THE JOURNAL OF Vol. 256, No. 20. Issue of October 25. pp ...THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 20.Issue of October 25.pp. 10671-10683, 1981 Prmted in U.S.A. Molecular

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 20. Issue of October 25. pp. 10671-10683, 1981 Prmted in U.S.A.

Molecular Mechanisms of Substitution Mutagenesis AN EXPERIMENTAL TEST OF THE WATSON-CRICK AND TOPAL-FRESCO MODELS OF BASE MISPAIRINGS*

(Received for publication, April 22, 1981)

Navin K. Sinha and Melanie D. Haimes From the Waksman Institute of Microbiology, Rutgers Uniuersity, Piscataway, New Jersey 08854

The proteins coded by bacteriophate T4 replication genes 32,41,43,44,45,61, and 62 together can replicate 4x174 DNA templates very efficiently. The fidelity of this in uitro replication reaction has been measured using an infectivity assay. The product molecules have the same specific infectivity as the template DNA. When an amber mutant DNA template is used, no increase in the frequency of revertants is seen even after more than 60 duplications in uitro.

By using imbalances in the concentrations of deoxynucleotide substrates, the error rate during DNA replication in vitro can be greatly increased. Control experiments indicate that the increased mutagenesis is not due to tee presence of dITP or dUTP as contaminants in the deoxynucleotide substrates used. The increase in the frequency of revertants is linearly related to the ratio of the correct and the incorrect deoxynucleotides. Determination of the DNA sequence of the revertants induced shows that a change in DNA sequence of the amber site predicted from the nucleotide bias occurs.

DNA synthesis in vitro resembles in vivo replication in that the error rate depends not only upon the base change required for reversion but also upon the neighboring DNA sequences. The error rate is estimated to be 5 X at am3 site, 6.4 X lo-‘ at am86 site, and less than 2.9 X at am9 site. Comparison of the frequency of G-T and A-C mispairs reveals that most AT += GC transition mutations occur through G-T mispairs. Measurement of the frequency of the mispairs required to induce transversion mutations reveals that these occur primarily through purine-purine mispairs. Tran- sition mutations are more frequent than transversion mutations at both the am3 and the am86 sites. These observations support the models for base pairing errors proposed by Watson and Crick ((1953) Nature 171,964-967) and Topal and Fresco ((1976) Nature 263, 285-289).

DNA replication in vivo is an extremely accurate process. An average rate of 2 X errors per nucleotide replicated has been estimated for T4 phage replication (1). The mechanisms involved in generating this accuracy are not fully un- derstood. Interactions between complementary bases alone are not sufficient to explain this accuracy. Behavior of certain

* This work was supported by grants from the Charles and Johanna Busch Foundation, Biomedical Research Support Grant of Rutgers University, and the United States Public Health Service Grant GM24391). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertzsement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

mutants in the gene for T4 DNA polymerase (gene 43) as general mutators and antimutators suggested that this protein plays a major role in determining the accuracy of DNA replication (2, 3). Estimates for the error rate during the duplication of natural DNA by T4 DNA polymerase alone are not available. The error rate during the copying of homopolymer templates by the T4 DNA polymerase is only 5 to 8 x

(4, 5). Therefore, it is unlikely that DNA polymerase done can account for all of in vivo accuracy.

Genetic studies in vivo have shown that some of the other enzymes of the DNA replication apparatus also affect mutation rates (6-9), presumably by affecting the accuracy of the replication complex. Consistent with this, it has been shown that the addition of DNA-binding protein to DNA polymerase increases the accuracy of DNA synthesis in vitro (10, 11). In addition to the involvement of the replication enzymes, a proper balanced ratio of deoxynucleotide substrates is also needed for maintaining high fidelity (6). A study of rII mutants revealed that reversion rates of different mutants requiring the same base pair change for reversion varied over a 5 X lo4- fold range (1). Therefore, the sequence of neighboring bases in DNA has a very significant effect on the mutation rate. A detailed analysis of the mechanisms by which all these factors affect fidelity of DNA replication has not been done so far.

In recent years, the products of T4 replication genes 32,41, 43,44,45,61, and 62 have been greatly purified (12-17). These have been used to assemble in vitro DNA replication apparatuses that can carry out extensive replication of both single- and double-stranded DNA templates by a process closely mimicking in vivo DNA replication (17-20). The fidelity of replication in vitro by these proteins is close to that seen during replication in vivo (17, 23, 62).

An observable increase in error rates due to imbalances in substrate concentrations during DNA synthesis in vitro using purified replication proteins have been reported by a number of workers, (4, 21-23, 62). By using appropriate nucleotide pool biases to force the reversion of sequenced amber mutants of 6x174, the frequency of the formation of a given mispair can be calculated (21-23, 62). Since we can synthesize both strands of 6x174 DNA simultaneously using the T4 replication apparatus, we can measure the frequencies of complementary mispairs leading to the same revertant (such as G-T and A-C mispairs required to generate an AT -+ GC transition).

Watson and Crick (59) had originally proposed that transition mutations may arise due to mispairings involving rare tautomers of the bases. This idea was further extended by Topal and Fresco (61), who proposed that there were other kinds of non-Watson-Crick base mispairs possible within the constraints of the DNA double helix. An examination of the structure of all these mispairs led to three predictions, two by Topal and Fresco and one by us: 1) Transitions should occur

10671

10672 Mechanism of Base Substitution Mutagenesis

primarily through G-T mispairs. 2) Transversions should occur primarily through purine-purine mispairs. 3) Transitions should be more frequent than transversions at a given site.

Here we present experimental data that c o n f i i these three predictions and provide strong support for the models of base mispairings proposed by Watson and Crick and Topal and Fresco. We have previously summarized some of these find- ings (62).

MATERIALS AND METHODS

Bacteria and Phages-Bacteriophage T4 amber mutants were grown on Escherichia coli CR63 (Su') and used to infect E. coli Dl10 (pol A- endo 1-, thy-, Su-) for the preparation of T4 replication proteins. Mutants of 6x174 phage were grown in E. coli HF4714

the strain HF4704 (Su-, thy-). (Su', thy-) and the number of revertants was estimated by plating on

Chemicals and Radioisotopes-Ribo- and deoxyribonucleoside triphosphates were obtained from the Sigma Chemical Corp., or from P-L Biochemicals. [methyl-3H]Thymidine and r3H]thymidine tri- phosphate were obtained from the New England Nuclear Corp. a-"P- labeled deoxynucleoside triphosphates and [3H]dUTP were purchased from the Amersham-Searle Corp. Bovine serum albumin used in the DNA transfection assays was either from the Biotest Serum Institute, Frankfurt, West Germany (a gift of Dr. Rolf Benzinger) or Pentex bovine serum albumin from the Miles Laboratories. Protamine sulfate solution used in transfection assays was a 1% solution from Eli Ldy and Co.

Purification ofDeoxynucleotides-Deoxynucleoside triphosphates used in this study were purified by chromatography on columns of DEAE-Sephadex A-25 using gradient elution with triethyl ammonium bicarbonate buffer. For purine deoxynucleotides, a buffer with a pH of 8.0 was used. To ensure the separation of dCTP from TTP (and dUTP), the pyrimidine nucleotides were chromatographed using a pH 9.4 buffer (24). In each case, a trace of radioactive contaminant was added to ensure that the fractions pooled in the end were completely free of any contaminants. For example, 50 pmol of dGTP was purified in the presence of 25 pCi (6.3 nmol) of L3H]dATP.

Measurement of Radioactiuity-The incorporation of radioactive precursors into DNA was followed by taking samples of the reaction mixtures and spotting onto glass fiber filters, which were successively washed at 4 "C with 5% trichloroacetic acid containing 0.1 volume of saturated sodium pyrophosphate (one wash), 1 M HC1 (four washes), and 95% ethanol (two washes). The fdters were then dried and counted in a toluene-based scintillation fluid.

Enzymes-Phage T4 coded replication proteins (the products of genes 32, 41, 43, 45, and the complex of 44 and 62 were purified as previously described (13-15). The gene 32 protein used in this study contained, as a minor contaminant, another replication protein, the product of gene 61, which is required for initiation of new okazaki pieces (15). Restriction endonucleases Hue IIZ, Hpa ZZ, and Hha I , and T4 and E. coli DNA ligases were purchased from the New England BioLabs and used as suggested by them. Pst I restriction enzyme was purified by successive chromatography on Bio-gel A- 0.5m, phosphocellulose and DEAE-cellulose columns essentially as described by Roberts et al. (25) for the purification of the enzyme Hue 11. The final yield from 10 g of cell paste was 35,000 units with 1 unit defined as the enzyme quantity needed to digest 1 pg of phage h DNA in 1 h at 37 "C in a 5Opl reaction volume. The enzyme was free of nonspecific endo- and exodeoxyribonuclease contaminants. It has remained stable for more than 3 years at -20 "C in a buffer containing 50% glycerol.

DNA Templates-Double-stranded closed circular form I (RFI) DNA from wild type and mutant phage 6x174-infected cells was prepared by a modikation of the method of Godson and Boyer (26). This DNA was further purified free of any contaminating DNA fragments by chromatography on columns of Sepharose 2B. Some 6x174 am3 RFI DNA was also obtained from the New England Biolabs and as gifts of Drs. Clyde Hutchison and Mike Bittner. Single- stranded DNAs from +X mutant am3 and its revertants were obtained by phenol extraction of phages that had been successively purified in cesium chloride step and equilibrium gradients. The DNA prepara- tions were stored at 4 "C in a buffer with 10 mid Tris-HC1, pH 8.1, 1 mM Nas EDTA.

Standard Conditions for DNA Synthesis in Vitro-The reaction mixture (60 pl/sample) contained 0.5 mM dithiothreitol, 33 mM Tris/ acetate, pH 7.8,67 mM potassium acetate, 10 mM magnesium acetate, 1 mM ATP, 0.2 mM each CTP, UTP, GTP, dATP, dCTP, dGTP, and

TTP. One of the four deoxynucleoside triphosphates was radioac- tively labeled. Replication proteins were gene 32 protein (200 pg/ml) with a trace of gene 61 protein, gene 41 protein (14 pg/ml), gene 43 protein (7 pg/ml), gene 45 protein (40 pg/ml), and the 44/64 protein complex (27 pg/ml). The DNA template was used at 1 to 2 pg/ml.

