DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli

The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G.C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.     

The Specificity of Topoisomerase-Mediated DNA Cleavage Defines Acridine-Induced Frameshift Specificity within a Hotspot in Bacteriophage T4

Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro. The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond. In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated. These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site. Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis. This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene. The model successfully predicted the results. When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions. DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site. These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.     

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.     

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complex Frameshift Mutations Mediated by Plasmid Pkm101: Mutational Mechanisms Deduced from 4-Aminobiphenyl-Induced Mutation Spectra in Salmonella

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in ~2,400 4-aminobiphenyl (4-AB) +S9-induced revertants of the -1 frameshift allele hisD3052 and of the base-substitution allele hisG46 of Salmonella typhimurium. Most of the mutations occurred at sites containing guanine, which is the primary base at which 4-AB forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100 and 99.9%, respectively, of the reversion events at the hisD3052 allele in the pKM101 plasmid-minus strains TA1978 (uvr(+)) and TA1538 (δuvrB). In strain TA98 (δuvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, ~85% of the revertants also contained the hotspot deletion; the remaining ~15% contained one of two types of mutations: (1) complex frameshifts that can be described as a -2 or + 1 frameshift and an associated base substitution and (2) deletions of the CC or GG sequences that flank the hotspot site (CCGCGCGCGG). We propose a misincorporation/slippage model to account for these mutations in which (1) pKM101-mediated misincorporation and translesion synthesis occurs across a 4-AB-adducted guanine; (2) the instability of such a mispairing and/or the presence of the adduct leads to strand slippage in a run of repeated bases adjacent to the adducted guanine; and (3) continued DNA synthesis from the slipped intermediate produces a frameshift associated with a base substitution. This model readily accounts for the deletion of the CC or GG sequences flanking the hotspot site, indicating that these mutations are, in fact, complex mutations in disguise (i.e., cryptic complex frameshifts). The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G·C -> T·A transversions) is consistent with the finding that 4-AB induced primarily G·C -> T·A transversions at the hisG46 base-substitution allele. The model also provides a framework for understanding the different relative mutagenic potencies of 4-AB at the two alleles in the various DNA repair backgrounds of Salmonella.     

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.     

DNA sequence analysis of spontaneous tonB deletion mutations in a polA1 strain of Escherichia coli K12.

By DNA sequence analysis, we have determined a spectrum of 61 spontaneous mutations occurring in the endogenous tonB gene in the polA1 strain of Escherichia coli. The overall mutation frequency was approximately 2.4-fold higher in the polA1 strain and this was attributable to enhanced rates of deletion and frameshift mutations. Among 39 deletions, a hot spot (17 mutations) was detected: a 13-bp deletion presumably directed by a 3-bp repeated sequence at its end points. The remaining 22 were distributed among 19 different mutations either flanked (16/19) or not flanked (3/19) by repeated sequences. Single-base frameshifts accounted for 8 mutations of either repeated (3/8) or nonrepeated (5/8) bases among which 6 were minus one frameshift. In contrast to previous reports, we did not frequently observe a 5'-GTGG-3' sequence in the vicinity of the deletions and frameshifts. The results presented here indicated an anti-deletion and anti-frameshift role for DNA polymerase I.     

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.     

Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis. In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative

The sequences of more than 600 frameshift mutations produced as a consequence of in vitro DNA replication on an oligonucleotide-primed, single-stranded DNA template by the Escherichia coli polymerase I enzyme (PolI) or its large fragment derivative (PolLF) were compared. Four categories of mutants were found: (1) single-base deletions, (2) base substitutions, (3) multiple-base deletions and (4) complex frameshift mutations that change both the base sequence and the number of bases in a concerted mutational process. The template sequence 5'-Py-T-G-3', previously identified as a PolLF hotspot for single-base deletions opposite G, is also a hotspot for PolI. A PolI-specific warm spot for single-base deletions was identified. Among base substitutions, transitions were more frequent than transversions. Transversions were mediated by (template)G.G, (template)G.A, and (template)C.T mispairs. Multiple-base deletions were found only after PolI replication. Although each of these deletions can be explained by a misalignment mediated by directly repeated DNA sequences, deletion frequencies were often different for repeats of the same length. Both PolI and PolLF produced many complex frameshift mutants. The new sequences at the mutant sites are exactly complementary to nearby DNA sequences in the newly synthesized DNA strand. In each case, palindromic complementarity could mediate the misalignment needed to initiate the mutational process. The misaligned DNA synthesis accounts for the nucleotide changes at the mutant site and for homology that could direct realignment of the DNA onto the template. Most of the complex mutant sequences could be initiated by either intramolecular misalignments involving fold-back structures in newly synthesized DNA or by strand-switching during strand-displacement synthesis. The striking differences between the specificities of complex frameshift mutations and multiple-base deletions by PolI and PolLF identify the existence of polymerase-specific determinants that influence the frequency and specificity of misalignment-mediated frameshifts and deletions.     

