©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Replication Error Rates for GdGTP, TdGTP, and AdGTP Mispairs and Evidence for Differential Proofreading by Leading and Lagging Strand DNA Replication Complexes in Human Cells (*)

(Received for publication, October 17, 1994; and in revised form, November 17, 1994)

Shunji Izuta John D. Roberts Thomas A. Kunkel (§)

From the Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have determined the fidelity of DNA replication by human cell extracts in reactions containing excess dGTP. Replication errors were scored using two M13 DNA substrates having the replication origin on opposite sides of the lacZ alpha-complementation gene. The data suggest that the average rates for replication errors resulting from G(template), TbulletdGTP, and AbulletdGTP mispairs are 25 times 10, 12 times 10, and 3 times 10, respectively. The data also suggest that error rates for both the (+) and(-) strands differ by less than 2-fold when they are replicated either as the leading or lagging strand. This is in contrast to the 33- and 8-fold differences observed earlier for GbulletdTTP and CbulletdTTP mispairs on the (+) strand when replicated by the leading or lagging strand complex (Roberts, J. D., Izuta, S., Thomas, D. C., and Kunkel, T. A.(1994) J. Biol. Chem. 269, 1711-1717). Thus, the relative fidelity of the leading and lagging strand replication proteins varies with the mispair and sequence considered. Misincorporation of dGTP preferentially occurs at template positions where dGTP is the next correct nucleotide to be incorporated. This ``next nucleotide'' effect is characteristic of reduced exonucleolytic proofreading and suggests that these replication errors are normally proofread efficiently. Fidelity measurements performed in the absence or presence of dGMP, an inhibitor of proofreading exonuclease activity, suggest that the leading strand replication complex proofreads some mispairs more efficiently than does the lagging strand replication complex.


INTRODUCTION

Studies with purified DNA polymerases performed during the last 25 years have been invaluable for understanding the basic principles for accurate DNA polymerization (reviewed in Echols and Goodman(1991), Kunkel(1992), and Johnson(1993)). These studies have shown that several discrimination steps in the reaction cycle determine the selectivity for correct nucleotide incorporation and the efficiency of exonucleolytic proofreading. They have also revealed that the fidelity of polymerization reactions can be highly variable, depending on the DNA polymerase under study, the type of error being considered (e.g. base substitution versus frameshift), the base composition of the mispair or misalignment, the symmetry of the error (e.g. TbulletdGTP (^1)versus GbulletdTTP or addition versus deletion intermediate), and the local sequence surrounding the error.

As complex as these model polymerization reactions are, replicating the entire genome of an organism is much more complicated. More than one DNA polymerase and several accessory proteins are required to replicate the two antiparallel strands coordinately. Thus, a full appreciation of how genomes are stably replicated and how instability may arise to generate disease requires a better understanding of the fidelity of this complex replication machinery. An important step toward achieving this understanding has been the development of systems that replicate double-stranded DNA in vitro. One system for studying human genomic replication depends on the SV40 origin of replication (for recent review, see Stillman(1994)). Circular double-stranded DNA substrates containing the SV40 origin can be fully replicated by the proteins present in human cells, with only the addition of SV40 large T antigen needed to initiate replication at the origin. These factors can be supplied either by crude extracts of human cells grown in culture or by reconstitution with purified proteins prepared from such extracts (Waga and Stillman, 1994). At least two DNA polymerases, alpha and , are among the host factors required for complete DNA replication (Lee et al., 1989; Weinberg and Kelly, 1989; Melendy and Stillman, 1991). Additional host proteins are required for specific initiation at the origin, for chain elongation on the leading and lagging strands, and for completion and separation of the daughter molecules.

As measured with DNA substrates containing reporter genes for scoring replication errors, SV40 replication in unfractionated cell extracts has been found to be highly accurate (Roberts and Kunkel, 1988; Hauser et al., 1988). Replication is in fact more accurate than DNA synthesis by either the 4-subunit DNA polymerase alpha-primase complex or DNA polymerase with its associated 3`5` exonuclease (Thomas et al., 1991). Further investigation of highly accurate replication thus requires reaction conditions that generate replication errors above the background frequencies of existing fidelity assays. One approach has been to replicate damaged DNA (Carty et al., 1992; Thomas and Kunkel, 1993; Thomas et al., 1993, 1994). Another strategy is to replicate undamaged DNA using a damaged dNTP (Pavlov et al., 1994). Still a third approach uses undamaged substrates in reactions containing unequal concentrations of dNTPs to force errors that revert specific pre-existing substitution (Roberts and Kunkel, 1988; Roberts et al., 1991) or frameshift mutations (Bebenek et al., 1992; Roberts et al., 1993).