Infectiuity Assays-After DNA synthesis in uitro, one of two procedures was used to terminate the reaction. In some experiments, 20 mM Na3 EDTA was added to stop DNA synthesis and the sample gently phenol-extracted once. Phenol was removed and the DNA purified by chromatography on a column of Sephadex G-50 (1 to 4 ml packed volume) in 10 m~ Tris-HC1, pH 8.1, 1 m~ NaCl EDTA. Fractions were collected from the bottom and those with DNA were located by counting an aliquot of each fraction. This method was too cumbersome when a large number of samples were involved. There- fore, in most experiments, DNA synthesis was terminated and the replication proteins inactivated simply by heating to 65 "C for 10 min. The DNA was cut into unit length linear molecules by the addition of 100 pg/ml of nuclease-free bovine serum albumin and excess of Pst Z enzyme (5 to 25 units in a 60-p1 reaction with 0.1 to 5 pg of DNA) and incubation at 37 "C for 2 h. At the end of this period, the samples were heated to 65 "C for 10 min to inactivate the restriction enzyme before ligation of the linear molecules to generate circles. For ligation by T4 DNA-ligase, 1 m~ ATP, 20 mM dithiothreitol, 100 pg/ml of nuclease-free bovine serum albumin and T4 ligase (0.5 unit/sample) were added. When E. coli DNA-ligase was used, P-NAD (60 p ~ ) , dithiothreitol (10 mid), ammonium sulfate (IO mM), nuclease-free bovine serum albumin (100 pg/ml) and ligase (0.5 unit/sample) were added. Ligation was done by incubation overnight at 15 "C. For infectivity assays this DNA was diluted in sterile 0.01 M Tris-HC1 buffer (pH 8.1).

Spheroplasts were made essentially as described by Henner et al. (27), except the cells were grown in H-broth rather than in Fraser and Jerrel's medium. Midway through this study we were unable to obtain the "Povite" bovine serum albumin used by Henner et al. (made by Biotest Serum Institute, Frankfurt), and as a result, we examined six batches of bovine serum albumin made by manufac- turers in the United States. Of these, the Pentex albumin (supplied by Miles Laboratories as a 30% solution gave spheroplasts with the highest transfection efficiencies (approximately 80% compared to spheroplasts made with "Povite" albumin). Therefore, this material was used in many of the experiments described here.

DNA and spheroplasts (0.4 ml each) were incubated at 37 "C for 12 min and plated after the addition of 2 drops of the appropriate indicator cells, 1 drop of 30% bovine serum albumin and 3 ml of sucrose-containing top agar (27). Total infective centers were estimated by using CR63 (Su') spheroplasts with HF4714 indicator cells and revertant DNA molecules were estimated using W3110 (Su-) spheroplasts and HF4704 indicator cells. Multiple dilutions (usually 20- and 100-fold for the estimation of revertants and 10"- and 10h-fold for the estimation of total infective centers were plated to make sure that the number of plaques observed was linearly related to DNA concentration. The transfection efficiency of different batches of W3110 (Su-) spheroplasts was fairly constant (varying over a 3-fold range). CR63 (Su') spheroplasts had more variable transfection efficiencies, usually 2- to 10-fold lower than the efficiency of W3110 spheroplasts. In order to correct for these differences in plating efficiencies, a set of four plates was also plated in parallel with wild type reference DNA. These were then used to correct any differences in the plating efficiency of the spheroplasts of the two bacterial strains. In the data below, the numbers for revertant infective centers given are the observed values. The numbers for total infective centers, however, were corrected for the lower plating efficiencies of CR63 (&+) spheroplasts. We have provided the actual number of revertants observed (the value given in all cases is an average of at least two separate platings for each DNA sample) so that a reader can judge for oneself how reliable the experiment was (see Tables I, 111 to VII).

Other Procedures-Acrylamide gel electrophoresis was done by the method of Loening (28) except sodium dodecyl sulfate was omitted. DNA fragments were eluted from the gel slices as described by Galibert et al. (29). Agarose gel electrophoresis was done as described by Greene et al. (30). Procedures used for electron microscopy have been previously described (15). DNA sequencing was done by the chain termination method of Sanger et al. (31) using 6x174 virion DNA as template and Hue 111 25 restriction fragment as a primer. The concentrations of deoxynucleoside tri-, di-, and monophosphates were estimated by thin layer chromatography on polyethyleneimine cellulose plates using ascending chromatography with lithium chloride solutions (32).

Mechanism of Base Substitution Mutagenesis 10673

A

5. In Vitro Replication

- - t- UNIT LENGTH LINEARS

J Ligation

Su- S p h e r o p l o s t y Spheroplosts

Revertants Total Infective Centers

15 MIN 30MIN 45 MIN nn- A B C A B C A B C

ROLLING - CIRCLES

1- MULTIMERS

- UNIT LENGTH CIRCLES

- UNIT LENGTH LINEARS

FIG. 1. An infectivity assay for measuring the fidelity of DNA synthesis in v i tm by phage T4 replication proteins. A, outline of the assay scheme. DNA is synthesized using a 9x174 amber mutant RF DNA as template. The rolling circles generated are cut into unit length linear molecules with sticky ends by treatment with Pst I restriction endonuclease. These pieces are ligated into circles by treatment with DNA-ligase. Spheroplasts of Su’ and Su- E. coli cells are transfected with this ligated DNA sample for the measurement of total and revertant infective centers, respectively. B, analysis of the DNA generated in the steps of above scheme by electrophoresis in agarose gels. DNA samples were withdrawn after 15 (1.9 duplications of the input DNA template), 30 (8.4 duplications), and 45 min (22 duplications) of in vitro DNA synthesis a t 37 “C under “standard” conditions. Synthesis was stopped by heating to 65 “C for 10 rnin and the DNA was cut with Pst I endonuclease. After inactivation of the restriction enzyme by heating for 10 min at 65 “C, the sample was ligated overnight with E. coli DNA-ligase. The conditions for DNA synthesis, Pst I cutting and ligase treatment are described under “Materials and Methods.” After the addition of 5% glycerol, 1% sodium dodecyl sulfate, and 0.005% bromphenol-blue, the samples of uncut, Pst I cut, and cut and ligated DNA were electrophoresed in a 0.8% agarose slab gel a t 100 V for 4 h at room temperature. Only the 32P-labeled DNA made in vitro is visible in this autoradiograph of the gel. Two size classes of DNA molecules appear to be generated by

RESULTS

Assay for Measuring Fidelity-The most sensitive assay available for measuring the fidelity of DNA synthesis in vitro consists of duplicating a mutant DNA template and measuring the reversion of the mutant codon during in vitro replication (33). We would have liked to measure the accuracy of DNA synthesis in vitro by T4 replication proteins with T4 DNA as template so that direct comparisons could be made with the accuracy seen during replication in vivo. However, due to the extremely poor transfection efficiency (less than and very large size of T4 DNA (1.1 X 10’ daltons), it is not possible to do this. In this study we have used DNA from the phage 4x174 as template because of three reasons. The very high transfection efficiency with E. coli spheroplasts (approximately with @X RF DNA in our hand) permits the detection of rare revertants. The genetics of this phage has been studied in considerable detail and a large number of mutants are available (34). Finally, the complete nucleotide sequence of the wild type DNA is known (35) and the changes involved in several of the mutants have beed identified (35- 38). Knowledge of the nucleotide substitutions needed for the reversion of a mutant codon allows attempts to force the reversion during in vitro DNA synthesis by using appropriate deoxynucleotide substrate imbalances.

The assay we have used is the same one that was used by Hibner and Alberts (23) and is shown in Fig. IA. This assay relies on the finding that with circular DNA templates the seven purified replication proteins of T4 synthesize rolling circles with long unbranched double-stranded tails, both strands of which have been synthesized in vitro (18). The rate of DNA synthesis is very rapid (greater than 400 nucleotides/ s) resulting in tails that contain a large number of copies of the template DNA in tandem (19). Upon cutting with a variety of restriction endonucleases, a pattern of DNA fragments that is identical with that generated by cutting the template DNA is generated (data not shown). Upon cutting with a restriction endonuclease for which there is a single recognition sequence per genome, these rolling circles are converted into unit length linear double-stranded molecules (39). Circles are obtained by ligation of the sticky ends of these DNA molecules. These circles are then tested for infectivity and reversion of a mutant codon present in the template DNA by transfection into Su’ and Su- E. coli spheroplasts, respectively.

In Fig. 1B are shown DNAs generated in some of the steps of this scheme. Because of their very large molecular weight, the rolling circles are seen not to enter the agarose gel. All the DNA product is seen to be digested into unit length material by treatment with Pst I restriction nuclease for which there is a single cutting site per +X genome. Upon ligation, all of the DNA is seen to change in electrophoretic mobility indicating a change in its conformation.

The Product Made in Vitro Is as Infectious as the Tem- plate DNA-For estimating the infectivity of the product molecules made in vitro, we have measured the amount of

ligation, unit length, and multimers. The ligase used in these studies also contains some DNA gyrase causing the introduction of supercoils in some of the unit length circles formed. At all times examined here, we find that the ligation step causes the formation of a significant amount of multimers even though the concentration of DNA molecules is quite low (4 pg/ml after 15 min, 13 pg/ml after 30 min, and 32 pg/ml after 45 min in the experiment shown). By purifying the multimers by sedimentation in neutral sucrose velocity gradients and measuring their infectivity, we have found that, on the average, approximately 16% of the total DNA is present as multimers and these contribute less than 3% to the total infectivity and the number of revertants (based on average of three separate determinations). Lanes A, uncut rolling circle DNA lanes B, Pst I cut DNA, and lanes C, Pst I cut and ligated DNA.

10674 Mechanism of Rase Substitution Mutagenesis

infectious DNA present after allowing various levels of DNA synthesis. In the experiment shown, the ligation step was omitted so that all the DNA molecules, both the template RF and the in vitro product, were present as unit length linear molecules. As is shown in Fig. 2, the number of infective centers observed closely parallels the amount of DNA present at all times, even though a large number of duplications of the template occurred during the course of the experiment. Thus, the product synthesized in vitro must be having the same specific infectivity as the template DNA.

Measuring the Reversion of a Mutant Codon during DNA Synthesis in Vitro-Since the product synthesized in vitro is fully infectious, one can measure the reversion of a mutant codon during replication in vitro. We have synthesized DNA in vitro using RF DNA molecules from three different +X174 amber mutants as templates and measured the reversion of these amber codons. Even after as many as 60 duplications of the template DNA in vitro, no increase in the frequency of am3 revertants is seen in this experiment (Table I).

