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)     

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.     

Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene

We have obtained via DNA sequence analysis a spectrum of 174 spontaneous mutations occurring in the lac I gene of Escherichia coli. The spectrum comprised base substitution, frameshift, deletion, duplication and insertion mutations, of which the relative contributions to spontaneous mutation could be estimated. Two thirds of all lacI mutations occurred in the frameshift hotspot site. An analysis of the local DNA sequence suggested that the intensity of this hotspot may depend on structural features of the DNA that extend beyond those permitted by the repeated tetramer at this site. Deletions comprised the largest non-hotspot class (37%). They could be divided into two subclasses, depending on whether they included the lac operator sequence; the latter was found to be a preferred site for deletion endpoints. Most of the deletions internal to the lacI gene were associated with the presence of directly or invertedly repeated sequences capable of accounting for their endpoints. Base substitutions comprised 34% of the non-hotspot events. Unlike the base substitution spectrum obtained via nonsense mutations, G . C----A . T transitions do not predominate. A new base substitution hotspot was discovered at position +6 in the lac operator; its intensity may reflect specific features of the operator DNA. IS1 insertion mutations contributed 12% of the non-hotspot mutations and occurred dispersed throughout the gene in both orientations. Since the lacI gene is not A + T-rich, the contribution of IS1 insertion to spontaneous mutation in general might be underestimated. Single-base frameshift mutations were found only infrequently. In general, they did not occur in runs of a common base. Instead, their occurrence seemed based on the "perfection" of direct or inverted repeats in the local DNA sequence. Three (tandem) duplication events were recovered. No repeated sequences were found that might have determined their endpoints.     

DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing: application to 1-nitropyrene-induced revertants

We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.     

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.     

Phenotypic manifestations of yeast transposon insertion in the LYS2 gene and deletions resulting from imprecise excision of the transposon

The lys2-32 mutant allele resulted from Ty1 element insertion was identified and cloned. The expression and reversions of lys2-32 localized in an autonomous plasmid were studied. The insertion was shown to inactivate LYS2 gene incompletely. Spontaneous reversions to complete or almost complete prototrophy were also obtained. About 50% of revertants retained the insertion. Others arise as a result of imprecise excision events leading to deletions of adjacent LYS2 sequences.     

Deletion induction in bacteria. I. The role of mutagens and cellular error-prone repair

The amber mutation trpD28 of Salmonella typhimurium shows a complex reversion pattern on anthranilate (AA)-supplemented minimal medium. Under such conditions it is possible to recover revertants of two phenotypes, prototrophs (MM+) and anthranilate utilizers (AA+), each phenotype brought about by several mutational events. Since one class of AA+ revertants is caused by deletion of the trpD28 mutation, this constitutes a useful system for quantitative studies of the effects of mutagenic agents and cellular factors on the production of deletions. In the present study we have tried to assess the relative contribution of chemical mutagens vs. cellular mutator factors in causing this class of mutations. Strains of S. typhimurium in which the spontaneous reversion rate of trpD28 was modified by pKM101, (strain SO1007), mutL (strain SO1018) and both (strain SO1008), as well as the wild type (strain SO939) were treated with nitrous acid (HNO2) and mitomycin C (MC), mutagens reported to induce deletions in bacteria. The results showed that while the absolute frequency of deletions increased exponentially with dose of mutagen in parallel with the total reversion frequency, the relative frequency (percent) of these mutations was characteristic for each strain and for the most part unaffected by the dose of mutagen. It appears that deletions, spontaneous or induced, occur as a fixed percentage of total mutations and are brought about by the cells' own repair capacity and characteristic DNA metabolism. Perhaps these mutations are the result of untargeted events during SOS misrepair.     

The Influence of Local DNA Sequence and DNA Repair Background on the Mutational Specificity of 1-Nitroso-8-Nitropyrene in Escherichia Coli: Inferences for Mutagenic Mechanisms

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/- delta uvrB) and MucA/B-mediated error-prone translesion synthesis (+/- pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (> 92%) which occurred at G.C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G.C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G.C-->T.A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G.C-->T.A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G.C-->T.A transversions by a factor of 30-60. In contrast, the -(G.C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G.C-->T.A transversions and -(G.C) frameshifts. Deletion mutations were induced in the delta uvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G.C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell.     

