Defect of an Escherichia Coli Atopa Mutant: R-Loop Formation Is a Major Problem in the Absence of DNA Topoisomerase I
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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3526-3530, April 1995 Genetics Overexpression of RNase H partially complements the growth defect of an Escherichia coli AtopA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I MARc DROLET*t, PAULINE PHOENIX*, ROLF MENZELt§, ERIC MASSE*, LEROY F. Liu§, AND ROBERT J. CROUCHS *Departement de Microbiologie et Immunologie, Universite de Montreal, C.P. 6128, Succursale Centre-Ville, Montreal, PQ, Canada, H3C 3J7; tBristol-Myers Squibb, Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543; §Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854; and 1Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Communicated by John R. Roth, University of Utah, Salt Lake City, UT, January 6, 1995 (received for review September 1, 1994) ABSTRACT Previous biochemical studies have suggested Hypernegatively supercoiled DNA can, under certain cir- a role for bacterial DNA topoisomerase (TOPO) I in the cumstances, be produced during transcription (9, 10). In the suppression of R-loop formation during transcription. In this twin-supercoiling domain model, the tracking of RNA poly- report, we present several pieces ofgenetic evidence to support merase generates positively supercoiled DNA ahead of the a model in which R-loop formation is dynamically regulated transcribing complex while leaving negatively supercoiled during transcription by activities ofmultiple DNA TOPOs and DNA in its wake (11). DNA gyrase is postulated to remove the RNase H. In addition, our results suggest that events leading overwound DNA ahead of the transcribing complex while to the serious growth problems in the absence of DNA TOPO TOPO I is thought to relax the 5' underwound. region. This I are linked to R-loop formation. We show that the overex- model predicts that transcription in the absence ofTOPO I will pression ofRNase H, an enzyme that degrades the RNA moiety result in hypernegatively supercoiled DNA. In E. coli strains of an R loop, can partially compensate for the absence ofDNA with a topA deletion, the production of pBR322 plasmid with TOPO I. We also note that a defect in DNA gyrase can correct almost twice the normal level of negative DNA supercoiling several phenotypes associated with a mutation in the rnhA has been linked to transcription (9, 10). Recent in vitro gene, which encodes the major RNase H activity. In addition, experiments show that transcription in the presence of DNA we found that a combination of topA and rnhA mutations is gyrase alone leads to the production of such hypernegatively lethal. supercoiled pBR322 molecules (12). In these experiments, the production of the hypernegatively supercoiled DNA topoiso- DNA topoisomerase (TOPO) I, originally known as o, is the mers involved the formation of R-looped DNA. It was also major DNA relaxing activity in Escherichia coli (1). The gene shown that E. coli DNA TOPO I efficiently inhibited the encodingE. coli DNA TOPO I topA was localized to the cys-trp production of hypernegatively supercoiled pBR322 and, region of the chromosome (2, 3). Deletions that remove both hence, the associated R loops. Moreover, pBR322 DNAs cysB and topA were identified in E. coli strains and it was first extracted from an E. coli topA mutant were found to contain assumed that topA was not an essential gene (3). A more R loops. Interestingly, electron microscopic studies have careful analysis showed that a deletion of the topA gene could shown that DNA TOPO I is concentrated in the actively only be inherited in strains that contained a second mutation transcribed regions of the E. coli chromosome (13). It is worth that would compensate for the loss oftopA (4, 5). These studies recalling that a small single-stranded region of DNA is re- identified mutations in both gyrA and gyrB that could com- quired in the substrate of DNA TOPO I and pointing out that pensate for the loss oftopA and demonstrated that these muta- such regions are likely to be present at the junction of R-looped tions result in reduced global DNA supercoiling. This lead to DNA segments. We have proposed (14) that a major role for a picture in which a "global balance" of DNA supercoiling is DNA TOPO I is to modulate the impact of transcription- essential and controlled by the competing activities of DNA induced negative supercoils on DNA structures such as R loops. gyrase and TOPO I. The in vitro experiments cited above demonstrate a rela- In vitro DNA TOPO I does not relax negatively supercoiled tionship between transcription in the presence of DNA gyrase DNA to completion (1). Other in vitro studies show that