A collection of 164 spontaneous lacI- mutations were recovered from a uracil-DNA glycosylase deficient (Ung-) strain of Escherichia coli and analyzed by DNA sequencing. As predicted by genetic studies, G:C----A:T transitions predominated among base substitution events. However, DNA sequence analysis indicated that these events did not occur at random. Of the 31 G:C----A:T transitions recovered, 24 involved cytosine residues located in the nontranscribed strand of the gene and 15 of the 31 transitions occurred at cytosines located on the 3' side of 3 or more A:T base pairs. These differentials likely reflect the more single-stranded character of the non-transcribed strand of the gene and of regions rich in A:T base pairs. In addition, mutation at the frameshift hotspot was altered in the Ung- strain, suggesting a role for DNA repair in the formation of structural intermediates that potentiate these events. Also, the analysis of non-hotspot frameshifts, deletions and duplications showed that many involved local DNA sequence. Specifically, several of the frameshift, deletion and duplication mutations occurred near the sequence 5'-CTGG-3'. Thus, DNA sequence analysis of mutational specificity in an Ung- strain has provided evidence that gene expression, DNA repair and DNA context can all potentially influence the classes and frequencies of spontaneous mutation.
Ultraviolet light (UV) induced mutations in the glnU and glnVa tRNA genes in Escherichia coli are thought to be targeted by UV photoproducts. In a previous study with a uracil-DNA glycosylase deficient strain, UV-induced glnU0 and glnV0 tRNA suppressor mutations became resistant to photoreactivation (PR) following thermal treatment. It was proposed that deamination of cytosine in the cytosine-containing cyclobutyl dimers at the sites of these suppressor mutations produced uracil residues in sequence upon PR. In the absence of glycosylase, the C----U conversion yielded the requisite G:C----A:T transitions. In the present study, this thermal resistance of UV-mutagenesis to PR is characterized. It is dependent on the initial UV-fluence and temperature of holding but not on the UmuC+ gene product. The data obtained yield an estimate of an activation energy of 17 +/- 3 kcal/mol for the deamination of cytosines contained in dimers. This compares to 29 kcal/mol for unaffected cytosines in DNA. In addition, an estimate of the probability of cyclobutyl dimer formation at the target sites for glnU0 and glnV0 suppressor mutations indicate that these lesions can not entirely account for the mutation frequencies recovered in the absence of PR. This is interpreted as an indication that, in addition to thymine-cytosine cyclobutyl dimers, other UV-induced lesions, possibly Thy(6-4)Cyt photoproducts, may also target glnU0 and glnV0 suppressor mutations.
The sequences of a collection of 261 spontaneous lacI- mutants recovered in a PolA- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5'-GTGG-3' sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5'-GTGG-3' sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5'-GTGG-3' sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.
Methylmethane sulfonate (MMS) produces DNA base lesions, including 3-methylcytosine (m3C), more effectively in single-stranded DNA. The repair of m3C in Escherichia coli is mediated by AlkB through oxidative demethylation and in the absence of repair, m3C leads to base-substitution mutations. We describe here results of experiments that were designed to investigate whether transcription of a gene in E. coli affects the process of mutagenesis by MMS and the roles played by AlkB and lesion bypass polymerase PolV. Using a genetic reversion assay, we have confirmed that MMS mutagenesis is suppressed by AlkB, but is enhanced by PolV. High transcription of the target gene enhances reversion frequency in an orientation-dependent manner. When the cytosines that are the likely targets of MMS were in the non-template strand (NTS), transcription increased the MMS-induced reversion frequency several fold. This increase was dependent on the presence of PolV. In contrast, when the same cytosines were present in the template strand, transcription had little effect on reversion frequency induced by MMS. These data suggest that MMS creates 3-methylcytosine adducts in the NTS and are consistent with an idea proposed previously that transcription makes the NTS transiently single-stranded and more accessible to chemicals. We propose that this is the underlying cause of its increased sensitivity to MMS and suggest that transcriptionally active DNA may be a preferred target for the action of alkylating agents that prefer single-stranded DNA.
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