In order to define SV40 replication fidelity with respect to the type, base composition, symmetry, and location of errors, we are performing experiments using a fidelity assay that detects a variety of substitution, deletion, and addition errors in a target sequence of several hundred base pairs. The first such study used reactions containing excess dTTP to force a specific subset of replication errors (Roberts et al., 1994). Two of twelve possible substitutions as well as single-nucleotide frameshifts were induced by this substrate imbalance. Errors were found throughout the 250-base pair target, but they were distributed non-randomly. Two hot spots were observed, one for a G A transition and one for the loss of a GbulletC base pair in a homopolymeric run. Examination of the fidelity of replication of the same sequence when copied as the leading or lagging strand suggested that the overall error rates for GbulletdTTP and CbulletdTTP mispairs as well as the error rates at the two hot spots depended on whether replication was performed by leading or lagging strand replication proteins.

The current study presents two sets of experiments intended to expand our understanding of the fidelity of the human replication apparatus. The first set describes replication fidelity in reactions containing excess dGTP to define a new set of substitution and frameshift error rates on the leading and lagging strands that are forced by this substrate imbalance. This pool bias provides information on 3 more of the 12 possible mispairs, and the observed error specificity further suggests that proofreading contributes to replication fidelity. The analysis also reveals a base substitution hot spot that is detected as a lagging strand error but not as a leading strand error. A similar observation was made in the earlier study with excess dTTP (Roberts et al., 1994), but it was for a different mispair at a different location. In both cases, the error specificity is consistent with the possibility that some mispairs are more effectively proofread during leading strand replication than during lagging strand replication. The second set of experiments was performed to examine this possibility.


EXPERIMENTAL PROCEDURES

Materials

Bacteriophage, bacterial strains, enzymes, and reagents were from previously described sources (Roberts and Kunkel, 1993; Roberts et al., 1994). Fractions CFI` and CFII were kindly provided by Thomas J. Kelly (The Johns Hopkins University).

Methods

All procedures, including performance and processing of replication reactions, transfections, plating, mutant scoring, plaque hybridization assays, and sequencing of mutants, were as described (Roberts and Kunkel, 1993; Roberts et al., 1994).

DNA Substrates

The M13mp2SV Ori left and Ori right vectors have been described (see Fig. 1in Roberts et al.(1994)). Both contain 7398 base pairs and carry the lacZ alpha-complementation target, comprising nucleotides -84 to +170 of the lacZ gene, where +1 is the first transcribed nucleotide. As a reference point for the distance from the SV40 origin to the target, we use the transition from discontinuous to continuous replication (nucleotide 5210 in SV40 DNA) (Hay and DePamphilis, 1982). To fully replicate the lacZ target, the closest replication fork emanating from the origin must proceed 594 base pairs for Ori left and 403 base pairs for Ori right.


Figure 1: Replication error spectra in reactions containing excess dGTP. The mutational spectra with both the Ori left and Ori right vectors are displayed above and below, respectively, the double-stranded sequence of the lacZ alpha region of M13mp2SV. Each mutation is shown as the mispair considered most likely to occur under the reaction conditions used. Only those mutations consistent with the pool bias are displayed. The underlined nucleotides in the sequence are sites at which mutations generated by mispairs with dGTP have previously been identified and are known to be detectable. Arrows on the right indicate the direction of synthesis for that strand. Open triangles represent the loss of a single nucleotide; closed triangles represent the addition of a single nucleotide. Because neither the nucleotide that is lost or added in homopolymeric runs nor the strand that represents the template strand for frameshift errors is known, the deletion or addition is centered under the runs in the (+) strand.



