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J. Biol. Chem., Vol. 275, Issue 23, 17677-17682, June 9, 2000

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Fidelity of Eucaryotic DNA Polymerase  Holoenzyme from Schizosaccharomyces pombe^*

Xiluo Chen, Shaojun Zuo^§, Zvi Kelman^§, Mike O'Donnell^¶, Jerard Hurwitz^§, and Myron F. Goodman^**

From the  Department of Biological Sciences and Chemistry, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, ^¶ Rockefeller University and Howard Hughes Medical Institute, New York, New York 10021, and ^§ Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, December 27, 1999, and in revised form, March 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The fidelity of Schizosaccharomyces pombe DNA polymerase  was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol  alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol  holoenzyme (HE) (pol  + PCNA + RFC) was 4.6 × 10⁴ for T·G mispairs, 5.3 × 10⁵ for G·G mispairs, and 4.5 × 10⁶ for A·G mispairs. The T·G misincorporation frequency for pol  without the accessory proteins was unchanged. The fidelity of pol  HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol  HE with E. coli pol III HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The enzymes principally responsible for catalyzing procaryotic and eucaryotic DNA replication share many common elements. Eucaryotic DNA polymerases  and  have the ability to proofread replication errors using pol¹-associated 3'  5'-exonuclease activity (1, 2), a property shared with Escherichia coli pols I-III (3). Each of these enzymes copies DNA with extremely low processivity, typically adding less than 30 nt before dissociating. There are closely analogous groups of eucaryotic and procaryotic polymerase accessory proteins that interact with the non-processive core pols, forming highly processive polymerase holoenzymes. E. coli pol III HE and pol II HE bind to the  dimeric sliding clamp (4-6), whereas eucaryotic pol  HE and pol  HE bind to the PCNA trimeric sliding clamp (7). The processivity clamps are loaded on and off the DNA by clamp loading complexes,  complex in E. coli (8-11) and RFC in eucaryotic cells (7, 12-14).

Extensive studies on the fidelity properties of core DNA polymerases have been reported over the past 3 decades focusing on biochemical and kinetic analysis of deoxynucleotide insertion specificity and the reduction in pol-generated errors by proofreading exonucleases (15-18), whereas there are but a paucity of experiments reporting on the fidelity properties of the more biologically relevant pol HE systems. Previous experiments employing pol HE systems have attempted to probe the fidelity of leading versus lagging strand synthesis using mutational reporter sequences (e.g. lacZ) (19, 20) and to visualize synthesis past DNA damage sites using two-dimensional gel electrophoresis (21, 22). We have recently generalized a gel kinetic assay originally designed to measure polymerase fidelity in the absence of proofreading (23, 24), enabling fidelity measurements to be made at arbitrary p/t DNA sites in the presence of proofreading and pol accessory proteins (25-27).

There are a variety of questions regarding the fidelity properties of holoenzymes that can be investigated systematically using gel kinetic methodology. Recently, measurements on the fidelity of calf thymus pol  were made in the presence and absence of PCNA at normal (28) and abasic (29) template sites. In this paper, we report on the base substitution error rate of the Schizosaccharomyces pombe pol  HE and core for comparison with data with the E. coli pol III HE-catalyzed error rates (26) determined in the same sequence context.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Proteins cloned S. pombe pol , PCNA, and RFC were purified as described (30). The enzyme reaction buffer contained 40 mM Tris·HCl, pH 7.8, 170 µg/ml bovine serum albumin, 0.5 mM dithiothreitol, and 7 mM MgCl₂. Bacteriophage T4 polynucleotide kinase was purchased from United States Biochemical Corp. or Amersham Pharmacia Biotech. T4 DNA ligase was purchased from Promega. E. coli single strand DNA-binding protein and bovine serum albumin were purchased from Amersham Pharmacia Biotech.

