Etude des mecanismes de surenroulement de l'ADN induit par la transcription chez Escherichia coli (French and English text)

Topoisomerases accomplish many functions in DNA metabolism. Topoisomerase I and gyrase play substantial roles in transcription. Transcription in the absence of topoisomerase I can result in the formation of hypernegatively supercoiled plasmid DNAs. Historically, hypernegative supercoiling has been linked to the coupled transcription and translation of plasmid-borne membrane-bound proteins such as TetA. According to the Twin-Domain Model, this anchorage blocks the RNA polymerase from rotating and in the absence of topoisomerase I, this results in an accumulation of negative supercoils. Hypernegative supercoiling of plasmid DNAs can also result from R-loop formation. R-loops occur preferentially when the transcript remains untranslated (rRNA) or when transcription and translation are uncoupled. R-loop formation is the greatest consequence of the absence of topA in E. coli.; In order to better understand the factors which regulate R-loop formation in E. coli, we sought to isolate multicopy suppressors of a topA mutation by using an in vivo cloning system with the mini-Mu phage. We isolated topB which encodes topoisomerase III and rnhA which encodes RNase HI. We found that topoisomerase III can relax transcription-induced negative supercoils in vivo and in vitro, that R-loops are hot-spots for relaxation by topoisomerase III and that the in vivo overexpression of this enzyme can prevent hypernegative supercoiling. The overexpression of both topoisomerase III and RNase HI has a synergistic effect on the growth of topA null mutants.; In order to study the importance of coupled transcription/translation in the inhibition of R-loop formation, we evaluated the sensitivity of hypernegatively supercoiled plasmids to RNase H1 overproduction and we used several methods to specifically or non specifically inhibit protein synthesis. We also performed Northen analysis to evaluate the effects of overproducing RNase HI on full-length RNA synthesis. Our results suggest that the inhibition of translation promotes hypernegative supercoiling in two ways: by freeing RNAs of ribosomes which can stimulate R-loop formation, and by increasing their half-life due to a shortage of ribonucleases caused by a sequestration of these enzymes to newly synthesized rRNA. Longer RNAs can promote hypernegative supercoiling by the "Twin-Domain" mechanism by increasing frictional drag, as shown by the fact that a mutation in the RNA polymerase which inhibits full-length RNA synthesis also abolishes this form of DNA. Hypernegative supercoiling can therefore exist in a free state inside the cell and can be a major cause of R-loop formation. RNase HI can eliminate hypernegative supercoiling by degrading the R-loop which constrains it, thus rendering it accessible for relaxation by DNA gyrase. Our results suggest that topoisomerase I, DNA gyrase, the ATP/ADP ratio, topoisomerase III, topoisomerase IV, RNA polymerase, RNase HI and other ribonucleases are trans regulators of hypernegative supercoiling whereas the length of the transcript plays a more cis role in the accumulation of hypernegatively supercoiled plasmid DNAs.