INTRODUCTION

DNA polymerase  (pol )¹ plays a central role in mammalian base excision repair (BER (1-5)). pol  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 (6, 7). The presence of both polymerase and lyase activities suggests that pol  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 (4, 6). pol  has also been implicated in "long-patch" BER (4) and may function in meiosis (8) and nucleotide excision repair (9, 10).

The biochemical activities of purified pol  are consistent with a role in gap-filling DNA synthesis. Early studies showed that pol  is non-processive on single-stranded DNA templates, prefers short-gapped DNA substrates, and is capable of filling gaps to completion (11-16). More recently, Wilson and colleagues (17) observed that pol  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  to these short-gapped substrates is also strongly enhanced by the presence of a 5-PO₄ (18). These experiments, together with recent structural data, suggest a model in which pol  binding to gapped DNA is mediated by interactions between the 8-kDa domain of pol  and the 5-PO₄ at the downstream margin of the gap (7, 18). Processive DNA synthesis on short (2-6-nt) gaps is consistent with roles for pol  in long-patch BER (4) and in the completion of gap-filling synthesis initiated by other cellular DNA polymerases (9, 10, 14-16).

Although DNA polymerization by pol  on single-stranded and short-gapped DNAs is understood in some detail, much less is known about pol  activity on its short-patch BER substrate, 1-nt gapped DNA. The model of pol  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 (18). This suggests that pol  may interact with 1-nt gapped DNA by a distinct mechanism.

To better characterize the parameters governing pol  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,dNTP^app) and nucleotide insertion fidelity of pol  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  DNA binding and provide biochemical evidence that 5-phosphorylated 1-nt gapped DNA is the preferred substrate for pol .

EXPERIMENTAL PROCEDURES

Recombinant rat DNA polymerase  was purified as described previously (19). All oligonucleotides were synthesized and high pressure liquid chromatography-purified by Operon Technologies. 5-³²P Labeling of the primers was performed with [-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 [-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 and saturating concentrations of dNTPs (data not shown).

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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 , 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  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 (20), 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_cat and K_m,dNTP^app values were determined using a non-linear curve fitting program (SigmaPlot). For mispairs, k_cat/K_m,dNTP^app values were determined from the initial slopes of Michaelis-Menten plots (20, 21), and the frequencies of misincorporation were calculated as described (22). To detect extension products resulting from dNMP misincorporation, it was often necessary to increase pol  concentrations and/or incubation times. As expected in steady state (22), V_max values were directly proportional to enzyme concentration (data not shown).

The processivity of pol  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  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.

RESULTS

To examine pol  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 X174 DNA used in previous fidelity studies (Refs. 20 and 23, 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 X174 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. 1A). Two gapped substrates were also constructed (Fig. 1, B and C). 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. 1B). A similar substrate with a 1-nt gap (designated gap-1) was constructed by hybridizing a 20-mer oligonucleotide to template residues 1-20 (Fig. 1C). Variants of gap-6 and gap-1 containing PO₄ moieties at the 5 margins of the gaps (designated P-gap-6 and P-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  on these DNA substrates. This approach is based on the observation of Singhal and Wilson (17) that pol  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 (17), although synthesis on our phosphorylated 6-nt gapped substrate was not strictly processive (Fig. 2, right).

Processivity of pol  on recessed (left), gap-6 (middle), and P-gap-6 (right) DNA substrates.

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Effect of DNA Substrate Structure on pol  Catalytic Efficiency

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,dCTP^app and k_cat values of 170 µM and 0.6 s¹ observed on the recessed primer-template are comparable with those reported by others for pol  (21, 24). We noted, however, that the K_m,dNTP^app values for pol  (40-170 µM; Table I and Ref. 21) 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 µM²; Refs. 20-22, and references therein). The unusually high K_m,dCTP^app values observed on the recessed and gap-6 primer-templates suggested that these DNAs were relatively poor substrates for pol .

Gel assay of nucleotide insertion kinetics by pol .

