N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli

274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.     

Disruption of the Rad52 Gene Alters the Spectrum of Spontaneous Sup4-O Mutations in Saccharomyces Cerevisiae

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae.

Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A.T----G.C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G.C----A.T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G.C----A.T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A.T----G.C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This "hot spot" was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.     

Mutational specificity of DNA precursor pool imbalances in yeast arising from deoxycytidylate deaminase deficiency or treatment with thymidylate

Disruption of the dCMP deaminase (DCD1) gene, or provision of excess dTMP to a nucleotide-permeable strain, produced dramatic increases in the dCTP or dTTP pools, respectively, in growing cells of the yeast Saccharomyces cerevisiae. The mutation rate of the SUP4-o gene was enhanced 2-fold by the dCTP imbalance and 104-fold by the dTTP imbalance. 407 SUP4-o mutations that arose under these conditions, and 334 spontaneous mutations recovered in an isogenic strain having balanced DNA precursor levels, were characterized by DNA sequencing and the resulting mutational spectra were compared. Significantly more (greater than 98%) of the changes resulting from nucleotide pool imbalance were single base-pair events, the majority of which could have been due to misinsertion of the nucleotides present in excess. Unexpectedly, expanding the dCTP pool did not increase the fraction of A.T----G.C transitions relative to the spontaneous value nor did enlarging the dTTP pool enhance the proportion of G.C----A.T transitions. Instead, the elevated levels of dCTP or dTTP were associated primarily with increases in the fractions of G.C----C.G or A.T----T.A. transversions, respectively. Furthermore, T----C, and possibly A----C, events occurred preferentially in the dcd1 strain at sites where dCTP was to be inserted next. C----T and A----T events were induced most often by dTMP treatment at sites where the next correct nucleotide was dTTP or dGTP (dGTP levels were also elevated by dTMP treatment). Finally, misinsertion of dCTP or dTTP did not exhibit a strand bias. Collectively, our data suggest that increased levels of dCTP and dTTP induced mutations in yeast via nucleotide misinsertion and inhibition of proofreading but indicate that other factors must also be involved. We consider several possibilities, including potential roles for the regulation and specificity of proofreading and for mismatch correction.     

Base alterations in yeast induced by alkylating agents with differing Swain-Scott substrate constants.

The base alterations induced by four alkylating agents, methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-methylurea (MNU), and N-nitroso-N-ethylurea (ENU), have been determined at the URA3 locus in the yeast Saccharomyces cerevisiae. The mutagen treatment was carried out on yeast cells in the logarithmic phase of growth. The mutants were selected by their resistance to 7.3 mM-5-fluoroorotic acid at pH 3.8. DNA sequence analysis was carried out by the dideoxy chain termination method. The alkylating agents were selected for their widely differing Swain-Scott substrate constants (s values), which are as follows: MMS, s = 0.83; EMS, s = 0.67; MNU, s = 0.42; ENU, s = 0.26. A higher s value is correlated with a higher ratio of 7-alkylguanine to O6-alkylguanine in native DNA in vitro. 125 forward mutations from URA3----ura3 were sequenced with marked differences in the mutational spectra being observed as the s value changed. Five hotspots were recorded for the four alkylating agents. They were all G.C----A.T transition mutations. There was one common hotspot for all of them; there were two additional ones for the two ethylating agents (ENU and EMS) and two different ones for MNU. Four of the five hotspots have the 5'-GG-3' sequence with the 3'-guanine mutated. It was seen that MMS, which has the highest Swain-Scott substrate constant, yielded the widest array of mutational types. As the substrate constants decreased, the types of mutations became more and more restricted to the G.C----A.T transitions and the A.T----T.A transversions. The transitions are consistent with the concept that mutations arise from O6-alkylation of guanine and alkylation of thymine. The transversions are consistent with the notion of N1-alkylation of adenosine or adenylic acid.     