the and the formation of hypernegatively supercoiled DNA, with specificity for negatively supercoiled DNA derives from a its associated R loops. The current study extends this work to requirement of a short single-stranded DNA region as part of suggest that R-loop formation is a significant in vivo conse- the enzyme-DNA complex (6). Since negative DNA super- quence of transcription in the absence of TOPO I. coiling favors the unpairing of DNA strands, DNA molecules with high negative superhelical density will be a better sub- strate for DNA TOPO I. Some in vivo studies also suggest that MATERIALS AND METHODS DNA TOPO I does not relax DNA efficiently: in one study a E. coli Strains. E. coli strains used are listed in Table 1. topA mutation had little effect on the rate of in vivo DNA Details of their construction by transduction using Plvir phage relaxation after the inhibition of DNA gyrase by coumermycin (18) are provided in the figures and tables. (7); and in a second in vivo study, DNA TOPO I produced only Media and Growth Conditions. Unless otherwise indicated, slow and partial DNA relaxation (8). However, a defect in liquid cultures were grown at 37°C in Vogel-Bonner (VB) DNA TOPO I can cause an in vivo increase in the level of minimal medium (19) supplemented with required amino negative supercoiling (5). Thus, these results suggest that DNA acids (50 ,ug/ml). When needed, thymine was added to 8 TOPO I functions to prevent DNA supercoiling from reaching ,ug/ml. LB (0.5% NaCl) and TB media were prepared as an unacceptably high level. described (20). Luria medium is identical to LB medium except The publication costs of this article were defrayed in part by page charge Abbreviations: IPTG, isopropyl o3-D-thiogalactoside; Ts, temperature payment. This article must therefore be hereby marked "advertisement" in sensitive; TOPO, topoisomerase. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3526 Downloaded by guest on October 1, 2021 Genetics: Drolet et at Proc. Natl. Acad Sci. USA 92 (1995) 3527 Table 1. E. coli strains Table 2. Coinheritance of Trp+ with topA20::TnlO in various recipients Strain Genotype Source or Ref. N99 rpsL galK2 NIH strain Fraction of Tetr Trp+ collection transductants N4177 N99 gyrB221(couR) 15 Recipient 300C 370C 420C gyrB203(Ts) RFM429 (gyrB225) 19/50 22/50 22/50 DM800 A(topA cysB)204 acrA13 3 RFM430 (gyrB+) 0/50 0/50 0/50 gyrB225 RFM431 0/50 19/50 NT RED31 Hfr KL99 topA20::TnlO R. Depew (16) (gyrB203, gyrB221) toc-1 RFM429 DM800 AtrpE cysB+ This work Phage Plvir was grown on the host strain RED31 (trp+ topA + topA20::TnlO) and used to transduce the indicated strains to proto- RFM430 N99 AtrpE This work trophy (Trp+) on minimal glucose plates at 30°C, 370C, and 420C. Isolated Trp+ transductants were purified for single colonies on the RFM431 N4177 AtrpE This work same medium at the same temperatures and then scored for the RFM443 N99, Alac74 This work coinheritance of tetracycline resistance Tetr (tetracycline at 25 ,ug/ml) RFM445 N4177, Alac74 This work on rich LB media at 37°C. Strain RFM429 was constructed from RFM475 RFM431 A(topA cysB)204 This work DM800 by the sequential transduction to Cys+TopA+ with phage trp+ Alac74 grown on N99, followed by transduction to AtrpE pyrF zci-TnlO by RFM480 RFM431 topA20::TnlO This work selection for tetracycline resistance using phage grown on PLK831, trp+ Alac74 and finally to AtrpEpyrF+ (tetracycline sensitive) by selection for Pyr+ AQ634 ilv metB his-29 trpA9605 T. Kogoma (17) on minimal plates containing tryptophan and phage grown on N99. pro thyA deoB (or C) RFM430 is an N99 derivative with the AtrpE allele introduced as described for RFM429. RFM431 is the N99 derivative N4177 again AQ666 AQ634 rnhA224 T. Kogoma (17) with the AtrpE allele introduced as described for RFM429. NT, no CAG18633 zag-3198::TnlOkan C. Gross transduction. MD310 AQ666 zag-3198::TnlOkan This work MD315 RFM430 rnhA224 This work The strain RFM431 contains mutations in thegyrB gene that zag-3198::TnlOkan confer courmermycin resistance (gyrB221) and cause temper- MD316 RFM430 This work ature sensitivity (gyrB203) (15). We reasoned that the Ts defect zag-3198::TnlOkan of the gyrB221 gyrB203 combination may result in a conditional MD317 RFM431 rhnA224 This work compensatory phenotype. The data shown in Table 2 demon- zag-3198::TnlOkan strates this. Tetracycline-resistant Trp+ transductants are re- MD318 RFM431 This work covered at 37°C but not at 300C or 42°C. Furthermore, a strain zag-3198::TnlOkan (RFM480) inheriting the topA::TnlO at 370C displays both a Ts C. Gross is at University of Wisconsin, Madison. character (it will not make colonies on LB plates at 420C) and a cold-sensitive character (it will not make colonies in 18 h on it contains 0.05% NaCl.