DNA Substrates-- The p/t DNA was made up of a synthetic 100-mer template annealed to complementary 30- or 35-mer primers or to a 35-mer primer containing a single noncomplementary base at its 3'-end. The 30-mer primer was annealed at the middle of the template leaving equal length (35 nt) ssDNA overhangs on each side. The matched 35-mer primer was annealed to the template leaving 35 nt of ssDNA at the 3'-end of the template and 30 nt of ssDNA at 5'-end. The mismatched 35-mer primer was identical to the matched 35-mer except that the nucleotide at the 3'-end contained an A in place of C. All oligomers were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer (Perkin-Elmer) and gel-purified. The 100-mer was synthesized as two half-length oligomers and then ligated together.

The sequences for the 30-mer primer/100-mer template were as follows:

View larger version (12K): [in this window] [in a new window]   SEQUENCE 1

where G is the target site where misincorporation frequencies were measured.

The sequences for the matched 35-mer primer/100-mer template were as follows:

View larger version (13K): [in this window] [in a new window]   SEQUENCE 2

The sequences of the mismatched 35-mer primer/100-mer template differed only in that A replaced C at the primer-3'-end.

Nucleotides-- dNTP substrates were purchased from Amersham Pharmacia Biotech. [-³²P]ATP (4500 Ci/mmol) was purchased from ICN Radiochemicals.

Methods

The primer was 5'-end-labeled with ³²P using T4 polynucleotide kinase in enzyme reaction buffer at 37 °C for 60 min. p/t DNA was annealed in enzyme reaction buffer using a ratio of 1 primer to 1.2 templates by heating to 90 °C and gradually cooling to room temperature. The concentration of p/t DNA after annealing was 100 nM (primer termini).

Assay for 3'-Exonuclease Activity of S. pombe pol -- 10 nM either matched or mismatched 35/100-mer DNA were incubated at 37 °C with 10 µg/ml (0.2 unit/µl) S. pombe pol  in reaction buffer in the presence and absence of all 4 dNTPs (0.5 mM each if present) in separate reactions containing 20 µl. One unit of pol  supports the incorporation of 1 nmol of dTMP under the conditions specified above. The 35-mer primers were 5'-end-³²P-labeled. The mismatched 35-mer primer (10 nM) was used as single-stranded DNA substrate and incubated at 37 °C with 10 µg/ml (0.2 unit/µl) S. pombe pol  in reaction buffer (20 µl). Aliquots (4 µl) were removed from each reaction and quenched by mixing with 10 µl of 20 mM EDTA, 95% formamide at different time points. Reaction products were separated on a 12% denaturing polyacrylamide gel run for 2 h at 2,000 V. The amount of primer extension catalyzed by pol  (gel bands above the primer band) or degradation catalyzed by pol  3'-exonuclease activity (gel bands below the primer band) was measured as percentage of total gel band intensity in each lane using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Processive Synthesis on 30/100-Mer p/t DNA-- p/t DNA was preincubated at 37 °C with different combinations of ATP and accessory proteins PCNA, RFC, and SSB in enzyme reaction buffer (16 µl) containing glycerol (4%) for 1 min. Following preincubation of enzyme reaction buffer (4 µl) containing glycerol (4%), the four dNTPs and pol  were added to initiate the reaction. Aliquots (4 µl) were removed at different times and mixed with formamide/EDTA (10 µl) to quench the reaction. Final substrate concentrations in the reactions were p/t DNA (10 nM), pol  (0.5 µg/ml), dATP (0.5 mM), dCTP (0.5 mM), dGTP (0.5 mM), dTTP (0.5 mM), ATP, and the accessory proteins, if present as follows: ATP (1 mM), RFC (54 µg/ml), (PCNA)₃ (90 nM), SSB (320 nM). Aliquots (4 µl) were removed from each reaction at different times and quenched by mixing with formamide/EDTA (10 µl).