The sensitivity of this assay is limited by the high frequency of revertants present in the am3 and am86 template DNAs. The frequency of revertants present in a given mutant DNA population is dependent upon a number of factors including the number of revertants in the starting population, the number of rounds of DNA replication during growth, the reversion rate of the mutant, and any differences in the growth rates of the mutant and the revertant. Despite attempts to minimize the frequency of revertants in the am3 DNA populations, we were unable to get DNAs much better than the one used in Table I because of the high reversion rate of this mutant. We, therefore, measured the reversion rates in uivo of a number of amber mutants of 4x174 in an attempt to find one that is substantially less mutable than am3 (and am86). We have found two mutants (am9 in gene G and am210 in gene B) that have a significantly lower reversion rate than am3 and am86. We, therefore, prepared R F DNA from the am9 (G-) mutant and it has only revertants present in it, thus increasing the sensitivity of this assay by a factor of 100. Using this DNA as template, we find that the in vitro replication reaction catalyzed by these seven proteins is extremely accurate. With this DNA template we seldom detect any revertants in the product molecules making accurate estimation of error rates impossible. We can only say that the error rate on this template is less than 2.9 X 10” (see Ref. 62).

Since DNA synthesis begins at random nicks in some of the RF template molecules, the leading strands can be either of

MINUTES OF INCUBATION AT 37.C

FIG. 2. Infectivity of DNA synthesized in vitro. DNA was made under “standard” conditions (see “Materials and Methods”) and samples withdrawn at the indicated times. The extent of DNA synthesis was determined by measuring the incorporation of [”HITTP into trichloroacetic acid-insoluble material. An aliquot of each sample was cut with Pst I endonuclease and the amount of infectious DNA was quantitated by transfection of E. coli spheroplasts. A- - -A, DNA concentration; M, infective centers.

TABLE I Reversion of +x174 amber codons during DNA synthesis in vitro Bacteriophage 4x174 replicative form DNA containing an amber

codon was used as template for DNA synthesis. After 45 min of synthesis at 37 “C under “standard” conditions, the sample was heated to 65 “C for 10 min to stop the reaction. An aliquot was withdrawn for the measurement of the extent of DNA synthesis and the rest of the sample was prepared for infectivity assays as described under “Materials and Methods.” DNA synthesis is expressed as the number of duplications of the input template DNA. Revertants were measured using E. coli W3110 spheroplasts and HF4704 indicator cells. Total infective centers were determined using E. coli CR63 spheroplasts and HF4714 indicator cells. All values given are averages of duplicate transfections. Each line in this table represents the result of an independent experiment done with an independently synthesized DNA sample and a different batch of spheroplasts. Therefore, these results provide an estimate for the range of variability in these infectivity experiments. The DNA synthesized in vitro is 2- to $-fold less infectious than untreated authentic 4x174 R F DNA. However,

+X RF DNA that has been cut and ligated in the absence of any DNA the infectivity of DNA made in vitro is comparable to that of in vivo

synthesis in vitro. The frequency of revertants in the parental DNA was 1.7 X for am3, 1.0 X for am86, and 1 X 10” for am9 DNA.

Infective centers Template Expen- DNA

DNA ment syn- Re- Frequency of revertants no. thesis ver- Total

tant.s

+X am3 (E-) 1 7.5 4 6.0 X lo” 6.7 X lo-‘’ 2 35.0 31 2.6 x lo6 1.2 x lo-> 3 41.9 24 1.11 x 10” 2.2 x 4 60.5 15 1.19 x lo6 1.3 x lo-’

+X am86 (A-) 1 16.1 14 1.26 x lo6 1.1 x lo-’’ 2 16.0 12 7.8 X 10% 1.5 X lo-; 3 33.3 8 6.2 X lo” 1.29 X lo-:

+X am9 (G-) 1 16.0 0 9.9 x 10‘ t l .O x 10“’ 2 28.1 0 3.44 x lo6 t2 .9 x 10“ 3 57.9 0 1.1 x 10‘’ t 9 .1 x 10” 4 78.1 0 1.69 x loti 4 . 9 x 10”

(+) or (-) polarity. An error during the synthesis of the leading strand will lead to the formation of a homoduplex revertant. Errors during the copying of the rolling circle tail (lagging strand synthesis) will lead to the formation of heter- oduplexes with approximately half of these molecules having the am codon on (+) strand and the other half having it on the (-) strand, depending upon which strand of the parental R F molecule contained the nick at which synthesis was initi- ated.

Correction mechanisms which use methylation in the parental strand of DNA to preferentially correct mismatches due to errors during DNA synthesis have been described (41 , 63). A heteroduplex correction mechanism can improve accuracy only if it can selectively remove errors in the newly made DNA strand. This requires some way of modifying the DNA so that the parental strand can be distinguished from the progeny DNA strand. Since both DNA strands are synthesized in vitro in our experiments and DNA modification enzymes are lacking, our estimates of the frequency of revertants should be unaffected by heteroduplex correction mechanisms of this type.

Studies with Deoxynucleotide Substrate Imbalances-An alternative method of increasing the sensitivity of this assay depends upon the assumption that all four deoxynucleotide precursors compete for the same binding site on T4 DNA polymerase. This has been rigorously proven for DNA polymerase 1 of E. coli only (42), but the observation that the frequency of misincorporation of an incorrect nucleotide by T4 DNA polymerase is a linear function of the ratio of the


incorrect and correct nucleotide substrates (4) suggests that this must be true for T4 DNA polymerase also. Since the complete nucleotide sequence of +X174 DNA is known, we have attempted to force the reversion of a mutant codon by biasing the concentration of nucleotide substrates. This method has only been applied in this study to am3 and am86 mutants. (For summary of data for am16 and am9 mutants, see Ref. 62)

Shown in Table I1 are all the possible reversion pathways at the am3 codon. Due to the location of the am3 codon on the +X DNA where genes D and E overlap, only those revertants that give both a functional gene D and gene E will survive. As can be seen from this table, two of the nine possible substitutions (pathways 7 and 9) will definitely yield nonviable products because they generate other nonsense codons either in gene D or in gene E. How many of the other 7 reversion pathways give viable products is not clear. Path- way 4 involving an AT + CG transversion results in the substitution of a serine codon for the amber terminator. Insertion of a serine in this position in the gene E protein by SUI suppressor strains (such as HF4714 on which this mutant is usually grown) results in a functional protein. Since this transversion does not affect the amino acid at this position in the gene D product, the reversion pathway 4 should yield a viable pseudorevertant. Reversion pathway 5 in Table I1 involving an AT -+ GC transition converts the TAG nonsense codon to TGG (trp) resulting in the generation of a true wild type revertant (37). Pathway 6 involving a TAG + TTG change has recently been shown to yield a viable revertant (40). The synthesis of both the strands of @X DNA in these experiments permits us to force the substitutions either on the viral (+) or on the complementary (-) strand. Nucleotide imbalances promoting reversions in either strand are shown in Table 11. In the experiments described below, we have concentrated on trying to force the reversion of the am3 codon by the reversion pathways 4, 5, or 6 of Table I1 because we knew that the products would be viable.

Nucleotide Imbalances Promoting Transition Mutagene- sis-Effects of alterations in the concentration of deoxynucleotide precursors on the yield of revertants induced during DNA synthesis in vitro is shown in Table 111. We find that lowering the concentration of dATP (by a factor of 10) relative to that of the other three dNTPs is mutagenic while lowering the concentration of one of the other three deoxynucleotides has little effect on the frequency of revertants seen (even though all platings were done in duplicate, the variability in

these experiments is such that only an increase in reversion frequency larger than 3-fold over background can be reliably detected). When the concentration of dATP is lowered it could be competing with any of the other three dNTPs. In order to determine which dNTP it was competing with, we raised (10-fold) the concentration of one dNTP at a time and the results show a sharp increase in reversion frequency when the concentration of dGTP was raised. This suggested that dGTP might be competing with dATP. This was confirmed by simultaneously lowering dATP concentration by a factor of 10 thus creating a G/A imbalance of 100. This yielded a 106-fold increase in reversion frequency in the experiment shown. Such dramatic increases in reversion frequencies only occurred when the ratio of dGTP relative to dATP was increased. Several other nucleotide imbalances were tested and these had little effect on the frequency of revertants as is shown in Table I11 (see also Table V).

The addition of 2 m~ dGTP to the reaction mixture after the termination of DNA synthesis had no effect on the error rate observed. Moreover, an increase (to 2 mM each) or a decrease (to 20 ,UM each) in the concentration of all four dNTPs together yielded no increase in mutagenesis. The reaction mixtures used in these experiments also contained 1 mM rATP and 200 ,UM each rCTP, rGTP, and rUTP. Since a hundredfold variation in the concentration of dNTPs relative to that of rNTPs had no detectable effect on the error rate, the rNTPs (or any impurities present in them) must not compete with the dNTPs and affect error rates.

An examination of the reversion pathways in Table I1 suggested that high G/A imbalances may be promoting A T + GC transitions either by the reversion pathway 2 or 5. These same pathways should also be promoted by high dCTP/low TTP imbalances. However, this was very ineffi- cient in promoting the reversion of am3 codon. At a 100-fold imbalance of dCTP/TTP, no significant increase in the frequency of revertants was seen. When we change the concentrations of deoxynucleotides in these experiments, we are not only changing the concentration of G relative to A but also G and A relative to C and T. However, when the ratio of G/A = 100, the G/T, G/C, C/A, and T/A ratios are only 10. As is shown in Table 111, these do not affect the reversion frequency significantly and only the G/A ratio appears to be significant (also see the data in Table V below).

Effects of the Addition of dITP or dUTP-The simplest interpretation of these results is that the amber codon (TAG) is being changed to either CAG or to TGG (true wild type) by

TABLE I1 Nucleotide substitutions possible at the amber 3 codon of +X1 74 DNA

Of the nine possible pathways of mutation at this site, at least three (pathways 4, 5, and 6 ) give viable revertants, and two (pathways 7 and 9) give nonviable revertants. The outcome of the remaining four is not known.

