Frameshift mutagenesis: the roles of primer-template misalignment and the nonhomologous end-joining pathway in Saccharomyces cerevisiae

Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs >3N from the yeast lys2ΔBgl and lys2ΔA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. -1 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.     

A Set of Lacz Mutations in Escherichia Coli That Allow Rapid Detection of Specific Frameshift Mutations

We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.     

Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors

We constructed a set of Escherichia coli strains containing deletions in genes encoding three SOS polymerases, and defective in MutS and DNA polymerase I (PolI) mismatch repair, and estimated the rate and specificity of spontaneous endogenous tonB(+)-->tonB- mutations. The rate and specificity of mutations in strains proficient or deficient in three SOS polymerases was compared and found that there was no contribution of SOS polymerases to the chromosomal tonB mutations. MutS-deficient strains displayed elevated spontaneous mutation rates, consisting of dominantly minus frameshifts and transitions. Minus frameshifts are dominated by warm spots at run-bases. Among 57 transitions (both G:C-->A:T and A:T-->G:C), 35 occurred at two hotspot sites. PolI-deficient strains possessed an increased rate of deletions and frameshifts, because of a deficiency in postreplicative deletion and frameshift mismatch corrections. Frameshifts in PolI-deficient strains occurred within the entire tonB gene at non-run and run sequences. MutS and PolI double deficiency indicated a synergistic increase in the rate of deletions, frameshifts and transitions. In this case, mutS-specific hotspots for frameshifts and transitions disappeared. The results suggested that, unlike the case previously known pertaining to postreplicative MutS mismatch repair for frameshifts and transitions and PolI mismatch repair for frameshifts and deletions, PolI can recognize and correct transition mismatches. Possible mechanisms for distinct MutS and PolI mismatch repair are discussed. A strain containing deficiencies in three SOS polymerases, MutS mismatch repair and PolI mismatch repair was also constructed. The spectrum of spontaneous mutations in this strain is considered to represent the spectrum of in vivo DNA polymerase III replication errors. The mutation rate of this strain was 219x10(-8), about a 100-fold increase relative to the wild-type strain. Uncorrected polymerase III replication errors were predominantly frameshifts and base substitutions followed by deletions.     

Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae

DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys(+) revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.     

Ionizing and ultraviolet radiation-induced reversion of sequenced frameshift mutations in Escherichia coli: a new role for umuDC suggested by delayed photoreactivation

The ultraviolet (UV) and gamma radiation-induced reversion of the trpA21, trpA9813, and trpE9777 sequenced-frameshift mutations were studied in Escherichia coli K-12 with or without the plasmid pKM101. Radiation induced the reversion of all 3 frameshifts, and pKM101 enhanced this reversion 10-50-fold. Factors influencing the differential radiation revertability of frameshifts are discussed. The two most revertable frameshifts, trpE9777 and trpA9813, were used as probes to understand the role of the umuDC genes in radiation-induced frameshift reversion. Unlike the UV radiation-induced reversion of base-substitution mutations, the reversion of these frameshifts was not enhanced in a uvrA umuC strain by photoreactivation after a post-UV-irradiation incubation. The UmuDC proteins are suggested to have functions in the radiation induction of frameshifts that are more complex than are their functions in the induction of base substitutions.     

Comparison of spontaneous and adaptive mutation spectra in yeast

Adaptive mutations occur in nongrowing populations of cells to overcome strong, nonlethal selection conditions. Several models have been proposed for the molecular mechanism(s) for this phenomenon inEscherichia coli, but the mechanisms involved in adaptive mutagenesis in the yeastSaccharomyces cerevisiae are largely unknown. We present here a comparison of the mutational spectra of spontaneous and adaptive frameshift reversion events in yeast. In contrast to results fromE. coli, we find that the mutational spectrum of adaptive mutations inS. cerevisiae is not similar to that seen in mismatch repair defective cells, but rather resembles the spontaneous mutational events that occur during normal growth.     