Gel Kinetic Fidelity Analysis-- A gel fidelity assay was used to determine the kinetics of incorporation of each of the four dNTPs opposite the target site (26, 31). Primer extension reactions for pol  in the absence of processivity accessory proteins were performed as follows. p/t DNA and the dNTP to be incorporated opposite the target were first mixed together in the reaction buffer. A mixture of S. pombe pol  and the running-start nt (dATP) in the same reaction buffer was then added to initiate the reaction. Reactions were run for 5 min to measure the incorporation of dCMP opposite G and for 20 min to measure the misincorporation of dTMP opposite G. The assay conditions for correct incorporation satisfied single-completed hit conditions, whereby most of the p/t DNA molecules that undergo extension encounter a polymerase only once (27, 31). However, multiple hit conditions were required to detect dTMP·G misincorporations for pol  in the absence of the processivity factors, and the minor modifications required to analyze properly multiple encounter kinetics were made as described in Ref. 27. The final concentrations were 1 mM dATP, 10 nM p/t DNA, 1.0 µg/ml S. pombe pol , and dNTP concentrations as indicated in the figures. Control reactions were run for 5 min using just the running-start dATP to verify that misincorporation of the running-start nt opposite the target G site did not occur.

Primer extension reactions for pol  in the presence of processivity accessory proteins were performed as follows. Solution A contained 33 nM p/t DNA, 150 µg/ml RFC, 270 nM (PCNA)₃, 1 µM SSB, 2 mM ATP, and 4% glycerol in enzyme reaction buffer. Solution B consisted of the enzyme reaction buffer containing various concentrations of the dNTP to be incorporated opposite target site. Solution C contained 0.5 µg/ml pol , running start dATP (188 µM), and 4% glycerol in the enzyme reaction buffer. The reaction was performed as follows: solution A (3 µl) was mixed with solution B (3 µl) and incubated at 37 °C for 1 min to allow RFC to load PCNA onto the DNA; then solution C (4 µl) was added to the mixture of A + B to initiate the primer extension reaction. The final concentrations in the 10-µl reaction mixture were 10 nM p/t DNA, 0.2 µg/ml pol , 75 µM dATP, 45 µg/ml RFC, 80 nM (PCNA)₃, 300 nM SSB, 0.6 mM ATP, and various concentrations of dNTP for incorporation opposite the target site. Control reactions were run with the running-start dATP only to ensure that it did not misincorporate opposite G. The reactions, run at 37 °C for 2 min for both correct incorporation and misincorporations opposite G, approximately satisfied single-completed hit conditions, in which about 20% of the primers were extended, so that no further corrections were required in the kinetic analysis. Reactions were quenched by addition of formamide/EDTA (20 µl) to the reaction mixture. The samples were heated to 100 °C for 6 min, placed on ice for 3 min, and then loaded on a 16% polyacrylamide denaturing gel. The gel was run at 2000 V for 4 h to separate reaction products.

Integrated polyacrylamide gel band intensities were measured on a PhosphorImager using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The nucleotide incorporation efficiency opposite the target site was obtained by measuring I_T/I_T1, where I_T is the integrated gel band intensities of primers extended to the target site and beyond, and I_T1 is the integrated gel band intensity of primers extended to the site just prior to the target site (26, 31).

A plot of the relative incorporation rate, I_T/I_T1 as a function of the dNTP substrate concentration, results in a rectangular hyperbola whose slope in the initial linear region is the apparent V_max/K_m. Apparent K_m and relative V_max values were obtained using a least squares fit to a rectangular hyperbola. The relative V_max value is equal to the maximum value of I_T/I_T1. In reactions where misincorporation opposite the target site was relatively inefficient, a plot I_T/I_T1 versus dNTP concentration showed little or no curvature, and apparent V_max/K_m values were obtained by a least squares fit of the data to a straight line. Apparent V_max/K_m values that were obtained under multiple-completed hit conditions were corrected to single-completed hit conditions as described in Ref. 27, but these corrections were essentially negligible. The misincorporation efficiency, f_inc, which is the inverse of the fidelity, is given by the ratio shown in Equation 1,

(Eq. 1)

where the subscripts W and R refer to wrong and right incorporations, respectively. Measurement errors for V_max/K_m are ± 30% and for f_inc are ± 40% (1 S.D.).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

pol , thought to be one of the principal eucaryotic replication polymerases (32), forms a processive holoenzyme complex in the presence of the trimeric PCNA sliding clamp and RFC, which is responsible for loading PCNA onto DNA (7). Eucaryotic pol  is analogous to E. coli pol III core which uses both the  sliding clamp and the clamp loading  complex to form the highly processive pol III HE. In this study, we have measured the fidelity of S. pombe pol  HE in the same sequence context that was used previously to study the fidelity of E. coli pol III HE (26). Although similar in some respects, the biochemical properties of the E. coli and S. pombe pol holoenzymes revealed several unanticipated differences, particularly with respect to the contribution of proofreading to fidelity.

