Abstract
Gyrase-mediated DNA cleavage on plasmid DNAs was measured in Escherichia coli treated with oxolinic acid. On pBR322 DNA, gyrase cleavage sites were concentrated in the region between the 3'-ends of the tetA and bla genes. The preferential cleavage in this region was dependent on RNA transcription and the divergent orientation of these two transcription units. The enhanced gyrase cleavage also required translation; chloramphenicol treatment or the insertion of a translation terminator within the 5'-proximal region of the tetA gene abolished the enhanced cleavage. We suggest that the enhanced gyrase cleavage may reflect the changes in local DNA supercoiling during RNA transcription as gyrase cleavage in vitro was shown to be sensitive to the supercoiling state of DNA. The effects of transcription and translation on gyrase cleavage can best be explained by the twin-supercoiled-domain model of transcription (Liu, L. F., and Wang, J. C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7024-7027).
Highlights
DNA cleavage on plasmid DNAs was measured in Escherichia coli treated with oxolinic acid
To test whether oxolinic acid-induced gyrase cleavage of DNA is sensitive to DNA conformation, purified E. coli gyrase was reacted with negatively and positively supercoiled plasmid
PBR322 DNA was preferentially linearized by gyrase in a reaction containing positively supercoiled pBR322 DNA and negatively supercoiled pJW270 DNA (Fig. 1, lanes f-j). These results suggest that the efficiency of gyrase cleavage induced by oxolinic acid is sensitive to the DNA supercoiling state and can be used as an indicator of the supercoiling state of DNA in vivo
Summary
Studies in eukaryotic cells have suggested that RNA transcription may require a topoisomerase activity [9,10,11,12,13,14,15,16] To explain these findings, a twin-supercoiled domain model has been proposed which describes the. The rotation of the DNA helical axis produces positive supercoils ahead of and negative supercoils behind the RNA polymerase complex This model has gained some support from recent studies on the formation of positively supercoiled plasmid DNAs in E. coli [18], yeast [19], and an in uitro transcription system [20]. Enhanced DNA cleavage was observed in the region between the 3’-ends of the two divergently transcribed genes
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