Codon and amino acid change Nucleotide imbalances promoting it during the synthesis of the +X nucleotide Reversion pathway

Gene D Gene E (+) strand (-) strand

586 T 1. T + A 2. T + C

3. T + G

4. A + C 587 A 5. A + G

6. A + T

7. G + A 588 G 8. G + C

9. G + T

GAA (Glu) GTA+ GCA (Ala) (Val)

GGA (Gly)

GTC (Val) GTA- GTG (Val) (Val)

GTT (Val)

AGA (Arg) GCA+ CGA (Arg) (Ala)

TGA (opal)

AAG (Lys) TAG- CAG (Gln)

GAG (Glu)

TCG (Ser) TAG+ TGG (Trp)

(amber) wild type TTG (Leu)

TAA (Ochre)

(amber)

TAG+ TAC (Tyr) (amber)

TAT (Tyr)

High A, low T High C, low T

High G, low T

High C, low A High G, low A

High T, low A

High A, low G High C, low G

High T, low G

High T, low A High G, low A

High C, low A

High G, low T High C, low T

High A, low T

High T, low C High G, low C

High A, low C


TABLE I11 Effect of imbalances in the concentrations of deoxynucleotide substrates on the error rate during DNA synthesis in vitro

DNA was synthesized under standard conditions for 45 min at product DNA synthesized under a certain condition relative to that 37 "C using +X am3 RF DNA as template. The initial concentration in the product DNA made under standard conditions. All other terms of deoxynucleotide substrates was varied as indicated. All other are as in Table I. Experiments I and I1 refer to independent experi- deoxynucleotides were kept at a concentration of 200 p ~ . At the end ments performed with different batches of spheroplasts on different of the DNA synthesis period, the DNA was prepared for infectivity days using independently synthesized DNA samples. The infective assay and the frequency of revertants in the product DNA was center values given were generated from 4 p1 of the cut and ligated determined as described under "Materials and Methods." Relative DNA per sample. error rate is defined as the ratio of the frequency of revertants in the

Condition DNA synthesis Infective centers

Revertants Total Frequency of revert- Relative rate,r

ants

Experiment I Standard (200 p~ each) 20 p~ each 2 mM each

20 ,UM dATP 20 p~ dCTP 20 p~ dGTP 20 p~ TTP

2 m~ dATP 2 mM dCTP 2 m~ dGTP 2 mM TTP

2 t n ~ dGTP, 20 p~ dATP 2 m~ dCTP, 20 p~ dTTP 2 mM TTP, 20 ,UM dCTP 2 mM dATP, 20 JIM dGTP

Experiment I1 Standard (200 p~ each) 50 ,UM dATP 50 ,UM TTP 50 ,UM dATP and TTP 50 ,UM dATP, dCTP, and TTP 50 ,UM dCTP, dGTP, and TTP

5 m~ dATP 5 m~ dCTP 5 m~ dGTP 5 m~ dGTP and dCTP

36.2 57 2.72 x lo6 2.1 x 1 .o 35.4 27 9.6 X lo5 2.81 X lo-' 1.3

0.6 0.59 1 8.4 X 10' 1.2 x

3.1 249 8.8 X lo5 2.83 X 10-~ 13.5

8.7 30 1.03 X lo6 2.9 X 1.4 2.4 10.7 100 1.98 X lo6 5 x

3.9 83 1.06 X lo6 7.8 X 1 0 - ~ 3.7

28.3 56 2.75 X 10' 2.0 x 0.95 43.2 36 3.74 x 10' 9.6 X 0.56 24.9 795 2.66 X 10' 2.99 X 10-~ 14.2 31.5 31 5.41 X lo6 5.7 x 0.3

3.6 1,781 1.02 x 10' 1.75 X 83.3 (106.0) 5.0 24 1.87 X 10' 1.28 X 10-~ 0.6 8.8 71 3.31 X lo6 2.15 X 1 0 - ~ 1.0 3.6 100 9.7 X lo5 1.03 X w 4 4.9

60.5 15 1.19 x lo6 1.3 X lo-$ 1.0 16.9 27 8.54 X lo5 3.2 X 2.5 8.6 11 9.4 x lo5 1.2 x 0.9

5.8 34 8.95 X lo5 3.8 X 2.9 10.3 10 3.4 X 105 2.9 X 2.3

30.9 9 1.27 X lo6 7.1 X lo-" 0.6 50.0 18 1.03 X lo6 1.7 X 10-~ 1.3 24.9 228 8.77 X lo5 2.6 X lo-' 20.0 15.9 117 8.26 X lo5 1.4 X lo-' 10.8

10.2 38 7.88 X lo5 4.8 X 3.7

5 m~ dGTP, 50 p~ dATP 7.6 597 4.27 X lo5 1.4 X 10-~ 107.7 (121.5) 5 mM dCTP, 50 PM TTP 13.2 8 3.21 X 105 2.5 X 10-~ 1.9 5 m~ dGTP and dCTP, 50 p~ dATP 1.2 161 2.18 X lo5 7.4 X 10-~ 56.9 (102.2)

5 m~ dGTP, 50 p~ dATP, dCTP, and 6.7 35 1 6.51 X lo5 5.4 X 41.5 (47.6)

5 m~ dATP, 50 ,UM dCTP, dGTP, and 5.5 5 4.76 X lo5 1.1 x 0.85

and TTP

TTP

TTP

Values for error rates given in the parentheses have been corrected for the contribution of parental DNA to the frequency of revertants.

a high G/A substrate imbalance. Moreover, G-T mispairs occur much more readily than A-C mispairs, at least at this site on +X174 DNA. However, the possibility existed that these results were due to some contaminant in the dGTP used. Since simply increasing the concentration of dGTP (without a change in the ratio of G/A) is not sufficient to give increased errors, any contaminant in dGTP must also compete with dATP. In order to examine the possibility that these results are simply due to the presence of dITP as a contaminant in the dGTP used, we added varying amounts of dITP to the synthesis mixture. The addition of dITP to 20 ~ L M (in the presence of 200 p~ of each of the other four dNTPs) had no effect on the extent of DNA synthesized. The product was fully infectious and no increase in the frequency of revertants was seen (Table IV). Addition of dITP t o 200 ~ L M did not affect the extent of DNA synthesis but the product was nonviable. Analysis of this DNA by electrophoresis in agarose gels re-

vealed rolling circles that were cut into unit length h e a r s by Pst I nuclease and ligated as usual by the DNA-ligase (data not shown). Therefore, the nonviability must be due to the inability of this DNA to carry out a successful cycle of transfection and growth. Since high levels of dITP give lethality rather than mutagenesis, our results cannot be due to contam- ination of dGTP by dITP.

We have also examined the effects of the addition of dUTP to the reaction mixture. At low concentrations, dUTP (up to 2 ~ L M along with 200 ~ L M of each of the other four dNTPs) had no effect on the viability and did not promote mutagenesis (Table IV). DNA synthesized in the presence of 20 ~ L M or higher concentration of dUTP is noninfectious. It is possible, therefore, that the lack of a marked increase in the frequency of revertants, when high dCTP/low TTP imbalances are used, might be due to the presence of a high level of dUTP as a contaminant in dCTP. However, two lines of evidence suggest


TABLE IV Effect of the addition of dZTP or dUTP on the fidelity of DNA

synthesis in vitro DNA was synthesized for 45 min at 37 “C on +X174 am3 RF DNA

template in the presence of the indicated concentration of dITP or dUTP. The frequency of revertants in the product DNA was measured as described under “Materials and Methods.”

Infective centers Condition DNA

synthesis Revert- Total revertants Frequency of

ants

Standard (200 p~ 15.3 16 2.84 X lo6 5.6 X

Standard + 2 p~ 14.3 21 2.61 X lo6 8.0 X

Standard + 20 p~ 17.3 22 1.98 x 10” 1.11 x

Standard + 200 p~ 12.6 0 2.75 x lo5 (3.6 x lo-&

Standard + 0.2 PM 13.8 14 2.48 x lo6 5.6 x lo-‘

Standard + 2 PM 12.8 15 1.29 X 10‘ 1.16 X

Standard + 20 p~ 17.5 o 1.85 X lo5 t 5 . 4 X

each dNTP)

dITP

dITP

dITP

dUTP

dUTP

dUTP

that the dCTP used in these experiments is relatively free of dUTP. First, we find that upon treatment with Pst I nuclease the DNA synthesized in the presence of a high concentration of dUTP gets degraded to small fragments rather than to unit length linear molecules (data not shown). This is because the Pst I restriction nuclease used in these experiments contains nucleases directed against uracil-containing DNA as contaminants in it. In contrast, DNA synthesized even in the presence of 10 mM dCTP was not degraded into small fragments but was only cut into unit length linear molecules. Second, the specific infectivity of the DNA made in the presence of high levels of dCTP was the same as that of the DNA under standard conditions of synthesis (Table 111). On the basis of these results, we conclude that the dCTP used must be relatively free of dUTP and that A-C mispairs simply must be much less frequent than G-T mispairs at the am3 site.

Error Frequency Is Linearly Related to the Ratio of the Correct and the Incorrect Deoxynucleotide Substrate-A plot of the increase in the frequency of revertants at the am3 site as a function of G/A and C/T deoxynucleotide substrate ratios shows that the frequency of revertants is linearly related to the G/A ratio (Fig. 3). The line has a slope of 1 on this log-log plot. Changes in C/T ratio are much less efficient in promoting the reversion of the am3 codon. Up to a C/T ratio of 100, no increase in the frequency of revertants was observed. At a 1000-fold ratio of dCTP/TTP, only a 7.4-fold increase in the reversion of am3 codon was seen. A similar linear increase in error rates in response to deoxynucleotide substrate imbalances during DNA synthesis with T4 replication proteins except in the presence of Mn2+, has also been reported recently by Hibner and Alberts (23). In nucleotide imbalance experiments with E. coli polymerase I11 holoenzyme, however, Fersht (22) observed that the frequency of revertants when plotted against the concentration of dGTP yielded a slope of 2, indicating that the error rate was related to the square of dGTP concentration. In contrast, with T4 enzymes the error rate is a function of G/A ratio rather than G2/A ratio.

This difference between our results from that of Fersht is probably due to the requirement for a higher concentration of dNTPs by E. coli enzymes to reach V,,, for the insertion of the next nucleotide. On the viral strand at the am3 site, the next correct base (at position 588) is a G also. Therefore, an increase in dGTP concentration affects not only the error rate by a G-T mispair formation at position 587, but also the rate

of its fiiation as a permanent error by controlling the rate of polymerization at position 588. The rate of synthesis at the next site is important because it affects the time that is available for the proofreading nuclease of the DNA polymerase to edit any mistakes made in the previous step (see Ref. 22, 23, 44, and 45 for a more detailed discussion). Therefore, the error rate at the am3 site during the synthesis of the (+) strand should be related to the square of dGTP concentration only till a saturating concentration of dGTP for DNA synthesis at position 588 on $X DNA has been reached. Beyond this, any further increase in dGTP concentration should only affect the rate of mispair formation at position 587 and the error rate should become a linear function of G/A ratio. The dGTP concentration needed to achieve V,,, at position 588 on $X DNA with the E. coli apparatus is not known but it has been reported that it is greater than 1 m~ (22). Therefore, the error rate at the am3 site in Fersht’s experiments would not be expected to become a linear function of dGTP concentration till it is higher than 1 m ~ . In contrast, with the T4 replication apparatus, certainly at concetrations above 200 IJM dGTP, the error rate is a linear function of dGTP concentration. There- fore, with the T4 apparatus V,,, at position 588 on the 4X DNA must be reached by 200 IJM dGTP (see also Ref. 23).

