In Vivo Technique for Determining Transcriptional Mutagenesis

Abstract

When an elongating RNA polymerase encounters DNA damage on the template strand of a transcribed gene it can either be arrested by or be transcribed through the lesion. Lesions that arrest RNA polymerases are thought to be subject to transcription-coupled repair, whereas that damage that is bypassed can cause miscoding, resulting in “mutations” in the transcript (transcriptional mutagenesis). We have developed a technique using a plasmid-based luciferase reporter assay to determine the extent to which a particular type of DNA base modification is capable of causing transcriptional mutagenesis in vivo. The system uses Escherichia coli strains with different DNA repair backgrounds and is designed to detect phenotypic changes caused by transcriptional mutagenesis under nongrowth conditions. In addition, this method is capable of indicating the extent to which a particular DNA repair enzyme (or pathway) suppresses the occurrence of transcriptional mutagenesis. Thus, this technique provides a tool with which the effects of various genes on non-replication-dependent pathways resulting in the generation of mutant proteins can be gauged.