A 30/100-mer p/t DNA Serves as a "Minimal" Substrate Supporting Processive Synthesis by S. pombe pol  HE-- We chose to measure the fidelity of S. pombe pol  HE when copying a synthetic 100-mer DNA template. The advantage of using relatively short synthetic oligonucleotide minimal p/t DNA is that pol fidelity can be measured at arbitrary template target sites in defined sequence contexts that can be easily varied. However, before such a system can be used, it is necessary to show that it recapitulates the properties observed with much longer biological substrates, e.g. SV40 DNA (33).

A time course showing extension of a ³²P-labeled primer is arranged in seven groups of lanes to test the effects of PCNA, RFC, and SSB on pol  processivity (Fig. 1). pol  copied the synthetic 100-mer DNA template in a completely distributive manner in the absence of PCNA (Fig. 1, groups 1 and 3). A marked stimulation in pol  processivity occurred in the presence of PCNA, PCNA + RFC, or PCNA + RFC + SSB (Fig. 1, group 2, group 4, and group 5, respectively). The observation that PCNA stimulated pol  synthesis in the absence of RFC suggests that the processivity clamp can load onto the short DNA by itself and stabilize the pol -p/t DNA complex. Threading of PCNA onto linear DNA in the absence of RFC has been reported previously for Saccharomyces cerevisiae PCNA (34). We observed that SSB strongly inhibited synthesis by pol  in the presence of PCNA (Fig. 1, group 6), but processive synthesis was restored by the addition of RFC (Fig. 1, group 5), clearly demonstrating that RFC was active in the assay.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Effects of PCNA, RFC, SSB on DNA synthesis by S. pombe pol  on a 30-mer primer/100-mer template. Primer extension reactions were run with pol , 4 dNTPs, and indicated (±) proteins and ATP on a 30/100-mer DNA. Four lanes in each group represent reaction products of 4 time points of 3, 8, 18, and 40 min. The left-hand lane, designated as 0, contained the band corresponding to the unextended 32P-labeled primer. Final concentrations in the reactions were p/t DNA (10 nM), pol  (0.5 µg/ml), dATP (0.5 mM), dCTP (0.5 mM), dGTP (0.5 mM), dTTP (0.5 mM); and the accessory proteins and ATP, if present, were as follows: ATP (1.0 mM), RFC (54 µg/ml), (PCNA)3 (90 nM), and SSB (320 nM).

The apparent stimulation of the RFC-dependent reaction by SSB (compare Fig. 1, groups 4 and 5) was caused most likely by the inhibition of a 3'-exonuclease contaminant present in our purified preparation of RFC. This adventitious 3'-exonuclease appeared to digest the primer extension products causing a uniform reduction in the gel band intensities in group 4 bands relative to either groups 5 or 2, while maintaining similar processivity patterns for these three groups.

The assays shown in Fig. 1 were performed in the presence of ATP (1 mM) required for RFC-mediated loading of PCNA onto p/t DNA (12, 35-37). Further characterization of the effects of PCNA and RFC on pol  synthesis was carried out by performing similar primer elongation experiments in the absence of ATP. We found that processive synthesis observed in the presence of PCNA (Fig. 1, group 2) was retained in the absence of ATP (data not shown), whereas ATP must be present to observe processive synthesis in presence of PCNA, RFC, and SSB (Fig. 1, groups 5 and 7). We conclude the following: (i) ATP is required for loading of PCNA onto DNA by RFC but that PCNA can also "thread" itself onto short linear p/t DNA in the absence of RFC, in a "passive" reaction not requiring ATP; (ii) SSB significantly inhibits the ATP-independent passive loading reaction but does not affect loading of PCNA by RFC.