DNA Sequence of the Revertants Induced by High G / A Imbalance-Substituting G for A could convert the amber codon TAG, to either CAG or to TGG (Table 11). Since the wild type sequence at this site is TGG (coding for tryptophan) it appeared unlikely that CAG (coding for glutamine) would yield a viable product. Therefore, we expected the revertants induced by high G/A imbalances to contain the codon TGG at the am3 site. In order to confirm this, we have sequenced the DNA from two of the revertants induced in vitro. The sequencing gel for the DNA of one of these revertants is shown in Fig. 4. Sequencing was done by the chain termination

g l L d 4 8 a LL IO-5l RATIO OF DEOXYNUCLEOTIDE IO 100 SUBSTRATES 1000

FIG. 3. Error rate during DNA synthesis in vitro is a function of the ratio of the incorrect and the correct deoxynucleotide substrates. DNA was synthesized in vitro for 45 min at 37 “C in the presence of different ratios of dGTP/dATP or dCTP/TTP as indicated. The frequency of um+ revertants in the product was measured. Deoxynucleoside triphosphates whose concentrations were not varied were present at 200 PM each. Different ratios of G/A were achieved by using 200 p~ each dGTP and dATP (G/A = 1); 240 p~ dGTP, 40 p~ dATP (G/A = 6); 560 p~ dGTP, 40 p~ dATP (G/A = 14); 1.6 mM dGTP, 40 p~ dATP (G/A = 40), and 3 mM dGTP, 40 p~ dATP (G/ A = 75). Variations in C/T ratios were achieved by using 200 PM each dCTP and TTP (C/T = 1); 200 p~ dCTP, 20 PM TTP (C/T = 10); 2 mM dCTP, 20 p~ TTP (C/T = 100); and 20 mM dCTP and 20 p~ TTP (C/T = 1OOO). At the end of the 45-min incubation period, a 3- pl sample was taken for the measurement of the extent of DNA synthesis. Remainder of the sample was prepared for infectivity assays. X-X, dGTP/dATP ratio varied; A-A, dCTP/TTP ratio varied.


method using the viral strand DNA as the template and the Hue I11 25 restriction fragment as the primer. Therefore, the sequence shown is that of the complementary (-) strand. The sequence CTA in the am3 mutant is seen to have been changed to CCA in the revertant, indicating an A T + GC transition at position 587 in the +X DNA. An identical result was obtained for the second revertant (data not shown). These two revertants were obtained from an experiment where a 187-fold increase in the frequency of revertants was observed. Therefore, the probability is extremely small (1 /34969) that two spontaneously occurring true revertants were picked ac- cidently. Since we sequenced the DNA of only two revertants i t is possible, that other substitutions capable of generating viable revertants also occurred (see the data in Table I 1 1 concerning the effects of G/T and G/C imbalances; also see the data in Table V).

A t t e m p t s to Promote Transuersions at the am3 Site-In addition to the true wild type, there are at least two other

Rever tan t FIG. 4. Sequence of 6x174 DNA at the amber 3 site in the

am3 mutant and its revertant. Hae 111 fragment 5 DNA was used as a primer with either the am3 or am3 revertant @X viral strand circles as template. DNA was synthesized with the large fragment of E. coli DNA polymerase I enzyme in the presence of ['2P]dATP, three unlabeled dNTPs, and the indicated dideoxynucleoside triphos- phate. Only a portion of the autoradiograph of the sequencing gel is shown. The sequence deduced for the minus strand of the am3 mutant and its revertant is shown. The revertant was induced by synthesizing DNA in vitro in presence of a dGTP/dATP ratio of 1OOO. The formation of the revertant is seen to have involved a T + C substitution at position 587 on the minus strand.

viable revertants possible at the a m 3 site. The formation of these revertants requires AT + CG or A T + TA transversions. These could occur through either an A-G or a C-T mispair (for pathway 4 ) or an A-A or a T-T mispair (for pathway 6 of Table 11). The substrate imbalances needed to promote these mutations are shown in Table 11. We have examined the effects of these imbalances on the reversion of the a m 3 mutant and the results are shown in Table V. To prevent A T + GC transitions through G-T mispairs that occur readily at this site, we have kept the concentration of dGTP equal to or lower than the concentration of dATP in these experiments. A slight, but consistent, increase in the frequency of revertants is seen when a high AG/T ratio (promoting both A-A and A-G mispairs) is used in these experiments. With a high A/T imbalance (promoting A-A mispairs) a level of increase smaller than that seen with AG/ T imbalance is usually seen (one of these experiments is shown in Table V; also see the data in Table 111, experiment 11). Since the increase in revertant frequency is so small, it is difficult to tell exactly how much of the 4- to 5-fold increase seen with AG/T imbalances is due to A-A and how much due to A-G mispairs. One would have to sequence a large number of these revertants to determine the exact contribution of these two mispairs to the observed increase in error rate. With a high C/AG imbalance (promoting C-T and C-C mispairs) or a T/AG imbalance (promoting T-T and C-T mispairs) no increase in the frequency of revertants was obtained even when an initial ratio of 250 was used. Therefore, C-T and T-T mispairs (that can also generate these two viable pseudore- vertants by the reversion pathways 4 or 6 of Table 11) must be less frequent than the corresponding purine-purine pairs at the position 587 on the @X174 DNA.

Effec t of Neighboring DNA Sequence on Error Rate- Mutation rates in uiuo can be strongly site-specific ( 1 , 4 6 , 4 7 ) . Significantly different reversion rates have been observed even for mutations that require the same base change for reversion. Like the am3 mutant, +X am86 (gene A) mutant also requires an AT + GC transition (TAG + CAG) for reversion to true wild type (38 ) . An examination of the effects of all possible substitutions at this site reveals that, in addition to the true wild type revertant, at least one other viable revertant requiring an A T + CG transversion is also possible. We have examined the effects of deoxynucleotide substrate imbalances promoting these changes on the reversion of am-

TABLE V Effect of deoxynucleotide substrate imbalances promoting transversions at the +X174 amber 3 site

Experiment no. Condition

Infective centers

Total DNA

synthesis Revert- F ~ ~ ~ ~ ~ ~ ~ f Relative error rate" ants ~~ ~

I Standard 25.0 36 1.21 x 10" 2.97 X 1 .o 2 mM dGTP, 20 PM TTP 6.6 293 7.4 x 1 0 4.0 X 13.3 (14.3) 2 mM dGTP and dATP, 20 p~ TTP 5.3 76 7.5 x 1 0

11. Standard 3.4 (4.0)

2 m~ dGTP and M T P , 20 p~ TTP 3.8 28 3.28 X 1 0 8.54 X lo-' 111. Standard 60.5 15 1.2 x lo6 1.3 X 1 .o

4.0 (4.7)

2 mM dCTP, 20 p~ dATP and dGTP 17.5 8 9.0 x lo5 9.0 x lo-" 0.7 2 mM dCTP, 200 p~ dATP and dGTP 13.2 1 1 6.5 X lo5

IV. 1.7 X

Standard 43.4 12 6.62 X lo5 1.81 X lo-' 1 .o 5 mM dCTP, 20 p~ dATP and dGTP 7.2 10 9.2 X lo5

V. Standard 1.1 x 0.6

34.8 1 6 4 1.9 x 10' 8.6 x lo-" 1 .o 2 mM dGTP and dATP, 20 p~ TTP 3.7 81 2.2 x 10" 3.7 x lo-$ 4.3 (5.4) 2 mM dATP, 20 pM TTP 6.3 198 7.8 X IO6 2.5 x lo-$ 3.0 (3.9) 2 mM TTP, 20 p~ dGTP and dATP 3.1 24 2.2 x 10" 1.1 x 1.3 (1.7) 2 mM dCTP, 20 p~ dGTP and dATP 4.0 26 2.3 X 10" 1.1 x 1.3 (1.6) 5 mM TTP, 20 PM dGTP and dATP 2.9 18 2.7 X 10" 6.7 x lo-" 0.8 5 mM dCTP, 20 p~ dGTP and dATP 4.2 30 2.6 X 10" 1.2 x 1.3 (1.5)

1.0 X 1 0 - ~ 41.9 24 1.1 x lo6 2.15 X 1.0

1.3 (1.4)

" Values for error rates given in the parentheses have been corrected for the contribution of parental DNA to the frequency of revertants. All other numbers are as in Table 111.


ber 86 codon during DNA synthesis in uitro. As shown in Table VI, a high dGTP/low dATP imbalance yields a significant increase in the frequency of revertants observed. The

TABLE VI Effect of deoxynucleotide substrate imbalances on the reversion of

G 1 7 4 am86 codon

Substrate imbalance" zis Revert-

DNA Infective centers

ants Total

Experiment I None A/C = 100 A/G = 100 A/T = 100 C/A = 100 C/G = 100 C/T = 100 G/A = 100 G/C = 100 G/T = 100 T/A = 100 T/C = 100 T/G = 100 GA/T = 100 C/GA = 100 C/GA = 100 G/A = 10 G/A = 10 G/A = 25 G/A = 25 G/A = 50 G/A = 50 G/A = 100

Experiment I1 None C/T = 100 G/A = 100 GA/T = 100 C/AG = 100

52.0 13.4 11.9 12.8 15.1 20.1 19.1 15.8 19.4 20.0 15.8 21.1 22.0

7.2 12.3 9.8

13.3 12.0 14.0 9.9

12.4 16.4 16.1

33.3 17.6 8.5 4.5

11.3

155 103 139 135 122 102 142 476 324 254 164 176 292 99 93

121 130 115 198 161 359 235 408

8 10 35 9 8

5.1 x LO6 4.4 x lo6 3.5 x IO6 7.8 x IO6 3.6 X 10" 4.0 X IO' 2.4 X lo6 4.1 X IO6 4.3 x lo6 3.2 X lo6 2.3 x 10' 2.9 x 10' 4.0 X lo6

2.9 X lo6 2.3 X lo6

2.2 x lo6

3.0 x lo6 3.3 x IO' 4.4 x lo6 3.3 x 10' 4.1 X 10' 2.5 x IO6 2.8 x lo6 6.2 X lo5 7.3 X lo5 4.7 x 105 3.6 X 105 4.2 X 105

Frequency of tive Rela-

revertants error rate

3.0 X

4.0 X lo-' 1.7 X 3.4 x 2.6 x 5.9 x

7.5 x 7.9 x 7.1 X 6.1 X 7.3 x

3.2 X 5.3 x 4.3 x 3.5 x 4.5 x 4.9 x 8.8 x 9.4 x

2.3 x lo-'

1.16 X 10-~

4.5 x lo-'

1.45 X lo-'

1.29 X 1.37 X 7.44 x 2.5 X 1.9 x

1 .o 0.8 1.3 0.6 1.1 0.9 2.0 3.9 2.5 2.6 2.3 2.1 2.4 1.5 1.1 1.8 1.4 1.2 1.5 1.6 2.9 3.1 4.8

1.0 1.1 5.8 1.9 1.5

~

other deoxynucleotide substrate imbalances used did not yield any significant (more than 3-fold over that seen with balanced substrates) increase in the frequency of revertants. This indi- cates that the frequency of A-C, A-G, and C-T mispairs must be lower than the frequency of G-T mispairs at this site. (Transitions are more frequent than transversions at the am86 site.) The data presented in Table VI also show that the reversion frequency at the am86 site increases linearly with the G/A ratio (in the range G/A = 25 to 100).

