title: Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.
abstract: R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA.
authors.affiliation: Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, H3C 3J7, Canada.
authors.forename: Valentine
authors.initials: V
authors.lastname: Usongo
authors.affiliation: Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, H3C 3J7, Canada.
authors.forename: Makisha
authors.initials: M
authors.lastname: Martel
authors.affiliation: Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, H3C 3J7, Canada.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Balleydier
authors.affiliation: Département de microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, P. Québec, H3C 3J7, Canada. Electronic address: marc.drolet@umontreal.ca.
authors.forename: Marc
authors.initials: M
authors.lastname: Drolet
mesh_terms: D049109:Cell Proliferation / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics*; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016914:Ribonuclease H / Q000172:deficiency / Q000235:genetics
chemical_list: D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; C489283:fimD protein, E coli; D036781:Fimbriae Proteins; D016914:Ribonuclease H; C078856:ribonuclease HI; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase
keywords: Gyrase; R-loops; RNase HI; Replication; Supercoiling; Topoisomerases