Fidelity Assay

The products of SV40 origin-dependent replication in vitro were introduced into Escherichia coli cells by electroporation to assess replication fidelity by scoring lacZ alpha-complementation mutant frequencies. Correct replication produces DNA that yields dark blue M13 plaques on indicator plates, whereas errors are seen as lighter blue or colorless plaques. Because the assay measures loss of a gene function that is not essential for phage production, a variety of mutations at different sites can be recovered and scored (Roberts and Kunkel, 1993). These include 482 substitution errors and the loss or gain of any of 174 different base pairs.

Fidelity on the Leading and Lagging Strands

Mutant frequency results with the Ori left and Ori right vectors can be compared to define fidelity on the leading and lagging strands. The approach has been described in detail elsewhere (Roberts and Kunkel, 1993; Roberts et al., 1994). Briefly, the logic is as follows. The Ori left substrate contains the SV40 origin a few hundred nucleotides to the left of the reporter gene, a distance that is short relative to the size of the vector. Because the rate of replication fork movement is known to be similar in both directions from the origin (Li and Kelly, 1985), the (+) strand of the lacZ gene is likely to be encountered first by the closest fork emanating from the origin and, thus, replicated as the lagging strand (see Fig. 2 in Roberts et al.(1994)). In contrast, because the Ori right vector contains the origin a few hundred nucleotides on the other side of the target, the (+) strand of the lacZ gene is inferred to be replicated as the leading strand. Comparative fidelity measurements (mutant frequency determinations followed by DNA sequence analysis of mutant collections) with the two vectors permit estimation of the fidelity of replication of the same sequence by either leading or lagging strand proteins, so long as the strand on which the error was made can be assigned. In this study, this is inferred to be those mutations consistent with dGTP misincorporation.


RESULTS

Replication Fidelity in the Presence of Excess dGTP

Replication reactions with M13mp2SV Ori left DNA were performed with HeLa cell extracts in the presence of excess dGTP. Agarose gel electrophoresis of the resulting replication products demonstrated that they were similar to those obtained from reactions containing equimolar dNTPs (not shown, but see Fig. 1in Roberts and Kunkel(1988)). Mutant frequency determinations for replicated DNA samples demonstrated that reactions containing less than a 100-fold dGTP imbalance did not increase mutant frequencies above that of DNA replicated with equimolar dNTPs (not shown). However, the mutant frequency of DNA replicated in HeLa cell extracts with a 100-fold excess of dGTP was increased (Table 1). We repeated the experiment with the Ori left vector and performed three similar experiments with the Ori right vector. Each time, similar results were obtained (Table 1). The observation that the increases in mutant frequencies were small despite the presence of a 100-fold excess of dGTP is consistent with our previous interpretation that replication in HeLa cell extracts is highly accurate (Thomas et al., 1991).



The reproducible increases in mutant frequency suggest that many of the mutants obtained from replication products may have resulted from incorporation of dGTP during replication. To examine this possibility, the DNA sequences of 104 mutants from excess dGTP-containing reactions with Ori left substrate and 180 mutants from reactions with the Ori right substrate were determined for nucleotides -84 through +170 of the lacZ alpha-complementation gene. Sequence changes were found in 76 Ori left mutants and 117 Ori right mutants (Table 2). The remainder had no change in the 254-nucleotide target sequence. We have previously reported (Roberts et al., 1994) that, with a dTTP bias, some mutants had changes between positions 170 and 479, the remaining downstream lacZ gene sequence in M13mp2. Although that may be the case here as well, we did not analyze sequences beyond position 170, as this would have more than doubled the sequencing effort.



Error Specificity

Sixty-six of the 76 mutants from the Ori left template contained a single-base substitution (Table 2), 54 of which were consistent with misincorporation of dGTP opposite template G (29 mutants), template T (23 mutants), or template A (2 mutants). For the Ori right vector, 101 of 117 mutants contained a single base substitution, 60 of which were consistent with misincorporation of dGTP opposite template G (36 mutants), template T (19 mutants), or template A (5 mutants). This pattern of substitutions is very different from that found in a previous study employing excess dTTP, where most substitutions were inferred to have resulted from dTTP opposite template G and C residues (Roberts et al., 1994). In addition to the substitutions, 7 frameshifts of 1 nucleotide were observed with the Ori left substrate (Table 2), 6 of which were deletions. With the Ori right substrate, 11 frameshift errors of 1 nucleotide were obtained; 6 were deletions, and 5 were additions.