Frameshift mutagenesis of lambda prophage by 9-aminoacridine, proflavin and ICR-191

Summary The changes in DNA base sequence induced in the lambda cI gene in an E. coli lysogen have been determined following mutagenesis by three acridine derivatives: 9-aminoacridine and proflavin, which bind reversibly to DNA; and ICR-191, which attaches covalently to DNA through a half-mustard group. For all three derivatives, most mutations are +1 and-1 frameshifts in runs of adjacent G:C pairs. The specificity of mutagenesis at various sites is similar for all three compounds. Prophage in mutL host cells, deficient in mismatch repair, are much more susceptible to mutagenesis by 9-aminoacridine. The induced mutations are also frameshifts, and the site specificity is the same as in lysogens of wild type cells. Thus, additions or deletions of single bases can be corrected by the mismatch repair system, but mismatch repair does not play an important role in determining the sequence specificity of the mutational events.     

The effect of oxidative metabolism on spontaneous Pol zeta-dependent translesion synthesis in Saccharomyces cerevisiae

DNA lesions can stall or block high-fidelity polymerases, thus inhibiting replication. To bypass such lesions, low-fidelity translesion synthesis (TLS) polymerases can be used to insert a nucleotide across from the lesion or extend from a lesion:base mispair. When DNA repair is compromised in Saccharomyces cerevisiae, spontaneous DNA lesions can lead to a novel mutational event in which a frameshift is accompanied by one or more base pair substitutions. These "complex frameshifts" are dependent upon the TLS polymerase Pol zeta, and provide a mutational signature for mutagenic Pol zeta-dependent activity. In the current study, we have found that a specific subset of the Pol zeta-dependent mutational events requires oxidative metabolism. These results suggest that translesion bypass of spontaneously oxidized DNA bases can be a significant source of mutagenesis in repair compromised cells.     

A DNA polymerase epsilon mutant that specifically causes +1 frameshift mutations within homonucleotide runs in yeast

The DNA polymerases delta and epsilon are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3' --> 5' exonuclease proofreading activity. Many errors arising during replication are corrected by these exonuclease activities. We have investigated the contributions of regions of Polepsilon other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polepsilon mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for +1 frameshifts while there was almost no effect on -1 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading. In combination with a deletion of the mismatch repair gene MSH2, the +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the -1 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes +1 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs). This is the first report of a +1-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polepsilon.     

A Major Role for Bacteriophage T4 DNA Polymerase in Frameshift Mutagenesis

T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.     

The interaction of the Escherichia coli mutD and mutT pathways in the prevention of A:T-->C:G transversions.

The Escherichia coli mutT mutator allele produces high frequencies of exclusively A:T-->C:G transversions. This is thought to be caused by a failure to prevent or remove A:G mispairs during DNA replication. The mutD5 mutator allele maps to the dnaQ locus which encodes the epsilon subunit of the DNA polymerase III holoenzyme. This subunit provides 3'-->5' exonuclease, proofreading, activity for removing mispaired nucleotides at the 3' end of the newly synthesized DNA strand. mutD5 has an altered epsilon resulting in reduced levels of proofreading and subsequent high mutation frequencies for all base-pair substitutions. We have analyzed the interaction between mutD5 and mutT-induced A:T-->C:G transversions by measuring reversion frequencies in mutD5 and mutT single mutator strains and mutD5mutT double mutator strains using the well-characterized trpA58 and trpA88 alleles. We find that the double mutator strains produce more A:T-->C:G substitutions than would be expected from simple additivity of the single mutator strains. We interpret this to mean that the two systems, at least in part, do act together to prevent the same mutational intermediate from producing A:T-->C:G transversions. It is estimated that over 90% of the mutT-induced A:G mispairs are corrected by proofreading at the trpA58 site while only about 30% are corrected at trpA88. Reversion frequencies in the mutD5mutT double mutator strains indicate A:G misincorporations occur about 100 x more frequently at trpA58 than at the trpA88 site. Using these and other data we also provide estimations of the fidelity contributions for mutT editing, proofreading and methyl-directed mismatch repair at the two trpA sites for both transversions and the transition that could be scored. In the case of A:T-->C:G transversions, both mutT editing and proofreading make major contributions in error reduction with mismatch repair playing a small or no role at all. For the A:T-->G:C transition, proofreading and mismatch repair were both important in preventing mutations while no contribution was observed for mutT editing.     