Estimation of the Frequencies of Various Mispairs at the am3 and am86 Sites-Since the DNA sequence changes at the mutant site predicted on the basis of deoxynucleotide substrate imbalances do occur and the error rate is a linear function of the substrate imbalance, these results can be used to estimate the frequency of mispairs leading to viable revertants at these sites. The revertants observed in these experiments arise from three sources; by duplication of pre-existing revertants in the template DNA, by mispairing on the viral

I TI4 I

IO"

t-

"2

0 IO 20 30 40 50 60

" l o 0 0

P "800 2 --- $

a I-

n I-

"400 %

"200

" 1 0 0

TIME (MINUTES)

FIG. 5. Kinetics of DNA synthesis, deoxynucleotide pool ratio change, and replication errors. DNA was synthesized at 37 "C on +X174 am3 RF DNA templates using standard conditions except for the presence of 2 m~ dGTP, 20 p~ dATP, and 200 PM each dCTP and TTP. DNA synthesis was followed by the incorporation of ["PI dATP into trichloroacetic acid-insoluble material. The change in dGTP/dATP ratio was followed by measuring the amounts of ["HI dGTP and [3ZP]dATP remaining at the indicated times by thin layer chromatography using polyethyleneimine cellulose plates. At the indicated times, a 60-p1 sample was taken for infectivity assays and the frequency of revertants in these was measured as described under "Materials and Methods." DNA synthesis is expressed as number of duplications of the input template DNA. O " - o , DNA synthesis; A-A, frequency of revertants; U - €I, dGTP/dATP ratio.

The substrate imbalance ratios indicated in this table were obtained by using 200 PM of each dNTP for balanced substrates. To obtain an imbalance ratio of 100, 2 mM of a given dNTP was used along with 20 p~ of the dNTP being biased against. G/A ratios of IO, 25, and 50 were similarly obtained by using 20 p . ~ dATP and 200 p ~ , 500 p ~ , and 1 mM dGTP, respectively. Unbiased nucleotides were at 200 p ~ . All other numbers are as in Table 111. Experiments I and I1 are separate experiments using the same batch of template DNA 1 year apart. It can be seen that these experiments are reasonably reproducible in our hands even when done with different batches of spheroplasts and replication proteins. It also shows that greater reproducibility is achieved if samples from the same experiment transfected into the same spheroplast in parallel are compared.

-

TABLE VI1 Estimation of error frequencies a t the am3 site of &X1 74

Substrate ratios" DNA synthesish Infective centers/&

s u - SU' Frequency of revert-

ants Estimated error frequency'

Balanced 13.1 5.1 x 10' dGTP/dATP = 5 14.2 3.1 X lo4 dGTP/dATP = 25 11.9 9.0 X lo4 dCTP/TTP = 5 20.1 4.2 X 10' dCTP/TTP = 25 14.0 4.0 X lo3 dCTP/TTP = 100 4.0 2.8 X 103

of the four dNTPs for the balanced reaction. dGTP/dATP ratios Substrate ratios indicated were obtained by using 200 PM of each

were varied by using 500 p~ or 2.5 m~ dGTP with 100 p~ dATP and 200 WM dCTP and TTP. dCTP/TTP ratios were changed using 500 pM, 2.5 m M , or 10 mM dCTP with 100 p~ TTP and 200 pM each dkTP and dGTP.

DNA synthesis is expressed as number of template equivalents synthesized in the 45-min incubation at 37 "C.

Since dGTP/dATP imbalances (promoting G-T mispairs) did yield a significant increase (more than 3-fold over that seen with

9.6 x 10' 5.3 x 1O-l' 1.1 x 10' 2.8 X 10-~ 5.6 x (G-T)

1 x 109 4.2 X 10"' 18.4 X 10" (A-C) 8.4 X 10" 1.1 X 10-~ 4.4 X (G-T)

8.5 x 10' 4.7 x lo-" t1 .9 X (AX) 4.1 X 10" 6.9 X 56.9 x (A-C)

balanced substrates) in the frequency of revertants, the error frequencies for G-T mispairs were estimated from the slope of the straight line obtained by plotting the increase in the frequency of revertants against G/A pool bias. For dCTP/dTTP imbalances (promoting A-C mispairs) the exact error frequencies cannot be estimated because no significant increase in the frequency of revertants was obtained with any of the imbalances used. The estimated error frequencies are only upper limits obtained by dividing the frequency of revertants observed by substrate imbalance ratio.


strand, and by mispairing on the complementary strand. Bias- ing one nucleotide substrate against another (such as high dGTP/low dATP used here) should promote mutagenesis on one side of the replication fork only (Table 11). Therefore, a 100-fold ratio of G/A should only increase the frequency of G- T mispairs by a factor of 100 based on a simple linear depend- ence of error rates on this ratio. Similarly, a 100-fold increase in C/T ratio should increase the frequency of A-C mispairs by a factor of 100. The frequency of a mispair (promoted by a certain imbalance) at a site can then be estimated by dividing the increase observed in the frequency of revertants by the substrate imbalance ratio used (see Tables VI1 and VIII). However, this is only accurate if the deoxynucleotide bias ratios remain unchanged during the course of the experiment.

By measuring the concentration of the substrates through- out such imbalance experiments, we have found that a sub- stantial percentage of the nucleotide being biased against (such as dATP present at an initial low Concentration of 20 p~ in the experiment shown) is used up to make DNA or is converted to dNMP during the course of a 45- to 60-min incubation so that a significant change in the ratio of the correct and incorrect nucleotides has occurred by the end of the experiment (Fig. 5). Most of this change occurs toward the end of the experiment. Initial 30 min of incubation in the experiment shown, resulted in about six duplications of the input template DNA (about 7 p~ dATP used). This changed the deoxynucleotide bias ratio only by a factor of 1.6. Between 30 and 60 min, only three more duplications of the template DNA occurred. This synthesis along with an increased turn- over of dATP changed the ratio of G/A by a factor of 5.4 in the last 30 min of the experiment (from 160 to about 900, lowering the concentration of dATP to about 2 p ~ ) . The frequency of revertants increased from at 0 min to 9.2 X

at 30 min. The large change in the G/A ratio that occurred between 30 and 60 min had only a small effect (less than 2-fold) on the frequency of revertants. If the change in substrate ratios in this experiment was completely ignored and the frequency of G-T mispairs estimated from the frequency of revertants seen in the 60-min sample, the mispair frequency would be only overestimated by about a factor of 2. Of course, in other experiments the error in estimation may be larger than this if a bigger change in the substrate imbalance occurred or more DNA was made toward the end of the experiment. Therefore, in order to avoid this problem alto- gether and obtain more accurate estimates of error rates, we have redone many of the deoxynucleotide imbalance experiments as described below.

The simplest method to avoid this complication is to raise the concentration of the dNTP being biased against to 100 p ~ . The results of an experiment where this was done is shown in Table VII. In our system, the maximum dNTP concentration that can be used before severe inhibition of DNA synthesis occurs is 5 mM for dATP and dGTP and 10 mM for dCTP and TTP. With this approach, therefore, the highest G/A or similar imbalance that can be used is 50-fold and the limit for GA/T or similar imbalances is 25-fold. With C/T, C/AG or similar imbalances, the limit is 100-fold. As can be seen from the data in Table VII, this results in an observable increase in errors at the am3 site for G-T mispairs only. The exact error frequency of other mispairs cannot be estimated.

An alternative method is to stop DNA synthesis after a 30- min incubation before any large change in substrate ratios has occurred. We have used this method to estimate error frequency for some of the mispairs leading to viable revertants at the am3 and am86 sites (Table VIII). At both sites, almost all AT -+ GC transitions occur through a G-T mispair. The error frequency at the am3 site is seen to be about 8-fold

TABLE VI11 Estimation of mispairing frequencies a t the 6x1 74 am3 and am86

Mutant Site

am3

am86

imbalance" Nucleotide

None G/A = 100 C/T = 500 AG/T = 100 C/AG = 250 T/AG = 250 None G/A = 200 C/T = 500 AG/T = 100 C/AG = 250 T/AG = 250

sites

promoted Mispair

G-T A-C A-A + A-G

None

C-T + C-C

None G-T A-C A-A + A-G

T-C + T-T

C-T + C-C T-C + T-T

2 k 0.6 X 10-~ 7 f 2 X

6.5 & 2.4 x 6.8 X

-5.6 X 7.6 k 2.4 X 1 9 X lo-"

3 f 0.9 X 10-~ 4 k 2.7 X

4 . 2 X 1 0 - ~ ~ 1 . 6 X

1.9 1.3 X 10-~ 1.47 k 0.7 X 6.4 X IO" 3.7 k 0.9 x 2.9 k 1.1 x

<7.4 x ~ 2 . 9 x 10"

3.1 f 2 X lo-' <1.2 X 10" 2.9 f 1.7 X 10-~ 4 . 2 X 10"

I .~ I

* These nucleotide imbalances were obtained by keeping the concentration of nucleotides being biased against at 20 p and raising the concentration of the indicated nucleotides to get the desired ratio. The nucleotides not involved in the imbalance were kept at 200 PM.

* These values were obtained from an experiment where DNA was synthesized for 30 min at 37 "C. The frequencies of revertants were derived from four platings on each spheroplast (Su- and Su') using two samples of DNA synthesized in parallel for each condition shown.

e The change in nucleotide ratios was checked by measuring the concentration of dNTPs that were being biased against. In all cases the change was less than 2-fold. No correction was made for the small change in ratios observed. The error rates given, therefore, may be slight (less than 1.5-fold) overestimates.

