Hypernegative supercoiling of the DNA template during transcription elongation in vitro

Supercoiled plasmid DNAs with negative superhelicity several times higher than normal have been isolated from Escherichia coli topA mutants. The formation of these hypernegatively supercoiled plasmid DNAs is apparently induced by transcription. We show that hypernegatively supercoiled plasmid DNAs isolated from topA mutants contain R-loop(s). To study the mechanism of formation of hypernegatively supercoiled plasmid DNA, we have been able to reproduce hypernegatively supercoiled DNA in vitro using purified RNA polymerase and DNA gyrase. The formation of hypernegatively supercoiled plasmid DNA template in vitro is shown to require transcription elongation and is tightly linked to R-loop formation. We propose that one of the roles of topoisomerase I is to suppress R-loop formation during transcription elongation.