Error Distribution

The distribution of the 18 single-base frameshifts and of single-base substitutions consistent with dGTP misincorporation is shown in Fig. 1. The mutants are not randomly distributed. For example, with the Ori left substrate (mutants shown above each line of primary sequence in Fig. 1), 9 of 23 mutants consistent with misincorporation of dGTP opposite template T are at one template site, position 121. In contrast, one or no mutants were observed at 39 of the 45 other sites where this transition substitution can be scored (underlined nucleotides in Fig. 1). Also, only one of 19 mutants consistent with misincorporation of dGTP opposite template T was observed at position 121 with the Ori right vector. Similarly, site- and vector-specific non-random mutant distributions were observed using a dTTP bias (Roberts et al., 1994). As would be expected for reactions performed with excesses of different dNTPs, the locations and types of errors found in that earlier study were very different from those shown in Table 2and Fig. 1.

The error rates on both the (+) and(-) strands for both vectors were calculated for GbulletdGTP, TbulletdGTP, and AbulletdGTP mispairs (Table 3). Rates are expressed per detectable nucleotide incorporated to correct for small differences in the number of detectable sites for each type of error on each of the two strands. For all three mispairs, note that the same sequence, whether a (+) or(-) strand, is replicated with similar accuracy regardless of the orientation of the origin relative to the lacZ target sequence. This is in marked contrast to previous observations with the same assay for reactions containing excess dTTP (Roberts et al., 1994). In that study, on the (+) strand, there were 33- and 8-fold differences in rates for GbulletdTTP and CbulletdTTP errors, respectively, between the Ori left and Ori right vectors.



Fidelity in Reconstituted Replication Reactions

In order to establish the reproducibility of these observations, we repeated the fidelity analysis by reconstituting replication reactions from fractions (designated CFI` and CFII) obtained from column chromatography of extracts (Wold et al., 1988). Because reconstituted reactions perform replication but not mismatch repair (Roberts et al., 1994), this analysis has the added advantage of examining replication fidelity in the absence of the mismatch repair that is known to occur in extracts (Thomas et al., 1991). Reconstituted replication reactions with excess dGTP were performed twice with each vector. All four mutant frequencies obtained were between 31 and 37 times 10. These values are slightly higher than those obtained with extracts (Table 1). DNA sequence analysis of collections of mutants showed error specificity similar to that observed with the extract (not shown). With both vectors, the majority of substitutions was again consistent with misincorporation of dGTP (29 of 34 with Ori left and 44 of 49 with Ori right) opposite template T or G. As with the extract, when the two vectors were compared, no significant differences in overall substitution error rates on the (+) or(-) strands were detected for either mispair.

Site Specificity of Misincorporation

To see whether substitutions occurred preferentially at template positions where proofreading might be less active, we analyzed the site specificity of misincorporation. A high concentration of dGTP could diminish proofreading at 5`-C-X-3` positions (where X is the site of the substitution), because misinsertions of dGTP would be fixed by incorporation of the next correct nucleotide (dGTP) prior to exonucleolytic removal. We therefore compared the error rate per detectable site for each of the three mispairs that were consistent with dGTP misincorporation at sites having a cytosine as a 5` template neighbor with error rates at sites having any of the other three nucleotides as a 5` template neighbor. Using the mutant collection obtained for reactions in the extract, dGTP was misincorporated opposite template G at 5`-C-G-3` sites at an 11-fold higher rate than at 5`-T-G-3`, 5`-A-G-3`, or 5`-G-G-3` sites (compare 5.0 versus 0.45 in Table 4). The same analysis performed for mutants obtained from the reconstituted reactions yielded values of 2.5 versus 0.05 mutants per site for 5`-C-G-3` and 5`-S-G-3`, respectively (Table 4), which is a 50-fold difference. Likewise, a 3-fold difference in error rate was observed for the TbulletdGTP mispair in reactions with extract (Table 4); this difference was also observed in the reconstituted reactions (not shown).



