shown). As a second indirect way of confirming structure, we examined the processivity of pol β on these DNA substrates. This approach is based on the observation of Singhal and Wilson (17Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar) that pol β is distributive on recessed primer-templates and short non-phosphorylated gapped DNAs but processive on short gaps containing 5′-phosphates. We observed the same trend on our substrates (Fig. 2). Average processivities on the recessed, gap-6, and P-gap-6 DNAs were 1.3, 1.1, and 3.5, respectively. Thus, our data confirm the results of Singhal and Wilson (17Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar), although synthesis on our phosphorylated 6-nt gapped substrate was not strictly processive (Fig. 2, right). We performed steady-state kinetic analyses of single-nucleotide addition (dCMP) opposite template G21 on the different DNA substrates (Fig. 3, dCTP reactions, and Table I). The K m,dCTPapp andk cat values of 170 μm and 0.6 s−1 observed on the recessed primer-template are comparable with those reported by others for pol β (21Boosalis M.S. Mosbaugh D.W. Hamatake R. Sugino A. Kunkel T.A. Goodman M.F. J. Biol. Chem. 1989; 264: 11360-11366Abstract Full Text PDF PubMed Google Scholar, 24Werneburg B.G. Ahn J. Zhong X. Hondal R.J. Kraynov V.S. Tsai M.D. Biochemistry. 1996; 35: 7041-7050Crossref PubMed Scopus (134) Google Scholar). We noted, however, that theK m,dNTPapp values for pol β (40-170 μm; Table I and Ref. 21Boosalis M.S. Mosbaugh D.W. Hamatake R. Sugino A. Kunkel T.A. Goodman M.F. J. Biol. Chem. 1989; 264: 11360-11366Abstract Full Text PDF PubMed Google Scholar) are substantially higher than those observed for other DNA polymerases on similar or identical primer-templates in similar steady-state kinetic assays (typically 0.1-10 μm 2B. D. Preston, unpublished data. ; Refs.20Preston B.D. Poiesz B.J. Loeb L.A. Science. 1988; 242: 1168-1171Crossref PubMed Scopus (685) Google Scholar, 21Boosalis M.S. Mosbaugh D.W. Hamatake R. Sugino A. Kunkel T.A. Goodman M.F. J. Biol. Chem. 1989; 264: 11360-11366Abstract Full Text PDF PubMed Google Scholar, 22Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (403) Google Scholar, and references therein). The unusually highK m,dCTPapp values observed on the recessed and gap-6 primer-templates suggested that these DNAs were relatively poor substrates for pol β.Table IEffect of DNA substrate structure on pol β catalytic efficiencyView Large Image Figure ViewerDownload Hi-res image Download (PPT)1-a Sequences and structures of the DNA substrates are shown in Fig. 1.1-b Calculated using total pol β protein concentration.1-c Values in parentheses indicate the number of independent experiments used for each analysis. Open table in a new tab 1-a Sequences and structures of the DNA substrates are shown in Fig. 1. 1-b Calculated using total pol β protein concentration. 1-c Values in parentheses indicate the number of independent experiments used for each analysis. In a manner reminiscent of its effect on processivity (Fig. 2 and Ref.17Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar) and DNA binding (18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar), 5′-phosphorylation of gap-6 resulted in a modest reduction in K m,dCTPapp and concomitant increase in overall catalytic efficiency (k cat/ K m,dCTPapp). A similar decrease in K m,dCTPapp and increase in catalytic efficiency occurred when the gap size was reduced from 6 to 1 nt in the absence of a 5′-PO4 (Table I; compare gap-6 with gap-1). Most striking, however, was the dramatic effect of 5′-phosphorylation on the 1-nt gapped substrate, where addition of a 5′-PO4 resulted in a 500-fold increase in catalytic efficiency (compare gap-1 to P-gap-1). Thus, the relative catalytic efficiencies of pol β on the different DNA substrates were P-gap-1 ≫ gap-1 ≈ P-gap-6 > gap-6 ≈ recessed. pol β was some 10,000 and 2,500 times more efficient on P-gap-1 than on the gap-6 and recessed DNA substrates, respectively. As noted above, this increase in catalytic efficiency resulted primarily from a decrease in K m,dCTPapp, although k cat values were also slightly higher on P-gap-1. The k cat value of 0.6 s−1 observed on the recessed DNA substrate is very similar to the value of 0.3 s−1 reported for a different pol β preparation on a different recessed primer-template (24Werneburg B.G. Ahn J. Zhong X. Hondal R.J. Kraynov V.S. Tsai M.D. Biochemistry. 1996; 35: 7041-7050Crossref PubMed Scopus (134) Google Scholar). The nucleotide insertion fidelity of pol β was determined on the same series of DNA substrates using a "standing start" (22Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (403) Google Scholar) kinetic fidelity assay (Fig. 3 and Table II). The frequencies of nucleotide misinsertions opposite the template G21 residue were similar for all substrates except P-gap-1. The fidelity of pol β on P-gap-1 was 100, 50, and 30 times higher for G-T, G-G, and G-A mispair formation, respectively, compared with the recessed substrate. G-T and G-A mispairs were formed ∼10-fold more readily than G-G mispairs on all of the DNA substrates studied.Table IIEffect of DNA substrate structure on pol β fidelity2-a Sequences and structures of the DNA substrates are shown in Fig. 1.2-b Values in parentheses indicate the number of independent experiments used for each analysis. Open table in a new tab 2-a Sequences and structures of the DNA substrates are shown in Fig. 1. 2-b Values in parentheses indicate the number of independent experiments used for each analysis. pol β plays a central role in mammalian short-patch BER (1Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 2Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (784) Google Scholar, 3Nealon K. Nicholl I.D. Kenny M.K. Nucleic Acids Res. 1996; 24: 3763-3770Crossref PubMed Scopus (59) Google Scholar, 4Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (662) Google Scholar, 5Friedberg E.C. Wood R.D. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 249-269Google Scholar). This suggests that a preferred substrate for pol β might be 5′-phosphorylated 1-nt gapped DNA. We examined the DNA substrate preferences of purified rat pol β in steady-state kinetic assays using synthetic DNAs of different structure. We show that pol β prefers 5′-phosphorylated 1-nt gapped DNA as substrate with relative catalytic efficiencies on P-gap-1 ≫ gap-1 ≈ P-gap-6 > gap-6 ≈ recessed (Table I). The efficiency of pol β on P-gap-1 DNA was 500–10,000 times higher than on the other DNA substrates examined. We also observed that the frequency of nucleotide misinsertion by pol β was 10–100-fold lower on P-gap-1 compared with the other DNA substrates (Table II). Singhal and Wilson (17Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar) showed that pol β switches from a distributive to a processive mode of DNA polymerization on short-gapped (2–6 nt) DNA substrates but only if the 5′-margin of the gap is phosphorylated. The very similar effects observed in our processivity experiments using different oligonucleotides (Fig. 2) indicate that this is an intrinsic property of pol β that has no obvious requirement for specific template sequences. Our steady-state kinetic analyses show that the catalytic efficiency and nucleotide insertion fidelity of pol β are also influenced by gap size and 5′-phosphorylation. Moreover, in contrast to what is obserbed for pol β binding to DNA (18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar), 5′-phosphorylation is required for both high catalytic efficiency and increased fidelity on 1-nt gapped DNA (TablesI and II). These data extend the model of Prasad et al. (18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar) by showing that 5′-PO4 residues must mediate a productive catalytic interaction between pol β and DNA even in 1-nt gaps. The relative low fidelity of pol β observed on the recessed primer-template (f ins = 10−3–10−4; Table II) is comparable with that reported by others on recessed DNA substrates (21Boosalis M.S. Mosbaugh D.W. Hamatake R. Sugino A. Kunkel T.A. Goodman M.F. J. Biol. Chem. 1989; 264: 11360-11366Abstract Full Text PDF PubMed Google Scholar, 23Roberts J.D. Kunkel T.A. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 217-247Google Scholar, 24Werneburg B.G. Ahn J. Zhong X. Hondal R.J. Kraynov V.S. Tsai M.D. Biochemistry. 1996; 35: 7041-7050Crossref PubMed Scopus (134) Google Scholar). However, our observation of similar fidelities on recessed and P-gap-6 DNAs appears to conflict with recent reports suggesting that pol β is less faithful during 5- and 6-nt gap-filling synthesis (17Singhal R.K. Wilson S.H. J. Biol. Chem. 1993; 268: 15906-15911Abstract Full Text PDF PubMed Google Scholar, 25Beard W.A. Osheroff W.P. Prasad R. Sawaya M.R. Jaju M. Wood T.G. Kraut J. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12141-12144Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). This apparent discrepancy may relate to the overall higher catalytic efficiency of pol β on phosphorylated short-gapped DNA (Table I), to template sequence effects, and/or to differences in the assays used to measure fidelity. Additional experiments are required to resolve this. Regardless, our data showing increased fidelity on P-gap-1 DNA indicate that pol β, and by inference BER, may be less error prone than once thought. Several mechanisms may contribute to the observed effects of gap structure on catalytic efficiency and fidelity. Based on the binding studies of Prasad et al. (18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar), it appears that the differences in catalytic efficiency on P-gap-1 and gap-1 are not due to differences in the levels of stable DNA binding (at least for pol β-DNA binary complexes detected by cross-linking and competition assays). An attractive general hypothesis is that the 5′-PO4 in a 1-nt gap somehow facilitates formation of a catalytically optimal pol β-DNA complex without affecting overall binding affinity. Amino acid changes at residues distant from the polymerase active site of pol β were recently shown to affect the fidelity of DNA synthesis (19Washington S.L. Yoon M.S. Chagovetz A.M. Li S.X. Clairmont C.A. Preston B.D. Eckert K.A. Sweasy J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1321-1326Crossref PubMed Scopus (55) Google Scholar). This indicates that molecular events at the active site respond to long range changes in the pol β protein. Thus, interactions between the DNA 5′-PO4 and the 8-kDa domain of pol β, which also occur at some distance from the active site (7Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12742-12761Crossref PubMed Scopus (273) Google Scholar), may remotely alter dNTP binding and/or protein conformational changes required for chemical catalysis (24Werneburg B.G. Ahn J. Zhong X. Hondal R.J. Kraynov V.S. Tsai M.D. Biochemistry. 1996; 35: 7041-7050Crossref PubMed Scopus (134) Google Scholar, 26Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (507) Google Scholar). Additional kinetic and structural studies will be required to delineate the contribution of these and other mechanisms to pol β substrate recognition and catalytic efficiency. It is particularly germane to examine the role of the 8-kDa domain in directing the interaction of pol β with P-gap-1 DNAs (7Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12742-12761Crossref PubMed Scopus (273) Google Scholar, 18Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar). In summary, we show that purified pol β exhibits relative high catalytic efficiency and fidelity on 5′-phosphorylated 1-nt gapped DNAin vitro. This suggests that a primary biochemical function of pol β in the mammalian cell is to fill 5′-phosphorylated 1-nt gaps. Gaps with this structure appear to be requisite intermediates in short-patch BER (1Singhal R.K. Prasad R. 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