Measuring Mutator Enzyme Activity Using an E. coli-Based Colony Formation Assay

Mutator enzymes alter the nucleotide sequences of DNA or RNA molecules; immune systems utilize them to destroy the integrity of pathogen genomes and to optimize immune mediators of the host. Their dysregulation has been linked to tumorigenesis in various tissues. Defining and comparing the activities of such mutator enzymes requires a robust versatile assay that is independent of their biological context as in vivo mutation rates are typically low. Here we provide detailed protocols for two widely used E. coli-based approaches that detect the activities of ectopically expressed cytidine deaminases on two distinct reporter genes: an extrachromosomal kanamycin-resistance gene or an endogenous chromosomal substrate, the rpoB gene-encoding RNA polymerase. The generation of mutations is in both cases measured in a colony formation assay. With appropriate modifications, these assays can be extended to study other mutator enzymes.