Mutagenic specificity of ultraviolet light

Genetic and sequencing studies of ultraviolet light (u.v.)-induced mutations in the lacI gene of Escherichia coli show the following: u.v. stimulates many types of mutations. In lacI, base substitutions account for 60 to 65% of the observed mutations, small frameshifts 30 to 35%, and deletions of more than several base-pairs approximately 5%. A comparison of the mutational spectrum of u.v.-induced mutations with those of other SOS-dependent mutagens and with the mutations produced by inducing the SOS system in the absence of mutagenic treatment indicates that most u.v.-induced base substitutions are "targeted", resulting from premutational lesions across from the site of the mutations. Among base substitutions, both transitions and transversions occur, although the most favored mutational sites involve G X C----A X T transitions. G X C----A X T transitions are induced preferentially at sites of adjacent pyrimidines. In one case the conversion of a site from -A-C-A- to -T-C-A- results in a 15-fold increase in u.v.-induced C----T transitions. Frameshifts at certain sites are well-induced by u.v., and the largest hotspot in the I gene involves the loss of an (sequence in text) base pair from a (sequence in text) sequence. Of 25 frameshifts detected by DNA sequencing, 23 mutations at seven different sites result from the elimination of a single base-pair, and two mutations result from the elimination of two base-pairs. No additions were detected. The use of a lacI-Z fusion system, which allows direct selection of frameshifts of either sign, reveals that throughout the entire gene frameshifts that eliminate a single base-pair (-1) predominate by a factor of 20 or more over frameshifts that add a single base-pair (+1). In one case a two-base-pair elimination occurs frequently, resulting in the loss of a -C-T- sequence (on one strand), or a -T-C- sequence, from a -C-T-C-T-C-T-C- sequence. For both frameshifts and base substitutions, some aspect of the larger surrounding sequence beyond the nearest neighbors can influence mutation rates by as much as 50-fold, thus determining which sites are seen as hotspots. The bearing of these and other data on the detailed mechanism of mutagenesis is considered in the Discussion.     

Multiple base-pair mutations in yeast

The nucleotide changes associated with both forward and reverse mutations at the CYC1 locus in the yeast Saccharomyces cerevisiae have been investigated by sequencing the mutated gene product, iso-1-cytochrome c and, more directly, by sequencing appropriate DNA segments. Although the majority of these mutations are the result of single base-pair changes, approximately 10% are the result of multiple mutations and these occur predominantly at certain sites and with certain patterns. Most multiple base-pair changes occur within 20 nucleotides of each other and are generally within six nucleotides. On the basis of the frequencies and patterns of mutations, these nucleotide changes are considered to have occurred as single, concerted events, rather than as multiple independent mutations. Analysis of these mutations indicates that multiple base-pair changes can arise by widely differing mechanisms. We have recognized the following classes of mutations: multiple base-pair changes that yield (1) direct repeats or (2) inverted repeats of local DNA sequences; (3) substitutions of two tandem base-pairs; (4) frameshift and contiguous single base-pair substitutions; and (5) recombination of the CYC1 gene with a non-allelic gene, resulting in alterations within contiguous segments that can be over 150 nucleotides in length. Some of the multiple base-pair changes do not fall into any of these categories. We suggest mechanisms to account for each of these five classes.     

Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity.

To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-). An analysis of Ung+ his-4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T----G:C transitions. However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C----A:T transitions and all four possible transversions also occurred. In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele. Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered. Furthermore, the reversion of trpE9777 was decreased by 90% in the Ung- strain. These findings demonstrate that in E. coli, thymine starvation can induce frameshift mutations and all types of base substitutions. The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion. We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis.     

Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.     

Spontaneous Mutation in the Escherichia Coli Laci Gene

To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene. This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites. Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts. The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions). All four transversions were observed. Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints. The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences. The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints. The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases. A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair. The overall effect was 260-fold but varied substantially among different classes of mutations. An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively. Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed.     

DNA sequence analysis of spontaneous mutations at the aprt locus of hamster cells.

To determine the nature of spontaneous mutational events in cellular genes in hamster cells, mutant adenine phosphoribosyltransferase (aprt) genes were cloned and the regions to which we mapped alterations were sequenced. A variety of nucleotide changes were found to occur in the 12 mutant genes analyzed. Most mutations were simple base-pair substitutions-transitions (both G X C----A X T and A X T----G X C) and transversions. The only multiple mutation was a simple transition next to a single-base-pair insertion. Of the 12 mutations, 4 were more complex, involving small deletions or duplications. Two of these were similar to previously described deletions in that they occurred between short direct sequence repeats. No hot spots were detected. Three independent mutations were characterized at one restriction endonuclease site, although no other mutations were detected in the nucleotides surrounding this site in other mutant strains. At a functional level, sequence changes were either in exons (resulting in missense and, in one instance, nonsense mutations) or at splicing sites.     

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.     

A:T --> G:C base pair substitutions occur at a higher rate than other substitution events in Pms2 deficient mouse cells

The mismatch repair pathway involves multiple proteins that are required to correct DNA polymerase generated mismatches before they become mutations. It has been shown recently, that the predominant base-pair substitution events leading to loss of endogenous Aprt activity in Pms2 null mouse cells are A:T --> G:C mutations (Oncogene 21 (2002) 1768, Oncogene 21 (2002) 2840). To determine if this observation could be explained by an increased rate of A:T --> G:C mutations relative to other base-pair substitutions, we developed a reversion assay to examine G:C --> A:T, C:G --> A:T, and A:T --> G:C mutations within mouse Aprt in a Pms2 null mouse kidney cell line. The results demonstrated a 6-50-fold increase in the rate of the A:T --> G:C mutations relative to the other base-pair substitutions. Additional work demonstrated that growth of the Pms2 null cells in antioxidant containing medium reduced the rate of the A:T --> G:C mutations. The results are discussed with regards to the role of mismatch repair proteins in preventing base-pair substitutions, including those induced by oxidative stress.     

Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast.

A collection of 384 mutations recovered in a tRNA gene (SUP4-o) following exposure of isogenic excision-repair-proficient (RAD1) or deficient (rad1) strains of the yeast Saccharomyces cerevisiae to sunlight was characterized by DNA sequencing. In each case, greater than 90% of the mutations were single base-pair substitutions with events at G.C pairs constituting most of the changes. However, more than half of these substitutions were transversions in the RAD1 strain whereas transitions predominated in the rad1 strain. Tandem double substitutions were recovered in both strains and the individual changes were exclusively G.C----A.T transitions. The majority of single substitutions, and all tandem double changes, were at base-pairs where the pyrimidine(s) was part of a dipyrimidine sequence and the site specificities were consistent with cyclobutane dimers and/or pyrimidine (6-4) pyrimidone photoproducts contributing to sunlight mutagenesis. Yet, the data also pointed to an important role for lesions that form at G.C pairs and give rise to transversions. Analysis of the strand specificity of sunlight mutagenesis indicated that transitions or transversions at G.C pairs occurred preferentially in SUP4-o at sites where a dipyrimidine or a guanine, respectively, was on the transcribed strand. These biases required a functional excision-repair system.     

Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes.

The majority of the mutations induced by ICR-170 in both the CYC1 gene (J. F. Ernst et al. Genetics 111:233-241, 1985) and the HIS4 gene (L. Mathison and M. R. Culbertson, Mol. Cell. Biol. 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs. However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a [sequence in text]. Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast. Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs. Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4. Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis. (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at [sequence in text] sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome. This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S. cerevisiae.     

Specificity of mutagenesis resulting from the induction of the SOS system in the absence of mutagenic treatment

Strains in which the E. coli SOS system is continuously induced, in the absence of mutagenic treatment, have been used to generate nonsense mutations in the lacl gene. The examination of over 600 independently occurring amber and ochre mutations reveals that inducing the SOS system stimulates specifically G:C----T:A and, to a lesser extent, A:T----T:A transversions. This specificity is similar to that seen for a number of carcinogens that form bulky adducts to DNA, such as benzo(a)pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), and which are dependent on the SOS system to mutagenize bacteria. However, G:C----T:A transversions resulting from SOS induction alone display a unique site specificity. One possibility is that the SOS-induced mutations result from cryptic, spontaneous lesions, such as apurinic sites, which cannot induce the SOS system themselves but which can target mutations once the SOS system is induced.     

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.     

Heat mutagenesis in bacteriophage T4: another walk down the transversion pathway.

Extracellular nonreplicating bacteriophage T4 particles accumulate mutations as functions of temperature, time, pH, and ionic environment via two mechanisms: 5-hydroxymethylcytidine deamination produces G.C----A.T transitions while a guanosine modification produces transversions. Neither frameshift mutations nor mutations at A.T base pairs are appreciably induced. We now show that heat induces G.C----T.A transversions which we suggest may arise via a G*.A mispair, in which G* is a modified guanosine that has experienced a glycosylic bond migration. The rate of this reaction at 37 degrees C is sufficient to present a genetic hazard, particularly to large genomes; thus, the lesion is probably efficiently repaired in cellular genomes.     

A role for Pms2 in the prevention of tandem CC --> TT substitutions induced by ultraviolet radiation and oxidative stress

DNA mismatch repair (MMR) is important for preventing base-pair substitutions caused by spontaneous or damage-related DNA polymerase errors. We have used a reversion assay based on mouse Aprt to investigate the role of MMR in preventing ultraviolet radiation (UV) and oxidative stress induced tandem CC --> TT base pair substitutions in cultured mammalian cells. The reversion construct used for this assay can detect both C --> T and CC --> TT mutational events. Most spontaneous mutations in Pms2-deficient cells were single C --> T substitutions (88%), with the remainder being tandem CC --> TT substitutions (12%). The percentage of tandem CC --> TT substitutions rose to 64% and 94% for Pms2-deficient cells exposed to UV and a mixture of hydrogen peroxide and metals (Cu/Fe), respectively. Exposure to hydrogen peroxide alone or metals alone did not induce the tandem substitutions, nor did treatment of the cells with the alkylating agent ethylmethane sulfonate, which induces G --> A substitutions on the opposite strand. Tandem CC --> TT substitutions were also induced by UV irradiation and the hydrogen peroxide/metal mixture in Pms2-proficient cells, but at frequencies significantly lower than those observed in the Pms2-deficient cells. We conclude that mismatch repair plays an important role in preventing tandem CC --> TT substitutions induced by certain genotoxin exposures.