ROLES OF DNA TOPOISOMERASES IN THE REGULATION OF R-LOOP FORMATION IN-VITRO

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transc ribed preferentially in its physiological orientation. Our results als o suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level . In the present report we investigated in greater detail the involvem ent of DNA topoisomerases in this process. By using an in vitro transc ription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs duri ng transcription in the presence of DNA gyrase. When RNase A is presen t, significant hypernegative supercoiling occurs only when the 567-bas e pair rrnB HindIII fragment is transcribed in its physiological orien tation. This result suggests that more stable R-loops are being produc ed in this orientation. Our results also suggest that DNA gyrase can p articipate in the process of R-loop elongation. The strong transcripti on-induced relaxing activity of E. coli DNA topoisomerase I is shown t o efficiently counteract the effect of DNA gyrase and thus inhibit ext ensive R-looping. In addition, we found that an R-looped plasmid DNA i s a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.