Adaptive mutations in Salmonella typhimurium phenotypic of purR super-repression

Under non-lethal selective conditions, a non-dividing or very slowly dividing microbial population gives rise to mutations that relieve selective pressures. This process is described as adaptive mutation. Salmonella typhimurium strain 5-28 has been used as a system for studying adaptive mutations in the chromosomal regulatory gene purR and its target, the purD operator. When this strain is plated on a minimal lactose medium, no apparent growth of parent lawn is observed, yet the revertant colonies accumulate over a period of time. Analysis of the purR mutational spectra showed that the frequencies of transitions and transversions were not significantly different among the growth-dependent and adaptive mutations. But the frequencies for five kinds of -1 frameshifts were significantly different between the growth-dependent and adaptive types. Among the growth-dependent mutations, most one-base deletions occurred in non-iterated bases and were distributed randomly. Among adaptive mutations, the frequency of one-base deletions in small mononucleotide repeats was higher and mutations were concentrated at three hotspots. One-base deletion in small mononucleotide repeats are generally believed to result from DNA polymerase slippage errors, which are not corrected by DNA repair machinery. We further investigated the role of DNA repair on adaptive mutation. Our results showed that the mismatch repair (MMR) might function less efficiently during adaptive mutation. However, DNA oxidative damage repair seemed no less effective in correcting errors under selective pressures than during non-selective growth.     

Development of a yeast system to assay mutational specificity

We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing. The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity. Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain. Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing. We have used this system to examine mutations arising spontaneously in the SUP4-o gene. The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division. Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid. Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation. Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size. These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.     

Molecular nature of 11 spontaneous de novo mutations in Drosophila melanogaster

To investigate the molecular nature and rate of spontaneous mutation in Drosophila melanogaster, we screened 887,000 individuals for de novo recessive loss-of-function mutations at eight loci that affect eye color. In total, 28 mutants were found in 16 independent events (13 singletons and three clusters). The molecular nature of the 13 events was analyzed. Coding exons of the locus were affected by insertions or deletions >100 nucleotides long (6 events), short frameshift insertions or deletions (4 events), and replacement nucleotide substitutions (1 event). In the case of 2 mutant alleles, coding regions were not affected. Because approximately 70% of spontaneous de novo loss-of-function mutations in Homo sapiens are due to nucleotide substitutions within coding regions, insertions and deletions appear to play a much larger role in spontaneous mutation in D. melanogaster than in H. sapiens. If so, the per nucleotide mutation rate in D. melanogaster may be lower than in H. sapiens, even if their per locus mutation rates are similar.     

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.     

Deletion induction in bacteria: I. The role of mutagens and cellular error-prone repair

The amber mutation trpD28 of Salmonella typimurium shows a complex reversion pattern on anthranilate (AA)-supplemented minimal medium. Under such conditions it is possible to recover revertants of two phenotypes, prototrophs (MM⁺) and anthranilate utilizers (AA⁺), each phenotype brought about by several mutational events. Since one class of AA⁺ revertants is caused by deletion of the trpD28 mutation, this constitutes a useful system for quantitative studies of the effects of mutagenic agents and cellular factors on the production of deletions. In the present study we have tried to assess the relative contribution of chemical mutagens vs. cellular mutator factors in causing this class of mutations. Strains of S. typhimurium in which the spontaneous reversion rate of trpD28 was modified by pKM101, (strain SO1007), mutL (strain SO1018) and both (strain SO1008), as well as the wild type (strain SO939) were treated with nitrous acid (HNO₂) and mitomycin C (MC), mutagens reported to induce deletions in bacteria. The results showed that while the absolute frequency of deletions increased exponentially with dose of mutagen in parallel with the total reversion frequency, the relative frequency (percent) of these mutations was characteristic for each strain and for the most part unaffected by the dose of mutagen. It appears that deletions, spontaneous or induced, occur as a fixed percentage of total mutations and are brought about by the cells' own repair capacity and characteristic DNA metabolism. Perhaps these mutations are the result of untargeted events during SOS misrepair.     

Microcalorimetry is a sensitive method for studying the effect of nucleotide mutation on promoter activity

Microcalorimetric method was successfully used to study the effect of nucleotide mutations on promoter activity and identify the important nucleotide necessary for the promoter function in Escherichia coli. The thermokinetic parameters, such as k, I and IC(50), were calculated from the metabolic power-time curves obtained by microcalorimetric measurement using the TAM air Isothermal Microcalorimeter (manufactured by Thermometric AB company of Sweden). Analysis of these data revealed that different nucleotide mutations in -10 box sequence of RM07 fragment had different effect on the promoter activity. Our research also suggest that the microcalorimetric method is a very sensitive and easily performed method for investigation of promoter mutation.     

Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli

1. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F(-)p(+) strain and Hfr strains carrying mutations L8 and L29. All of the 19 p(-) recombinants tested proved to suffer no transient repression, whereas all of the eight p(+) recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the beta-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of beta-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of beta-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.     

Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector.

Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.     

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.