Assays for transcriptional mutagenesis in active genes.
Cells exposed to DNA-damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Despite the relatively high efficiency of the different DNA repair pathways, some lesions remain in DNA. During transcription, RNA polymerase can bypass DNA damage on the transcribed strand of an active gene. This bypass can be at the origin of the production of "mutated" mRNA because of the transcriptional miscoding (transcriptional mutagenesis) due to the altered pairing specificities of the lesion. In vivo consequences of transcriptional mutagenesis on normal cell physiology have not well been documented because of the lack of a robust system allowing for its study. We describe here a procedure that we developed using a plasmid-based luciferase reporter assay to analyze the transcriptional mutagenesis events induced by different types of DNA lesions. Introduction of the DNA lesion to be studied at a specific site on the plasmid is based on the synthesis of a complementary strand of a circular, single-stranded DNA (ssDNA) from a DNA lesion-containing oligonucleotide. Once obtained, this construct can be transformed into different Escherichia coli strains that can express the luciferase gene under nongrowth conditions. Quantification of luciferase activity and sequencing of luciferase cDNAs allow for the characterization of transcriptional mutagenesis both quantitatively and qualitatively.
Brégeon
Damien
D
Laboratoire Instabilite genetique et cancer, Institut Gustav Roussy, PR2, Villejuif cedex, France.
Doetsch
Paul W
PW
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics*; D016296:Mutagenesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers; D004270:DNA, Circular