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Table I. Effect of DNA substrate structure on pol  catalytic efficiency Kinetic experiments were run, quantified, and analyzed as outlined under "Experimental Procedures." Fig. 3 shows representative gels used for analysis. a Sequences and structures of the DNA substrates are shown in Fig. 1. b Calculated using total pol  protein concentration. 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. 17) and DNA binding (18), 5-phosphorylation of gap-6 resulted in a modest reduction in K_m,dCTP^app and concomitant increase in overall catalytic efficiency (k_cat/K_m,dCTP^app). A similar decrease in K_m,dCTP^app 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  on the different DNA substrates were P-gap-1  gap-1  P-gap-6 > gap-6  recessed. pol  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,dCTP^app, although k_cat values were also slightly higher on P-gap-1. The k_cat value of 0.6 s¹ observed on the recessed DNA substrate is very similar to the value of 0.3 s¹ reported for a different pol  preparation on a different recessed primer-template (24).

Effect of DNA Substrate Structure on pol  Fidelity

The nucleotide insertion fidelity of pol  was determined on the same series of DNA substrates using a "standing start" (22) 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  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 II. Effect of DNA substrate structure on pol  fidelity Experiments were run as described under "Experimental Procedures." Fig. 3 shows representative gels used for analysis. Mispair formation frequencies were calculated from the initial slopes of Michaelis-Menten curves using the formula: fins = (kcat/Km,dNTPapp)incorrect/(kcat/Km,dNTPapp)correct (21,22), where "correct" corresponds to extension in the presence of dCTP to form the G-C base pair (Table I). a Sequences and structures of the DNA substrates are shown in Fig. 1. b Values in parentheses indicate the number of independent experiments used for each analysis.

DISCUSSION

pol  plays a central role in mammalian short-patch BER (1-5). This suggests that a preferred substrate for pol  might be 5-phosphorylated 1-nt gapped DNA. We examined the DNA substrate preferences of purified rat pol  in steady-state kinetic assays using synthetic DNAs of different structure. We show that pol  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  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  was 10-100-fold lower on P-gap-1 compared with the other DNA substrates (Table II).

Singhal and Wilson (17) showed that pol  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  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  are also influenced by gap size and 5-phosphorylation. Moreover, in contrast to what is obserbed for pol  binding to DNA (18), 5-phosphorylation is required for both high catalytic efficiency and increased fidelity on 1-nt gapped DNA (Tables I and II). These data extend the model of Prasad et al. (18) by showing that 5-PO₄ residues must mediate a productive catalytic interaction between pol  and DNA even in 1-nt gaps.

The relative low fidelity of pol  observed on the recessed primer-template (f_ins = 10³-10⁴; Table II) is comparable with that reported by others on recessed DNA substrates (21, 23, 24). However, our observation of similar fidelities on recessed and P-gap-6 DNAs appears to conflict with recent reports suggesting that pol  is less faithful during 5- and 6-nt gap-filling synthesis (17, 25). This apparent discrepancy may relate to the overall higher catalytic efficiency of pol  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 , 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. (18), 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 -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 -DNA complex without affecting overall binding affinity. Amino acid changes at residues distant from the polymerase active site of pol  were recently shown to affect the fidelity of DNA synthesis (19). This indicates that molecular events at the active site respond to long range changes in the pol  protein. Thus, interactions between the DNA 5-PO₄ and the 8-kDa domain of pol , which also occur at some distance from the active site (7), may remotely alter dNTP binding and/or protein conformational changes required for chemical catalysis (24, 26). Additional kinetic and structural studies will be required to delineate the contribution of these and other mechanisms to pol  substrate recognition and catalytic efficiency. It is particularly germane to examine the role of the 8-kDa domain in directing the interaction of pol  with P-gap-1 DNAs (7, 18).

In summary, we show that purified pol  exhibits relative high catalytic efficiency and fidelity on 5-phosphorylated 1-nt gapped DNA in vitro. This suggests that a primary biochemical function of pol  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 (1-4) and may exist in other pathways where involvement of pol  is implicated (4, 8-10).