S. pombe pol  Base Substitution Fidelity-- The nucleotide misincorporation value, f_inc, which is the reciprocal of the fidelity, was determined using a gel kinetic assay suitable for measuring fidelity in the presence of proofreading and polymerase processivity proteins (27, 31). The assay measured the relative rates of incorporating either a right (R) or wrong (W) nucleotide opposite a template target site base. Integrated gel band intensities corresponding to primers extended opposite a template target site and beyond were compared with extended primers terminating 1 base before the target site and were plotted as a function of dRTP and dWTP substrate concentrations to determine V_max/K_m values, in accordance with Equation 1 (27, 31) (see "Experimental Procedures").

Base substitution fidelity measurements were performed using pol  alone (Fig. 2) and pol  HE, i.e. pol  + PCNA, RFC, and SSB (Fig. 3). A ³²P-labeled primer was extended by incorporation of four running-start As prior to reaching the template target site G where fidelity was measured. A sketch of the p/t DNA sequence is shown at the top of Fig. 2.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Fidelity of S. pombe pol . Primer extension reactions were carried out with dATP (1 mM), p/t DNA (10 nM), S. pombe pol  (1.0 µg/ml), and various concentrations of dCTP or dTTP. Reactions with dCTP and dTTP were incubated for 5 and 20 min, respectively. A running-start dATP (1 mM) control reaction was run for 5 min, in the absence of a target dNTP substrate. The unextended 32P-labeled primer band is shown at the left-hand side of the gel. A sketch of the p/t DNA is shown at the top of the figure. Gel band intensities were measured using a PhosphorImager, and their ratio IT/IT1 was plotted versus dNTP concentration. The misincorporation efficiency finc was computed using Equation 1 (see "Experimental Procedures"). The template sequence corresponding to individual primer extension bands is indicated at the right-hand side of the gel.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Fidelity of S. pombe pol  HE. Primer extension reactions were carried out with dATP (75 µM), p/t DNA (10 nM), S. pombe pol  (0.2 µg/ml), RFC (45 µg/ml), (PCNA)3 (80 nM), SSB (300 nM), ATP (0.6 mM), and various concentrations of dNTP for incorporation opposite the target site. All reactions were run for 2 min. A running-start dATP (75 µM) control reaction was incubated for 2 min, in the absence of a target dNTP substrate. The unextended 32P-labeled primer band is shown at the left-hand side of the gel. A sketch of the p/t DNA is shown at the top of the figure. Gel band intensities were measured using a PhosphorImager and their ratio IT/IT1 was plotted versus dNTP concentration. The misincorporation efficiency finc was computed using Equation 1 (see "Experimental Procedures"). The template sequence corresponding to individual primer extension bands is indicated at the right-hand side of the gel. The dNTP concentration values have been rounded to two significant figures.

pol  incorporated four running-start As and then C opposite the target G in a thoroughly distributive manner in the absence of the processivity proteins (Fig. 2, dCTP lanes). The appearance of faint primer extension bands corresponding to misincorporation of T opposite G allowed us to compute a T·G misincorporation ratio of 5.6 × 10⁴ (Fig. 2, dTTP lanes). G·G and A·G misincorporations were not detectable (data not shown).

Each of these misincorporations was, however, readily detected for the pol  HE (Fig. 3), which synthesized DNA processively (compare Figs. 3 and 2). Note the presence of the high intensity primer extension bands terminating opposite the target site G and continuing further downstream (Fig. 3). The T·G error rate of 4.6 × 10⁴ was similar to that for pol  alone, suggesting that the processivity proteins have essentially no effect on the fidelity for this mispair. The error rates for G·G and A·G mispairs were 5.3 × 10⁵ and 4.5 × 10⁶, respectively.

