Abstract
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.
Highlights
R-loop formation during transcription elongation. tide to themembrane is important for this transcriptioneffect on DNA supercoiling [14, 22]
Transcription Elongation Increases Heterogeneity in Topoisomer Distribution of the DNA Template in the Presence of Topoisomerases-To test the effect of transcription on template supercoiling, pBR322 DNA with a native superhelical density was transcribed in uitro by E. coli RNA polymerase in the presence of various ratios of E. coli DNA topoisomerase I toM. luteus DNA topoisomerase I1 (DNA gyrase)
Our in vitro results indicate that plasmid DNA supercoiling is very sensitive to the gyrase/omega ratio during transcription elongation
Summary
In the first set of experiments, plasmid DNAs were treated with RNase A (5 pg/ml) with or without RNase H (2 units) in a buffer containing 10 mM Tris (pH 8.0), 100 mM NaCl, 10 mM MgCIz, and 0.1 mM EDTA for 15min a t 37 "C.The samples were phenol extracted once and analyzed by agarose gel electrophoresis (1%) with or without chloroquine. The second set of experiments was similar to the first one except that after RNases treatment, the samples were treated with calf thymus topoisomerase I1 in the presence of 1mM ATP for were extracted with phenol once and analyzed by agarose gel 20 min a t 37 "C.
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