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.     

Base composition of mononucleotide runs affects DNA polymerase slippage and removal of frameshift intermediates by mismatch repair in Saccharomyces cerevisiae

The postreplicative mismatch repair (MMR) system is important for removing mutational intermediates that are generated during DNA replication, especially those that arise as a result of DNA polymerase slippage in simple repeats. Here, we use a forward mutation assay to systematically examine the accumulation of frameshift mutations within mononucleotide runs of variable composition in wild-type and MMR-defective yeast strains. These studies demonstrate that (i) DNA polymerase slippage occurs more often in 10-cytosine/10-guanine (10C/10G) runs than in 10-adenine/10-thymine (10A/10T) runs, (ii) the MMR system removes frameshift intermediates in 10A/10T runs more efficiently than in 10C/10G runs, (iii) the MMR system removes -1 frameshift intermediates more efficiently than +1 intermediates in all 10-nucleotide runs, and (iv) the repair specificities of the Msh2p-Msh3p and Msh2p-Msh6p mismatch recognition complexes with respect to 1-nucleotide insertion/deletion loops vary dramatically as a function of run composition. These observations are relevant to issues of genome stability, with both the rates and types of mutations that accumulate in mononucleotide runs being influenced by the primary sequence of the run as well as by the status of the MMR system.     

Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'----5' exonuclease (proofreading) activity

Escherichia coli strains carrying the temperature-dependent dnaQ49 allele are strong mutators at 37 degrees C. Since the dnaQ49 gene encodes the epsilon subunit of DNA polymerase III, it is thought that the large number of errors results in part from impaired proofreading activity during DNA replication. We have examined dnaQ49-induced reversion patterns of defined trpA alleles to determine the kinds of errors produced by dnaQ49 at 30 degrees C and 37 degrees C. We found that at 37 degrees C dnaQ49 produced all types of base-pair substitutions in addition to frameshifts with transitions generally occurring more frequently than transversions. This generalized mutator activity is very similar to that displayed in rich medium by mutD5, another mutator allele at the dnaQ locus. However, when dnaQ49 strains were cultured at 30 degrees C, not only were reversion frequencies much lower than at 37 degrees C, but in addition, the spectrum was altered. Transversions became proportionally more prevalent in the reversion spectra at the lower temperature. We suggest the possibility that at 37 degrees C dnaQ49 results in defective proofreading and methyl-directed postreplicative mismatch repair, while at 30 degrees C mismatch repair is fully and proofreading partially restored.     

Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.

Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect. We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae. In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines. These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene. Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides. Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.     

High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12.

Slipped-strand mispairing (SSM) may play an major role in repetitive DNA sequence evolution by generating large numbers of short frameshift mutations within simple tandem repeats. Here we examine the frequency and size spectrum of frameshifts generated within poly-CA/TG sequences inserted into bacteriophage M13 in Escherichia coli hosts. The frequency of detectable frameshifts within a 40 bp tract of poly-CA/TG is greater than one percent and increases more than linearly with length, being lower by a factor of four in a 22 bp target sequence. The frequency increases more than 13-fold in mutL and mutS host cells, suggesting that a high proportion of frameshift events are normally repaired by methyl-directed mismatch repair. Of the 87 sequenced frameshifts in this study, 96% result from deletion or insertion of only or two 2 bp repeat units. The most frequent events are 2 bp deletions, 2 bp insertions, and 4 bp deletions, the relative frequencies of these events being about 18:6:1.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces cerevisiae

Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting -1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a -1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.     

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.