Frequencies of the various mispairs from these dNTP imbalances were calculated as follows: The fluctuation in the frequency of revertants in these pool bias experiments (from experiment to experiment even with the same pool bias) can be upto 3-fold (see Tables I, 111, V, and VI). Therefore, for the nucleotide imbalances that gave a greater than 3-fold increase in the frequency of revertants, the mispair frequency was computed from the observed values by dividing the increase in reversion frequency observed by the nucleotide imbalance ratio. For those imbalances that gave less than a 3-fold increase in the frequency of revertants (over the reversion frequency with balanced substrates), we have simply divided the observed frequency of revertants by the imbalance ratio used and provide an upper estimate for the frequencies of these mispairs. This assumes that the error frequency for these mispairs increases linearly with the imbalance ratio used.

higher than at am86 site. Transition mutations appear to occur more readily than transversions at both sites. At least at the am3 site, transversions appear to occur more frequently through a purine-purine than through a pyrimidine-pyrimidine mispair. Also, see the data for the reversion of the 4x174 mutant am16 (gene B) which requires a TAG + GAG transversion for its reversion (Ref. 62). Here also all AT "+ CG transversions occur through A-G mispairs and not C-T mispairs.

DISCUSSION

Fidelity of D N A Synthesis Catalyzed by Phage T4 Repli- cation Proteins in Vitro-We have measured the fidelity of DNA synthesis in vitro catalyzed by seven T4 phage-coded proteins using an infectivity assay. It was found that more errors occur when substrate imbalances promoting the reversion of a mutant codon in the DNA template are present. The frequenceis of various mispairs were estimated by dividing the observed increase in the frequencies of revertants by the substrate imbalance ratios used (Tables VI1 and VIII). The results of these substrate imbalance experiments are fairly reproducible as judged by a comparison of independent experiments done on different days with different batches of spheroplasts (Tables I, 111, V, and VI). The frequencies of revertants obtained for a given experimental condition always


fall within a 2- to %fold range. Therefore, our estimates for error rates are only accurate to this degree. The results presented here show that the fidelity of DNA synthesis in vitro by these seven proteins together is quite good and appears to be substantially better than that seen during the copying of homopolymer templates by T4 DNA polymerase alone (4, 5).

Comparison of Fidelity of DNA Synthesis in Vivo and in Vitro-It is difficult to assess if the accuracy seen in vitro is comparable to that seen during DNA replication in vivo because T4 replication proteins never replicate 4x174 DNA in uiuo. The error rate at the am3 site (5.0 X in vitro is substantidy higher than that at most sites in T4 genome in vivo (1.2 X lo-'). The error rate at the am86 site (6.4 X and particularly at the am9 site (<2.9 X is closer to the average error rate during T4 replication in uiuo. Even though the error rate at the am9 site is close to the average error rate during DNA synthesis in uiuo, we still do not know if all the features involved in generating the fidelity of replication in uiuo are present during DNA synthesis in vitro by these seven purified proteins. Such conclusions will have to wait till a method permitting the determination of error rates during the duplication of T4 DNA in vitro is developed.

The fidelity of DNA synthesis in vitro may well be lower than that during replication in uiuo. Several possible expla- nations for such a difference can be offered. The "standard" conditions used in this study were designed to maximize the rate of DNA synthesis (19). It might be possible to increase the fidelity in DNA synthesis further by changing these conditions. The data in Table I11 show that the rate of DNA synthesis, in the range seen in these experiments, has little effect on error rate. An 8-fold change in DNA synthesis rate while keeping the nucleotide imbalance constant at G/A = 10 had little effect on error rate (revertant frequencies of 2.83 X

There may be other replication proteins that increase fidelity in uiuo. Recently, the products of genes 39,52, and 60 have been purified as a tight complex having a topoisomerase activity (48, 49). It remains to be seen if the addition of this complex has any effect on the fidelity of replication. It has also been proposed (50, 51) that the replication complex in uiuo is coupled to an aggregate of the enzymes involved in the synthesis of the deoxynucleotide precursors. This coupling may be important for high fidelity. Finally, postreplication repair enzymes that selectively correct errors in the newly replicated strands may be involved in uiuo. Evidence for such enzymes has been obtained in E. coli (41, 52, 63). These enzymes use the presence of methyl groups on the parental DNA strand to distinguish the newly made progeny strand from the old strand. The existence of a T4-coded methylase (53) raises the possibility that such an error correction mechanism may also operate in T4. Error correction mechanisms like this can increase accuracy by as much as a 1000-fold. An increase of this magnitude would be enough to bring accuracy observed in vitro to the levels seen in uiuo.

Role of the Other Replication Proteins in Increasing the Fidelity of DNA polymerase-Since the error rate for T4 DNA polymerase alone during duplication of natural DNA templates is not known, we cannot tell how much of an increase in fidelity the addition of the other proteins makes. If it is assumed that the error rates are the same on homopolymer templates and 4x174 DNA, then a comparison ofthe frequency of A-C and C-T mispairs on homopolymer tern- plates by T4 DNA polymerase alone (4, 5) with that on +X template by the combination of seven proteins suggests that accuracy is increased at least 300-fold. Which protein contrib- utes how much to this increase and the mechanisms involved

and 2.99 X

remain to be determined. The other replication proteins could increase the accuracy of DNA polymerase either by increasing the discrimination between the correct and the incorrect deoxynucleotide substrates in the initial polymerization step or by increasing the efficiency of the proofreading step.

Both genetic and biochemical evidence suggesting that the gene 32 protein, a helix-destabilizing protein, plays a significant role in increasing the accuracy of T4 DNA polymerase are already available (6-10). The mechanism(s) involved, however, is not known. It is quite likely that this protein contrib- utes to the accuracy by changing the structure of template DNA. It is known to melt intrastrand hydrogen bonds and make single-stranded DNA more rigid (54). This might allow for a more strict pairing of bases during the initial polymerization step. This protein is also known to reduce stacking interactions between adjacent bases in a single-stranded DNA (55). Topal et al. have proposed that the gene 32 protein may reduce incorporation of an incorrect nucleotide that has been stabilized by stacking interactions with the residue at the primer terminus (56). Gene 32 protein may also increase the efficiency of the proofreading step by a general destabilization of the primer terminus, thus facilitating the detection of an incorrectly paired base. In addition to its interactions with single-stranded DNA, this protein has also been shown to directly interact with T4 DNA polymerase (57). This inter- action may also be important in increasing the accuracy of DNA polymerase.

Recently, it has been shown that the addition of the polymerase accessory proteins 44/62 and 45 in presence of ATP increases the discrimination between adenine and Z-amino- purine deoxynucleoside triphosphates (17). This increase in selectivity was shown to be due to an increase in the 3l-5' exonuclease activity. An increase in the proofreading exonuclease activity and fidelity is also seen with many antimutator DNA polymerases. However, there is one very significant difference. Increased Proofreading activity in the antimutator polymerases results in a marked decrease in the rate of DNA synthesis. Both correct and incorrect deoxynucleotides are repeatedly polymerized and excised resulting in little net synthesis. In contrast, the addition of 44/62 and 45 proteins not only increases the proofreading exonuclease activity of the DNA polymerase but also substantially increases the rate of DNA synthesis (17,39,58). Therefore, these proteins must not only increase the 3/45' exonuclease activity but also increase its specificity so that the correct deoxynucleotides are not removed from the primer terminus very often and a decrease in the rate of DNA chain growth due to indiscrimi- nate nucleolytic attack is avoided.

Mispairs Involved in the Generation of Transition Muta- tions-The existence of rare tautomeric forms of the bases led Watson and Crick (59) to propose a mechanism for the formation of transition mutants. They proposed that this is mediated by A-C or G-T base pairs. Since these base pairs occur rarely and are resolved into one mutant and one normal progeny duplex in the next replication cycle, it has not been possible to determine how often one or the other of these two mispairs is involved in the generation of a given transition. Our ability to force the induction of transition mutants by deoxynucleotide substrate imbalances during DNA synthesis in vitro reveals that almost all transitions at both am3 and am86 sites involve G-T mispairs. This is presumably due to the much greater stability of a G-T mispair relative to an A-C mispair. At least in tRNA anticodon-anticodon complexes, a G-U pair is much longer lived than an A-C pair (60). Studies with T4 DNA polymerase and poly(dA) .poly(dT) template also reveal a much smaller discrimination between dGTP and dATP (ratio of K, = 11) than between dCTP and TTP (Km


ratio = 267) suggesting that G-T pairs form much more readily than A-C pairs ( 1 1 , also see Ref. 56). More recent studies with helical polynucleotides containing an occasional mispair also show that a G-U pair is substantially more stable than an A- C base pair.'

If mispairs involving rare tautomeric bases were the inter- mediates leading to transition mutations, as originally proposed by Watson and Crick, then, one would predict a higher frequency for G-T mispairs compared to A-C mispairs, simply because G-T mispairs have three hydrogen bonds while A-C mispairs have only two (see Fig. 1 of Ref. 61 for structure of these mispairs). Since the tautomerism frequencies for the four bases are roughly the same (61 ) , one would expect that G-T and A-C mispairs would be formed more or less at the same rate. However, due to differences in their stabilities it would be expected that the DNA polymerase would recognize a G-T mispair as a properly paired intermediate for a longer time than an A-C mispair. The exact difference in the stabilities of G-T and A-C mispairs are not known. Therefore, it is not possible to tell if the magnitude of difference we are observing (approximately 80-fold at the am3 site) is what would be expected on the basis of free energy difference between these two mispairs. The exact magnitude of the difference in the frequency of G-T and A-C mispairs at different DNA sites may well turn out to be somewhat different than the one we have measured depending upon stacking and even longer range interactions.

Mispairs Involved in the Generation of Transversion Mu- tants-Generation of transversion mutations requires a purine-purine or a pyrimidine-pyrimidine base pair. Either of these would result in a serious distortion of the Watson-Crick double helix. Topal and Fresco have proposed a model of base pairing that allows for some of these without distorting the double helical structure of DNA (61). According to this model, all purine-purine pairs (A-A, A-G, and G-G) can occur if one of the two purine-purine bases is in syn form and the other one has undergone a tautomeric shift. Pyrimidine-pyrimidine pairs (C-C, C-T, and T-T) can not be accommodated in the double helix by this mechanism. Therefore, an AT --f CG transversion can occur only through an A-G mispair. Simi- larly, an AT + TA transversion can only occur through an A- A mispair. We have tried to test this prediction by promoting the formation of pseudo-wild type revertants requiring transversions at am3 site. An increase in the frequency of revertants at the am3 site was seen with a high AG/T imbalance (promoting A-A and A-G mispairs) but not with C/AG or T/AG imbalances (promoting C-C, C-T, and T-T mispairs). The effect was not very dramatic, resulting in only a 4- to 5-fold increase in the frequency of revertants with a AG/T ratio of 100. These results are consistent with the Topal-Fresco model. A more definite proof can be obtained with 4x174 mutants that are known to require a transversion for their reversion. At least two such mutants, am16 in gene B and ts116 in gene A, are presently available (36). Results obtained with the am16 mutant, which requires a TAG + GAG transversion for reversion, suggest that at this site also transversions occur primarily through purine-purine mispairs (62).

