Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

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.     

Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung- strain of Escherichia coli

In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E. coli was examined. In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34. In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites. This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase. Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced. This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition. Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence. It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.     

Metal-induced mutagenesis in the lacI gene of Escherichia coli

Mutagenesis in the lacI gene of Escherichia coli has been examined in cells grown in the presence of beryllium, manganese or chromium compounds, metals with suspected mutagenic or carcinogenic potential. 2--3-fold increases in mutation frequency were produced by BeCl2, MnCl2 and K2Cr2O7. Among the cells grown in the presence of Be2+, the frequency of amber and ochre mutants was 3-fold higher than the spontaneous background, suggesting that at least part of the increased mutagenicity was due to base-substitution mutations. The specificity of base-substitution mutations induced by Be2+ and Mn2+ in the lacI gene was analyzed. Among the amber mutations induced in cells grown in the presence of Be2+, an increase in G:C----A:T transitions was detected. In contrast, following growth in Mn2+, no increase in amber and ochre mutation frequencies was observed, and the mutational spectrum resembled that obtained spontaneously indicating that mutations induced by Mn2+ in the lacI gene involve changes that do not yield nonsense mutations. These results suggest that metals may exert a number of different mutagenic effects and that these effects vary for each metal.     

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.     

The distribution of UV damage in the lacI gene of Escherichia coli: correlation with mutation spectrum.

We have determined the UV (254 nm) damage distribution in the transcribed and non-transcribed strands of the i-d region of the Escherichia coli lacI gene. The locations of replication blocking lesions were revealed as termination sites of T7 DNA polymerase and/or T4 DNA polymerase 3'-5' exonuclease. Termination products, i.e. both cyclobutane pyrimidine dimers and 6-4 photoproducts, were resolved and analysed on an automated DNA sequencer. These two major photoproducts are not randomly distributed along the gene, but occur in clusters, in longer runs of pyrimidines. We also have compared the UV damage distribution with the previously reported UV-induced base substitutions in the same region. Mutational hotspots, in both repair-deficient and repair-proficient strains of E. coli, are all located in stretches of pyrimidines, and thus correlate well with the distribution of photolesions. One mutational hotspot in the wild-type strain may reflect the high frequency of closely opposed lesions. To explain the other mutational hotspots, we propose that the repair of UV lesions is impaired due to the local conformation of the DNA, which might deviate from the B-form. This hypothesis is supported by the excess of mutational hotspots in pyrimidine runs in the Uvr+ strain compared to Uvr-. Runs of pyrimidines thus represent both damage- and mutation-prone sequences following UV treatment.     

Construction and characterization of an Escherichia coli strain with a uncI mutation.

A strain of Escherichia coli with a mutation in the promoter proximal gene ( uncI ) of the unc operon has been constructed by using a new gene replacement method. The mutation is a deletion of a defined sequence of 196 base pairs. It was constructed by homologous integration and segregation of a ColE1-derived recombinant plasmid containing the mutation, in a temperature-sensitive polA strain. The mutant strain is phenotypically unc+ but has a reduced growth yield compared to a normal sibling strain.     

Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli

Summary The frequency of lac ^- mutations induced in an F lacI ^S plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, pro lac recipients, is 2-3 fold higher than that typical of non-mating cells which contain the plasmid. These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer. We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication.     

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.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

Distribution and specificity of mutations induced by neocarzinostatin in the lacI gene of Escherichia coli.

Although neocarzinostatin (NCS) attacks DNA almost exclusively at adenine and thymine residues in vitro, exposure of Escherichia coli to this antitumor drug resulted in a high frequency of mutations at guanine:cytosine base pairs in the lacI gene. Thus, NCS-induced base substitution mutations do not appear to result from the major DNA lesions that have been biochemically characterized. The overall distribution of nonsense mutations produced by NCS was distinctly nonrandom, consisting in part of a few "hotspots" and a large number of "coldspots." The existence of these coldspots implies that untargeted mutagenesis does not make a significant contribution to the mutations induced by this SOS-dependent mutagen.     

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12.

Summary A strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.