Monophosphate Effect on Replication Fidelity

These differences are consistent with diminished proofreading at sites having a neighboring 5` template cytosine under conditions of high dGTP concentration. At one such site, T on the (+) strand (Fig. 1), the misincorporation of dGTP is 12-fold more frequent with the Ori left substrate (error rate of 38 times 10) than with the Ori right substrate (error rate of 3.1 times 10). In an attempt to examine proofreading at this site, we took advantage of a previous observation that SV40 replication fidelity can be reduced by addition of a known inhibitor of exonucleolytic proofreading, dGMP (Roberts and Kunkel, 1993). We performed parallel replication reactions with and without added dGMP. T C transitions at position 121 were detected by plaque hybridization with a synthetic oligonucleotide (Roberts et al., 1994). The results (Table 5, top) again reveal a different error rate for misincorporation of dGTP at position 121 between the Ori left and Ori right vectors in the absence of dGMP (Ori left, 19 times 10; Ori right, 1.6 times 10). Moreover, the presence of 2 mM dGMP increases the error rate at position 121 by 6-fold with the Ori left substrate and by 13-fold with the Ori right substrate.



In the previous study, employing a dTTP pool bias (Roberts et al., 1994), we observed a situation similar to that just described. A template G at position 145 on the (+) strand was found to be a hot spot for misincorporation of dTTP but only when replicated as the lagging strand. The sequence at this site is 5`-A-G-3` (nucleotide 145 underlined), and the next correct nucleotide to be incorporated is dTTP. This site is thus suitable for a second test of the contribution of proofreading to fidelity but for a mispair having the reciprocal symmetry (GbulletdTTP rather than TbulletdGTP). Therefore, we performed parallel reactions containing excess dTTP with and without added dGMP, and monitored G A transitions at position 145. The results in the absence of dGMP (Table 5, bottom) confirm our previous observation that position 145 is replicated less accurately by the lagging strand apparatus (the Ori left substrate) than by the leading strand apparatus (the Ori right substrate). The addition of dGMP to the reaction increased the error rate with both substrates. An independent repeat of this analysis yielded a similar result (data not shown).


DISCUSSION

From this study we can infer error rates during replication in human cell extracts for the three mispairs involving misincorporation of dGTP. These and earlier results with excess dTTP (Roberts et al., 1994) provide replication error rates for 6 of the 12 possible single-base mispairs, in the following order of highest to lowest error rate: GbulletdTTP approx CbulletdTTP approx GbulletdGTP approx TbulletdGTP >> TbulletdTTP approx AbulletdGTP. This same relative order and similar error rates are obtained during replication in extracts and in reconstituted reactions known to lack mismatch repair activity (Roberts et al., 1994), demonstrating the reproducibility of the observations and suggesting that this specificity reflects the average base selectivity and proofreading potential of the human replication apparatus. The error specificity pattern is not characteristic of that obtained with DNA polymerase alpha, (plus proliferating cell nuclear antigen), or during gap-filling synthesis templated by the same (+) strand lacZ sequence used here (Thomas et al., 1991). Also note that the replication error rates for two of the four transversion mispairs examined, CbulletdTTP and GbulletdGTP, are similar to those for the two transition mispairs, GbulletdTTP and TbulletdGTP. These transition mispairs are among the most common DNA polymerase misinsertion and mispair extension errors (for review, see Echols and Goodman(1991) and Johnson(1993)). Specificity differences between purified DNA polymerases and the multiprotein replication apparatus could reflect modulation of polymerase fidelity by other proteins. For example, the single-stranded DNA-binding protein reportedly increases the accuracy of DNA synthesis by DNA polymerase alpha (Carty et al., 1992), and the 3`5` exonuclease of human DNA polymerase is regulated by accessory proteins such as human single-stranded DNA-binding protein, proliferating cell nuclear antigen, and replication factor C (Lee, 1993).

As in our earlier study of replication fidelity with excess dTTP (Roberts et al., 1994), errors resulting from the presence of excess dGTP do not occur equally at all template positions. This non-random error distribution represents preferential misincorporation of dGTP opposite certain template T and template G nucleotides (Fig. 1) with an overall preference for those sites followed by a template C (incoming correct dGTP, Table 4). This sequence preference is a hallmark of suppression of exonucleolytic proofreading, wherein dGTP misincorporations are not removed because the high concentration of dGTP also favors polymerization of the next correct nucleotide prior to excision of the error. This neighboring nucleotide effect is seen here for both GbulletdGTP and TbulletdGTP errors (Table 4) but was not seen in an earlier study of GbulletdTTP and CbulletdTTP errors. These data imply that, at least under the reaction conditions used here, which are required to observe errors with the highly accurate replication machinery, proofreading of all replication errors is not suppressed equally by a high concentration of the next correct dNTP.