To slip or skip, visualizing frameshift mutation dynamics for error-prone DNA polymerases

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol mu and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol mu switches to dNTP-stabilized misalignment. pol beta generates -1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.     

Spontaneous and induced mutability or frameshift strains of Salmonella typhimurium carrying uvrB and polA mutations

Three strains Salmonella typhimurium carrying frameshift mutations affecting the histidine genes (hisC3076, hisD3052 and hisC207) showed increased sensitivity to mutagenesis by ICR-191 (as judged by measuring back mutation to prototrophy), if they were made deficient in excision repair by deleting the uvrB gene. One frameshift strain, hisC3076, also showed increased sensitivity to mutagenesis by ICR-191 when it carried either of two different polA alleles, whereas the hidD305 and hisD207 frameshifts reduced sensitivity to mutagenesis in the presence of these alleles. Studies of spontaneous back mutation to prototrophy revealed siginificant mutator effects of the polA1 mutation on reversion of the hisD3052 frameshift and of the polA3 mutation on reversion of the hisC3076 frameshift. Other smaller mutator effects of the polA alleles on reversion of the his mutations may also be present. In an attempt to explain the complex interactions between different polA alleles and different frameshift mutations, it is tentatively suggested that deletion frameshift may arise mainly during DNA replication, while addition frameshifts may arise mainly during post-replication repair.     

Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.     

Defect in excision repair alters the mutational specificity of PUVA treatment in the lacI gene of Escherichia coli

The sequences of 152 lacI- mutations obtained following exposure of Escherichia coli UvrB- strain NR3951 to ultraviolet light in the presence of 8-methoxypsoralen (PUVA treatment) were compared to the spectrum of mutation induced by PUVA treatment in a Uvr+ strain, NR3835. Mutations recovered following PUVA treatment of the UvrB- strain were quite different from those recovered in the Uvr+ strain. In addition, they occurred at a restricted number of unique sites. For example, A.T----T.A base substitutions at position 141, minus G frameshifts at positions 586/587/588 and deletions of 15 base-pairs from position 78 to 92 accounted for 50% or more of mutations recovered in each of the above mutational classes. This altered mutational specificity was accompanied by a failure to recover mutations frequently identified following PUVA treatment of the Uvr+ strain. These mutations include spontaneous-hotspot frameshifts involving the gain or loss of a tetramer 5'-CTGG-3' repeated three times at position 620 to 631; and minus A.T base-pair frameshifts recovered at potential T-T crosslink sites. These results indicate that while crosslinks may play a substantial role in the induction of mutation in the Uvr+ strain, they do not contribute substantially to mutagenesis in the UvrB- strain. In addition, the data also suggest that excision repair may not always occur in an error-free manner.     

8-oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSalpha

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase α can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSα mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSα and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.     

A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli

Spontaneous frameshift mutations are an important source of genetic variation in all species and cause a large number of genetic disorders in humans. To enhance our understanding of the molecular mechanisms of frameshift mutagenesis, 583 spontaneous Trp+ revertants of two trpA frameshift alleles in Escherichia coli were isolated and DNA sequenced. In order to measure the contribution of methyl-directed mismatch repair to frameshift production, mutational spectra were constructed for both mismatch repair-proficient and repair-defective strains. The molecular origins of practically all of the frameshifts analyzed could be explained by one of six simple models based upon misalignment of the template or nascent DNA strands with or without misincorporation of primer nucleotides during DNA replication. Most frameshifts occurred within mononucleotide runs as has been shown often in previous studies but the location of the 76 frameshift sites was usually outside of runs. Mismatch repair generally was most effective in preventing the occurrence of frameshifts within runs but there was much variation from site to site. Most frameshift sites outside of runs appear to be refractory to mismatch repair although the small number of occurrences at most of these sites make firm conclusions impossible. There was a dense pattern of reversion sites within the trpA DNA region where reversion events could occur, suggesting that, in general, most DNA sequences are capable of undergoing spontaneous mutational events during replication that can lead to small deletions and insertions. Many of these errors are likely to occur at low frequencies and be tolerated as events too costly to prevent or repair. These studies also revealed an unpredicted flexibility in the primary amino acid sequence of the trpA product, the alpha subunit of tryptophan synthase.     