It is important to emphasize that the inability to detect G·G and A·G misincorporations in the absence of PCNA, RFC, and SSB does not imply that the 4-subunit pol  core has higher fidelity than pol  HE. Rather, the absence of target site misincorporation bands for the case of pol  alone was caused by the distributive nature of the enzyme. The residence time on the p/t DNA was simply too short to allow pol  to catalyze the most difficult misincorporation events during a single pol-p/t DNA encounter. The data indicated that the apparent K_m values for incorporation of C opposite G were 72 µM for pol  and 1.6 µM for pol  HE (Figs. 2 and 3). The K_m values for misincorporation of T opposite G were ~6900 µM for pol  and only 370 µM for pol  HE (Figs. 2 and 3). The observation that the apparent K_m values were much higher for both right (C·G) and wrong (T·G) incorporations for pol  alone is consistent with reduced processivity. Thus, a much higher concentration of dNTPs is required to attain one-half V_max when the pol -p/tDNA dissociation occurs rapidly, as is the case for a highly distributive synthesis. Because f_inc (Equation 1) is expressed as the ratio of V_max/K_m for wrong versus right incorporations, the sensitivity of the gel kinetic assay is reduced in proportion to the reduction in (V_max/K_m)_R for pol  in the absence of processivity factors. Thus, the assay is roughly 100-fold more sensitive for pol  HE, enabling detection of misincorporation ratios on the order of about 10⁶ to 10⁷.

S. pombe pol -associated 3'  5'-Exonuclease Activity Is a Weak Proofreader-- We measured 3'  5' proofreading exonuclease activity for the 4-subunit pol  core under synthesizing and non-synthesizing conditions on p/t DNA, using either matched or mismatched primer-3'-ends (Fig. 4A). Excision of the primer-3'-end containing an A·G mismatched base pair occurred more rapidly than removal of a C·G correctly matched pair both in the presence and absence of dNTP substrates. However, the pol  exonuclease activity appeared extremely weak. Removal of a terminal A·G mismatch was detectable in a 3-min incubation in either the presence or absence of dNTP substrates (Fig. 4A), whereas in the presence of dNTPs, a low level of incorporation of a next correct dGMP·C onto an A·G mismatched base pair was observed in a 7-min incubation.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   3'  5'-exonuclease activity of S. pombe pol . A, 10 nM each of matched (left) and mismatched (right) 35/100-mer DNA were incubated at 37 °C with S. pombe pol  (10 µg/ml), for the times indicated, in reaction buffer in the presence or absence of 4 dNTPs. The lane designated as 0 contains the unextended primer. B, 35-mer primer ssDNA (10 nM) was incubated at 37 °C with S. pombe pol  (10 µg/ml) in the absence of dNTP substrates. The left-hand lane designated as 0 contains the band corresponding to the unextended 32P-labeled primer.

The S. pombe pol  exonuclease-to-polymerase ratio, is about 1 to 30. That is, the rate of extending a correct dCMP·G terminus is roughly 30 times greater than the rate of removal of a dAMP·G mismatched terminus in the absence of dNTP substrates. Indeed, the excision of dAMP from a terminal A·G mispair was remarkably inefficient with greater than 90% of the input p/t DNA remaining following a 40-min reaction. In contrast, degradation of ssDNA occurred more rapidly than p/t DNA (Fig. 4B). The degradation reaction appeared to be distributive, showing removal of about 6 nt during a 6-min reaction.

The extremely low nuclease/polymerase ratio suggests that 3'-exonuclease of the pol  may not be effective in eliminating nucleotide substitution errors. We tested this supposition by measuring f_inc (dTMP·G) for pol  HE at different concentrations of the next correct dGTP substrate. We found no measurable change in the T·G misincorporation ratios (f_inc = 4.6 × 10⁴) for pol , when varying dGTP concentrations between 0 and 160 µM (data not shown). Since a decrease in fidelity with increasing next correct dNTP concentration is a well established hallmark of proofreading (38, 39), the absence of a dependence of fidelity on dNTP concentration implies that the 3'-exonuclease of pol  may not be effective in eliminating polymerase-catalyzed base substitution errors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

pol  is believed to be the primary replicative enzyme in eucaryotic cells responsible for carrying out processive DNA synthesis in the presence of PCNA, RFC, and RPA (32). Despite the importance of this enzyme, little is known regarding its fidelity properties in vitro and in vivo. In this paper, we have used a gel kinetic assay (24, 26, 31) to measure fidelity at an arbitrary template G site using the pol  HE purified from S. pombe (30).

