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.