An Oak Ridge legacy: the specific locus test and its role in mouse mutagenesis.

Mutations in the genes encoding single-strand DNA-specific exonucleases (ssExos) of Escherichia coli were examined for effects on mutation avoidance, UV repair, and conjugational recombination. Our results indicate complex and partially redundant roles for ssExos in these processes. Although biochemical experiments have implicated RecJ exonuclease, Exonuclease I (ExoI), and Exonuclease VII (ExoVII) in the methyl-directed mismatch repair pathway, the RecJ- ExoI- ExoVII- mutant did not exhibit a mutator phenotype in several assays for base substitution mutations. If these exonucleases do participate in mismatch excision, other exonucleases in E. coli can compensate for their loss. Frameshift mutations, however, were stimulated in the RecJ- ExoI- ExoVII- mutant. For acridine-induced frameshifts, this mutator effect was due to a synergistic effect of ExoI- and ExoVII- mutations, implicating both ExoI and ExoVII in avoidance of frameshift mutations. Although no single exonuclease mutant was especially sensitive to UV irradiation, the RecJ- ExoVII- double mutant was extremely sensitive. The addition of an ExoI- mutation augmented this sensitivity, suggesting that all three exonucleases play partially redundant roles in DNA repair. The ability to inherit genetic markers by conjugation was reduced modestly in the ExoI- RecJ- mutant, implying that the function of either ExoI or RecJ exonucleases enhances RecBCD-dependent homologous recombination.     

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.     

Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro.

We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The resulting lacZalpha peptide fusion protein remained functional. Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.     

Influence of DNA repair on mutation spectra in Salmonella

This paper reviews the influence of DNA repair on spontaneous and mutagen-induced mutation spectra at the base-substitution (hisG46) and -1 frameshift (hisD3052) alleles present in strains of the Salmonella (Ames) mutagenicity assay. At the frameshift allele (mostly a CGCGCGCG target), ΔuvrB influences the frequency of spontaneous hotspot mutations (−CG), duplications, and deletions, and it also shifts the sites of deletions and duplications. Cells with pKM101+ΔuvrB spontaneously produce complex frameshifts (frameshifts with an adjacent base substitution). The spontaneous frequency of 1-base insertions or concerted (templated) mutations is unaffected by DNA repair, and neither mutation is inducible by mutagens. Glu-P-1, 1-nitropyrene (1NP), and 2-acetylaminofluorene (2AAF) induce only hotspot mutations and are unaffected by pKM101, whereas benzo(a)pyrene and 4-aminobiphenyl induce only hotspot in pKM101⁻, and hotspot plus complex in pKM101⁺. At the base-substitution allele (mostly a CC/GG target), the ΔuvrB allele increases spontaneous transitions in the absence of pKM101 and increases transversions in its presence. The frequency of suppressor mutations is decreased 4× by ΔuvrB, but increased 7.5× by pKM101. Both repair factors cause a shift in the proportion of mutations to the second position of the CC/GG target. With UV light and γ-rays, the ΔuvrB allele increases the proportion of transitions relative to transversions. pKM101 is required for mutagenesis by Glu-P-1 and 4-AB, and the types and positions of the substitutions are not altered by the addition of the ΔuvrB allele. Changes in DNA repair appear to cause more changes in spontaneous than in mutagen-induced mutation spectra at both alleles. There is a high correlation (r²=0.8) between a mutagen's ability to induce complex frameshifts and its relative base-substitution/frameshift mutagenic potency. A mutagen induces the same primary class of base substitution in TA100 (ΔuvrB, pKM101) as it does in Escherichia coli, mammalian cells, or rodents as well as in the p53 gene of human tumors associated with exposure to that mutagen. Thus, a mutagen induces the same primary class of base substitution in most organisms, reflecting the conserved nature of DNA replication and repair processes.