Processive Synthesis by pol  Using a Synthetic p/t DNA Oligomer-- It is convenient to synthesize relatively short DNA templates to investigate DNA polymerase fidelity using defined sequence contexts. However, prior to performing a fidelity analysis using S. pombe pol  HE on a 30/100-mer p/t DNA, it was necessary to demonstrate that the PCNA sliding clamp stimulated pol  processivity, dependent on the presence of RFC and ATP, since the presence of ATP is required for loading of PCNA onto DNA by the RFC clamp loading complex (12, 35-37). This requirement is potentially important because PCNA can also diffuse onto linear but not circular DNA in the absence of RFC and ATP (34).

Synthesis by pol  alone was distributive on the 30/100-mer p/t DNA with the addition of about 6 nt following a 3-min reaction and increasing to just 7 nt at 8, 18, and 40 min (Fig. 1, group 1). The enzyme remained active during the 40-min time course as shown by the increased primer extension band intensities at the later time points. In contrast, synthesis by the pol  HE was much more processive, with the addition of 35 nt to reach the end of the template strand well within the first time point taken at 3 min (Fig. 1, group 5). Processive synthesis does not occur in the absence of either PCNA or ATP (Fig. 1, groups 3 and 7, respectively). One can also clearly observe the PCNA-independent passive clamp loading reaction, with full-length synthesis also occurring in less than 3 min (Fig. 1, group 2). However, it is important to note that the passive clamp loading reaction failed to occur in the presence of SSB (Fig. 1, group 7), ensuring that our fidelity measurements made with pol  HE in the presence of SSB, required PCNA, RFC, and ATP to carry out processive primer elongation. Experiments in which RPA (human or S. pombe RPA) was substituted for E. coli SSB showed no significant differences in either the rates or fidelity of DNA synthesis (data not shown). Therefore, a specific requirement for eucaryotic SSB has not been demonstrated in our in vitro model system and remains an open question requiring further investigation.

Fidelity of S. pombe pol  HE-- Nucleotide misincorporation values for S. pombe pol  were found to be f_inc = 4.6 × 10⁴ (T·G), 5.3 × 10⁵ (G·G), and 4.5 × 10⁶ (A·G) (Fig. 3 and Table I). The pol  HE error rates can be compared with values obtained with E. coli pol III HE and proofreading-defective E. coli pol III HE (26) containing the  sliding clamp (analogous to PCNA),  clamp loading complex (analogous to RFC), and SSB in the same p/t DNA sequence context (Table I). The fidelity of pol  HE is considerably lower than pol III HE for each mispair. The reduction in fidelity compared with pol III HE is 82-fold (T·G), 76-fold (G·G),² and 11-fold (A·G).

View this table: [in this window] [in a new window]   Table I Comparison of misincorporation efficiencies for DNA polymerase holoenzymes Misincorporation efficiency, finc (see Equation 1), opposite target G site catalyzed by S. pombe pol  HE, E. coli pol III HE, and E. coli pol III HE. E. coli pol III HE is devoid of proofreading exonuclease activity. The p/t DNA sequence is shown at the top of Fig. 2.

The higher nucleotide misincorporation rates for pol  HE appear to be attributable almost entirely to a severely compromised ability to proofread insertion errors made by the polymerase catalytic subunit. Indeed, a comparison of f_inc for pol III HE (containing the  polymerase subunit in the absence of the  proofreading and  subunits) shows that the nucleotide misinsertion rates for pol  and pol III are essentially the same (Table I). The reduction in fidelity for pol  HE compared with pol III HE is only 2.6- and 1.5-fold for T·G and G·G mispairs, respectively, whereas pol  HE may be slightly (1.2-fold) more accurate in forming A·G mispairs. These small differences are not statistically significant.

The apparent "absence" of effective proofreading for S. pombe pol  in the in vitro experiments is quite puzzling. By using the same assay and p/t DNA sequence to measure E. coli pol III fidelity, we observed an 8-fold reduction in fidelity as proofreading of mispaired A·G termini were reduced in the presence of high concentrations of a next-correct "rescue" dNTP (26). The "next nucleotide" reduction in fidelity is a well established hallmark of a proofreading polymerase (38, 39) and confirms that the gel kinetic assay can be used to analyze the effects of proofreading on fidelity. We observed no significant differences in pol  HE fidelity using a wide concentration range of next-nucleotide dNTP (data not shown), and we concluded, therefore, that S. pombe pol  is unable to effect a significant reduction in polymerase insertion errors. The p/t DNA sequence, requiring incorporation of four As prior to reaching the target G site, was chosen to maximize proofreading, i.e. "all things being equal" proofreading is most effective in removing misinserted nucleotides adjacent to relatively unstable DNA regions (40, 41). This latter point serves to emphasize the inability of pol  to carry out effective error correction.

