Bacterial Topoisomerase I Growth Complementation Assay.

Type IA topoisomerases carry out essential functions in all free-living organisms including bacteria. The type IA topoisomerase I encoded by topA gene in Escherichia coli has been shown to play an important role in maintaining optimal local and global level of DNA supercoiling and prevent R-loop accumulation in transcription. Deficiency in E. coli topoisomerase I activity associated with various topA mutations present in different mutant strains have been shown to result in temperature-sensitive or cold-sensitive growth, as well as hypersensitivity to killing by stress challenges. We describe here growth complementation assays utilizing these E. coli topA mutant strains that can be employed to assess the role of specific residues or domains in the relaxation activity of plasmid-encoded recombinant bacterial topoisomerase I used for complementation. We also provide the protocol for complementation assay measuring survival following lethal stress challenge that was used to study the specific protein-protein interaction between the C-terminal domain of topoisomerase I and RNA polymerase of E. coli.