Open AccessA pSC101‐ par sequence‐mediated study on the intracellular state of supercoiling of the pBR322 genome in Escherichia coli DNA topoisomerase I deletion mutant
Author(s) -
Ishii Satoshi,
Murakami Tetsuya,
Shishido Kazuo
Publication year - 1992
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1992.tb05076.x
Subject(s) - mutant , topoisomerase , dna supercoil , pbr322 , intracellular , biology , genetics , mutation , dna , plasmid , microbiology and biotechnology , chemistry , gene , dna replication
In Escherichia coli DNA topoisomerase I deletion mutant DM800, transcription of the tetracycline‐resistance gene ( tet ) in the pBR322 genome is though 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 Eco RI site upstream, or the Aval 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 Eco RI site as well as the Ava I 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 Ava I site) ahead of the tet transcripts, is not so positively supercoiled and preferential interaction of gyrase with the Eco RI‐ 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.