Topal-Fresco model of base substitution mutagenesis made a second prediction also. Since transversions require two different kinds of equilibria (syn-anti as well as tautomeric) while transitions need only one (tautomeric), they predicted that at any given site on DNA, transitions would be more frequent than transversions. Our results also show that this prediction is met at both am3 and am86 sites. The mutant am16 appears to be the only exception to this rule among the

' J. Fresco, personal communication.

five amber mutants we have so far examined (Ref. 62).' The frequencies of mispairs at the am16 site were determined to be 1.6 X for G-T and 1.2 X for A-G (62). At both am3 and am86 sites, G-T mispairs (leading to AT + GC transitions) are the most common error. Similar results have also been obtained at other sites on the @X174 DNA.' Recent results from the laboratory of L. Loeb, using DNA polymerase I, also show that transitions are more common than transversions (43 ) .

Effects of Base Stacking Interactions with the Primer Teminal Residue and the Proofreading Exonuclease Activity of the DNA Polymerase-Finally, it should be noted that in this study we have only looked at the effect of deoxynucleotide imbalances on the reversion of @X mutants am3 and am86. The influence of the availability of the next correct nucleotide or the primer terminal residue on the error rate was not considered in any detail. Intuitive reasoning dictates that both these factors must play some role, at least, in the generation of mispairs at the nucleotide insertion step as well as at the proofreading step. In our hands, at the am3 site, the concentration of the next available nucleotide (dGTP during the formation of G-T mispairs) played a much smaller role on error rate than was seen by Fersht (22) with the E. coli replication complex. We do not yet know if this reflects a definite difference in the mechanisms of accuracy control of the T4 and the E. coli replication apparatuses.

We are currently doing a detailed series of studies with five sequenced amber mutants of @X174, using not only wild type, mutator, and antimutator DNA polymerases, but also two different mutator forms of gene 32 protein, in order to better understand the relative contributions of base pairing, base stacking, and proofreading functions on replication accuracy.

Acknowledgments-We are grateful to Drs. R. Benzinger. M. Bitt- ner, R. Chambers, N. Godson, C. Hutchison, and M. Topal for helpful advice and gift of materials.

REFERENCES 1. Drake, J. W. (1970) The Molecular Basis ofMutation, Holden-

2. Speyer, J. F., Karam, J. D., and Lenny, A. B. (1966) Cold Spring

3. Drake, J. W., and Allen, E. F. (1968) Cold Spring Harbor Symp.

4. Hall, Z. W., and Lehman, I. R. (1968) J. Mol. Biol. 36,321-333 5. Loeb, L. A,, Weymouth, L. A,, Kunkel, T. A., Gopinathan, K. P.,

Beckman, R. A,, and Dube, D. K. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,921-928

6. Bernstein, C., Bernstein, H., Mufti, S., and Strom, B. (1972) Mutat. Res. 16, 113-119

7. Koch, R. E., McGaw, M. K., and Drake, J. W. (1976) J. Virol. 19,

8. Watanabe, S. M., and Goodman, M. F. (1978) J. Virol. 25, 73-77 9. Mufti, S. (1979) Virology 94, 1-9

10. GiUin F. D., and Nossal, N. G. (1976) J. Biol. Chem. 251, 5224,

11. Kunkel, T. A,, Meyer, R. R., and Loeb, L. A. (1979) Proc. Natl.

12. Barrv, J., and Alberts, B. (1972) Proc. Natl. Acad. Sci. U. S. A .

Day, San Francisco

Harbor Symp. Quant. Biol. 31,693-697

Quant. Biol. 33,339-344

490-494

5225-5232

Acad. Sci. U. S. A. 76,6331-6335

69; 2717-2721 13. Morris. C. F.. Hama-Inaba. H., Mace. D.. Sinha. N. K.. and

Alberts, B. M . (1979) J. Bcol. Chem. 254,6787-6796

Chem. 254,6797-6802

254,9565-9572

14. Morris, C. F., Moran, L. A,, and Alberts, B. M. (1979) J. Biol.

15. Bittner, M., Burke, R. L., and Alberts, B. M. (1979) J. Biol. Chem.

16. Nossal, N. G. (1979) J. BioZ. Chem. 254,6026-6031 17. Liu, C., Burke, R. L., Hibner, U., Barry, J., and Alberts, B. M.

18. Morris, C., Sinha, N., and Alberts, B. (1975) Proc. Natl. Acad. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,469-487

N. K. Sinha, unpublished results.


Sci. U. S. A . 72,4800-4804

Chem. 255,4290-4303 19. Sinha, N. K., Morris, C. F., and Alherts, B. M. (1980) J. Biol.

20. Nossal, N. G. (1980) J. Biol. Chem. 255, 2176-2182 21. Kunkel, T. A,, and Loeb, L. A. (1979) J. Biol. Chem. 254, 5718-

22. Fersht, A. R. (1979) Proc. Natl. Acad. Sci. U. S. A . 76,4946-4950 23. Hibner, U., and Alberts, B. (1980) Nature 285, 300-305 24. Brown, N. G., and Smith, M. (1977) J. Mol. Biol. 116, 1-30 25. Roberts, R. J., Breitmeyer, J . B., Tahachnick, N. F., and Myers,

26. Godson, G. N., and Boyer, H. W. (1974) Virology 62, 270-275 27. Henner, W. D., Kleber, I., and Benzinger, R. (1973) J. Virol. 12,

28. Loening, U. E. (1967) Biochem. J. 102,251-257 29. Galibert, F., Sedat, J. W., and Ziff, E. B. (1974) J. Mol. Biol. 87,

30. Greene, P. J., Betlach, M. C., Boyer, H. W., and Goodman, H. M.

31. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl.

32. Randerath, K., and Randerath, E. (1967) Methods Enzymol. 12A,

33. Weymouth, L. A., and Loeh, L. A. (1978) Proc. Natl. Acad. Sci.

3 4 . Benbow, R. M., Hutchison, C. A., Fahricant, J . D., and Sinshei- mer, R. L. (1971) J. Virol. 7, 549-558

35. Sanger, F., Coulson, A. R., Friedman, T., Air, G. M., Barrell, B. G., Brown, N. L., Fiddes, J. C., Hutchison, C. A,, 111, Slocombe, P. M., and Smith, M. (1978) J. Mol. Biol. 125, 225-246

36. Smith, M., Brown, N. L., Air, G. M., Barrell, B. G., Coulson, A,, Hutchison, C. A., 111, and Sanger, F. (1977) Nature 265, 702- 705

37. Barrell, B. G., Air, G. M., and Hutchison, C. A., I11 (1976) Nature 264,34-41

38. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J . C., Hutchison, C. A,, 111, Slocombe, P. M., and Smith, M. (1977) Nature 265,687-695

39. Alberts, B., Morris, C., Mace, D., Sinha, N., Bittner, M., and Moran, L. (1975) in DNA Synthesis and its Regulation (Gou- lian, M., Hanawalt, P., and Fox, C. F., eds) pp. 241-269, W. A. Benjamin, Inc., Menlo Park, CA

40. Kunkel, T. A., and Loeb, L. A. (1980) J. Biol. Chem. 255, 9961-

5725

P. A. (1975) J. Mol. Biol. 91, 121-123

741-747

377-407

(1974) MethodsMol. Biol. 7, 87-111

Acad. Sei. U. S. A . 74,5463-5467

323-347

U. S. A . 75, 1924-1928

9966 41. Wagner, H., and Meselson, M. (1976) Proc. Natl. Acad. Sci. U. S.

42. Englund, P. T., Huberman, J. A., Jovin, T. M., and Kornberg, A.

43. Loeh, L. A,, Kunkel, T. A., and Schaaper, R. A. (1980) ZCN- UCLA

44. Clayton, L. K., Goodman, M. F., Branscomb, E. W., and Galas, D.

45. Galas, D. J., and Branscomb, E. W. (1978) J. Mol. Biol. 124,653-

46. Benzer, S. (1961) Proc. Natl. Acad. Sci. U. S. A . 47,403-415 47. Coulondre, C., and Miller, J. H. (1977) J. Mol. Biol. 117,525-575 48. Stetler, G. L., King, G. J., and Huang, W. M. (1979) Proc. Natl.

Acad. Sci. U. S. A. 76, 3737-3741 49. Liu, L. F., Liu, C-C., and Alberts, B. M. (1979) Nature 281, 456-

461 50. Wovcha, M. G., Tomich, P. K., Chiu, C-S., and Greenberg, G. R.

(1973) Proc. Natl. Acad. Sei. U. S. A . 70,2196-2200 51. Collinsworth, W. L., and Mathews, C. K. (1974) J. Virol. 13,908-

915 52. Radman, M., Villani, G., Boiteux, S., Kinsella, A. R., Glickman,

B. W., and Spadari, S. (1979) Cold Spring Harbor Symp. Quant. Biol. 43,937-948

A . 73,4135-4139

(1969) J. Biol. Chem. 244,3038-3044

Symp. Mol. Cell. Biol. 19, 735-751

J. (1979) J. Biol. Chem. 254, 1902-1912

687

53. Brooks, J., and Hattman, S. (1973) Virology 55,285-288 5 4 . Delius, H., Mantell, N. J., and Alberts, B. M. (1972) J. Mol. Biol.

55. Jensen, D. E., Kelly, R. C., and von Hippel, P. H. (1976) J. Biol.

56. Topal, M. D., DiGuiseppi, S., and Sinha, N. K. (1980) J. Biol.

57. Huberman, J., Kornberg, A., and Alherts, B. (1971) J. Mol. Biol.

58. Mace, D. (1975) Ph.D. thesis, Princeton University, Princeton,

59. Watson, J . D., and Crick, F. H. C. (1953) Nature 171,964-967 60. Grosjean, H. J., Henau, S. D., and Crothers, D. M. (1978) Proc.

61. Topal, M. D., and Fresco, J . R. (1976) Nature 263,285-289 62. Sinha, N. K., and Haimes, M. D. (1980) ZCN-UCLA Symp. Mol.

63. Glickman, B. W., and Radman, M. (1980) Proc. Natl. Acad. Sci.

67,341-350

Chem. 251, 7215-7228

Chem. 255, 11717-11724

62, 39-52

NJ

Natl. Acad. Sei. U. S. A. 75,610-614

Cell. Biol. 19, 707-723

U. S. A . 77, 1063-1067

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THE JOURNAL OF Vol. 256, No. 20. Issue of October 25. pp ...THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 20.Issue of October 25.pp. 10671-10683, 1981 Prmted in U.S.A. Molecular