A pSC101-par sequence-mediated study on the intracellular state of supercoiling of the pBR322 genome in Escherichia coli DNA topoisomerase I deletion mutant

Abstract

In Escherichia coli DNA topoisomerase I deletion mutant DM800, transcription of the tetracycline-resistance gene (tet) in the pBR322 genome is thought to create and maintain two domains of positive supercoils ahead, and negative supercoils behind, the transcription complex. To assess the actual intracellular state of twin-supercoiled domains, par sequence (365 bp) of plasmid pSC101, which shows a high affinity for DNA gyrase, was inserted into the EcoRI site upstream, or the AvaI site downstream, of the tet gene on the pBR322 genome. Analysis of the oxolinic acid-induced sites of cleavage by gyrase in DM800 revealed that the pBR322 derivatives are highly preferentially cleaved at the par sequence of the EcoRI site as well as the AvaI site and efficiently linearized when compared with pBR322. Assessment of the state of negative supercoiling of the pBR322 derivatives isolated suggested that the DNA (containing the AvaI site) ahead of the tet transcripts, is not so positively supercoiled and preferential interaction of gyrase with the EcoRI-par sequence does not result in removing negative superhelical turns so effectively as DNA topoisomerase I does on pBR322 DNA in the isogenic wild-type cells.