The absence of a next-nucleotide effect is consistent with our observation that pol  has an extremely weak associated 3'-exonuclease activity (Fig. 4). This activity barely degraded p/t DNA containing mismatched primer 3'-ends in the absence of dNTP substrates (Fig. 4A), and although it was able to degrade the single-stranded 35-mer primer somewhat more effectively (Fig. 4B), its activity was far lower than that observed using either E. coli pol III core or HE (data not shown). The pol -catalyzed DNA degradation rate was essentially unchanged in the presence of PCNA (data not shown). A small increase in the rate of DNA hydrolysis was, however, observed in the presence of PCNA + RFC, which can be attributed to a low level of nuclease contamination in our most highly purified RFC fraction (data not shown).

In an earlier in vitro study, Kunkel and co-workers (42) were also unable to demonstrate a proofreading contribution to accuracy when using calf thymus pol  to copy a lacZ reporter gene sequence in the absence of processivity subunits, although pol  proofreading was readily apparent using the same gap filling assay. Although we are unaware of any in vivo mutational data for proofreading-deficient S. pombe mutants, there are such data in S. cerevisiae showing that proofreading for pols  and  can substantially reduce base substitution errors (43, 44). In view of this dichotomy, it seems reasonable to speculate that associated exonuclease activity of pol  may be masked in vitro. In this regard, it is interesting to note that the S. pombe and S. cerevisiae pol s differ in their subunit structure. In S. cerevisiae, this complex has been shown to be a dimer of the three-subunit complex (M_r 125,000, 58,000, and 55,000 (45, 46)) whereas the S. pombe pol  has been shown to be a dimer of the four-subunit complex (M_r 125,000, 55,000, 54,000, and 22,000 (30)). Whether the unique 22-kDa subunit found in S. pombe pol , the product of the non-essential cdm1⁺ gene (47) affects the proofreading function of the S. pombe pol in vitro, remains to be explored. In addition, perhaps some other protein cofactor may be required to stimulate proofreading. As indicated here, the gel fidelity assay might prove useful as a means to identify and purify proofreading stimulatory factors from cell lysates. On the other hand, the presence of an alternative excision repair pathway in S. pombe that has been shown to excise mispaired bases (48), in addition to damaged DNA bases, raises the possibility that this repair pathway might compensate for a lack of effective proofreading by pol.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM21422 (to M. F. G.), GM38559 (to J. H.), and GM38839 (to M. O'D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Professor of the American Cancer Society.

^** To whom correspondence should be addressed: Dept. of Biological Sciences, SHS Rm. 172, University of Southern California, University Park, Los Angeles, CA 90089-1340. Tel.: 213-740-5190; Fax: 213-740-8631; E-mail: mgoodman@mizar.usc.edu.

Published, JBC Papers in Press, March 23, 2000, DOI 10.1074/jbc.M910278199

² In the fidelity comparison for G·G misincorporations, the template used for E. coli pol III HE contains the base A in place of C immediately downstream from the target G site because pol III HE can incorporate dGMP opposite the downstream C by a primer-template slippage mechanism called "dNTP-stabilized" misalignment (26). In contrast, S. pombe pol  HE misincorporates dGMP directly opposite G when C is located at the 5'-side of the target.

	ABBREVIATIONS

The abbreviations used are: pol, DNA polymerase; HE, holoenzyme comprised of DNA polymerase + processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp, and replication factor C (RFC) clamp loading complex for S. pombe, and  sliding clamp and  clamp loading complex for E. coli; SSB, E. coli single strand DNA-binding protein; RPA, replication protein A, eucaryotic single strand DNA-binding protein; p/t DNA, primer-template DNA; nt, nucleotide; ssDNA, single-stranded DNA.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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