We noted that several 1-nucleotide frameshift errors at repetitive sequence positions were generated in reactions containing excess dGTP. For example, four mutants contained an extra GbulletC base pair in a three-base run of GbulletC base pairs at positions 88-90 and another had one fewer GbulletC base pair (Fig. 1). These mutations in repetitive sequences were not observed in either of two previous replication fidelity studies (Thomas et al., 1991; Roberts et al., 1994) or among background mutations from unreplicated DNA (Thomas et al., 1991), suggesting that they are dGTP-induced replication slippage errors. Models for their generation have been described (Bebenek and Kunkel, 1990; Roberts et al., 1993; Roberts et al., 1994).

SV40 replication in vitro is a complex reaction that utilizes a variety of enzymatic activities and accessory proteins (for review, see Stillman(1994)). These studies have shown that there is an enzymological asymmetry at the replication fork. A major objective of comparing error specificity with the two M13mp2SV substrates was to determine if asymmetric replication enzymology on the leading and lagging strands results in unequal error rates. Initial observations revealed that replication errors inferred to result from misincorporation of dTTP opposite template G and C on the (+) strand occurred at 33- and 8-fold higher rates, respectively, when this strand was replicated as the lagging strand as compared with the leading strand (Roberts et al., 1994). In the present study, we were particularly interested in determining if lagging strand replication is also less accurate with a completely different set of replication errors, those resulting from dGTP misincorporation. Here the pattern that emerged is remarkably different from the earlier study. Although average rates for dGTP misincorporation opposite template T and G were slightly higher for the lagging as compared with the leading strand replication complex (Table 4), the differences were 2-fold or less. This suggests that the relative fidelity of the leading and lagging strand replication complexes varies depending on both the template sequence and the mispair considered.

The next nucleotide effect shown in Table 4suggests that exonucleolytic proofreading modulates site-specific misincorporation of dGTP. One possible explanation for a difference in error rates between the leading and lagging strand replication complexes for the same mispair in the same sequence context is a difference in proofreading activity. To determine if this could explain site-specific differences, we looked for deoxynucleoside monophosphate inhibition of exonuclease activity at positions 121 and 145 on the (+) strand, hot spots for two different substitution errors by the lagging strand complex but not the leading strand complex. The addition of dGMP, a known inhibitor of exonucleolytic proofreading (for review, see Kunkel(1988)), increased the error rate at both sites with both the Ori left and Ori right substrates (Table 5). If one assumes that the addition of dGMP does not affect the inherent base selectivity of the insertion step, then the dGMP-dependent increase in error rate suggests that misinsertions are indeed occurring that, in the absence of monophosphate, are removed by the exonuclease. Because fidelity in the absence of dGMP is higher for both errors during leading strand replication, this suggests that leading strand misinsertions are more effectively excised than are the lagging strand errors that are readily detected even when dGMP is absent. This provides one mechanism that can explain site- and mispair-specific differences in the fidelity of leading and lagging strand replication. Because the assignment of the leading and lagging strand DNA polymerases during eukaryotic replication is not yet definitive (for review, see Linn(1991)), proofreading on the two strands could be carried out by the exonucleases tightly associated with DNA polymerase or , by an exonuclease that copurified with DNA polymerase alpha-primase (Bialek and Grosse, 1993), or by a separate exonuclease.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Molecular Genetics, E3-01, National Institute of Environmental Health Sciences, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-2644; Fax: 919-541-7613.

(^1)
The abbreviations used are: TbulletdGTP, misincorporation of dGTP opposite template T; GbulletdGTP, misincorporation of dGTP opposite template G; AbulletdGTP, misincorporation of dGTP opposite template A; dNTP(s), deoxynucleoside triphosphate(s).


ACKNOWLEDGEMENTS

We thank William C. Copeland and Kenneth R. Tindall for critical comments on the manuscript.


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