Transcription increases methylmethane sulfonate-induced mutations in alkB strains of Escherichia coli.

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.

DNA sequence context affects UV-induced mutagenesis in Escherichia coli.

We investigated the effect of altering the DNA sequence surrounding a mutable target site on the production of ultraviolet light (UV) induced mutations. Site-directed base substitutions were incorporated on both sides of a TAA sequence encoding a UAA nonsense defect in the tyrA14 allele of Escherichia coli. This allele is readily revertable by UV and a total of eight different base substitution mutations can be recovered. Five different strains harboring DNA sequences allowing the formation of 5'-TT, 5'-CT and 5'-TA* photoproducts were constructed and exposed to UV. DNA sequence analysis was used to determine the spectrum of the revertants that were recovered. The results showed that changes at the 3'-base of a TT site were predominantly T to C transitions and T to A transversions. However, unlike the TT site, a 5'-CT site produced a relatively high frequency of T to G transversions. In addition, T to A transversions that could not have been targeted by a cyclobutane-type or [6-4]-type pyrimidine dimer were produced; this result suggested that these mutations may be targeted by a TA* photoproduct. Also, a distinct strand bias was noted for two mechanistically identical base substitutions in a strain having a palindromic target sequence; this result may reflect an unequal damage distribution or processing of photoproducts as a consequence of asymmetric DNA replication. Finally, our results show that DNA sequences expected to allow the greatest density of UV-induced DNA damage produce the highest mutation frequencies. Overall, these findings provide new insights regarding the role of DNA photoproducts in UV mutagenesis.

Genetic analysis of the anti-mutagenic effect of genistein in Escherichia coli.

Genistein, the main isoflavone in soy, has received considerable attention for its potential anti-carcinogenic properties. In a previous report, we investigated the possible role of genistein in anti-mutagenesis, using an Escherichia coli reversion assay system. Genistein reduced ENU-induced mutagenesis in a dose-dependent manner and the reduction of mutation frequency was differential among several categories of mutation. Most notable was a loss of transversion mutations, which require SOS functions. In this report, we further investigated the anti-mutagenic effect of genistein using a genetic approach. E. coli strains having alterations in genes involved in SOS-mutagenesis were examined, as were strains having defects in proteins that might serve as potential targets for genistein. The results showed that ENU-induced mutations produced in recA730 and lexA(Def) strains, both expressing a constitutive SOS response, were reduced by genistein to a lesser extent than in the wild-type strain. The effect of genistein was not entirely abolished, however. ENU mutagenesis in a umuC derivative, which reflects predominantly transition mutations, was unaffected by genistein. ENU-induced mutations in strains having defects in topA, gyrA, typA or uspA were not different than the wild-type, suggesting that these gene products were not involved in genistein's anti-mutagenic effect. In addition, we determined the distribution of genistein in various cellular fractions using HPLC. These studies revealed that genistein could be recovered from E. coli cells grown on agar media containing genistein; the intracellular concentration was similar to that in the agar plates. Further, most of the genistein recovered was associated with proteins in the cytosolic fraction and little partitioned in the membrane fraction. In vitro studies showed that genistein could be precipitated from a protein (BSA) containing solution. Finally, we examined the effect of genistein on formation of the RecA filament on ssDNA in vitro and observed an inhibition at high concentrations of genistein. In total, these results suggested that genistein may reduce SOS-dependent mutagenesis by reducing the interaction of RecA protein with ssDNA. As a consequence, genistein could cause a reduction in (1) the overall SOS response (confirmed using beta-galactosidase assays) and (2) trans-lesion DNA synthesis by DNA polymerase V.

Neighboring base identity affects N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli.

This study investigated the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU). A set of bacterial strains having all possible bases neighboring an ochre (TAA) nonsense mutation in the tyrA gene of Escherichia coli were employed and true reversions of the nonsense mutation were induced by two separate doses of ENU. Base substitution mutations were investigated by direct sequencing methods. These studies revealed that 1) mutations occurring at 5'-purine-T sites were produced better, on average, than mutations involving 5'-pyrimidine-T sites, and 5'-TT sites contributed the least to the formation of mutations, 2) the order of preference for A:T to G:C transitions was 5'-GT>5'-AT, 5'-CT>5'-TT, and 3) A:T to C:G transversions at the first position of the codon (GAA mutations) were produced best at 5'-AT sites, while A:T to T:A transversions at the third position (TAT mutations) occurred more often at 5'-GT sites. These findings suggest that the occurrence of a specific mutation may reflect the sequence-dependent probability of DNA damage at a particular site as well as factors involving preferential DNA repair or differential base selection by DNA polymerase.