Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3526-3530, April 1995 Genetics

Overexpression of RNase H partially complements the growth defect of an AtopA mutant: R-loop formation is a major problem in the absence of DNA 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 (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 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 in the rnhA has been linked to transcription (9, 10). Recent in vitro , 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 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 (2, 3). Deletions that remove both hence, the associated R loops. Moreover, pBR322 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. Brain heart infusion (BHI) medium LB plates at 300C). Similar observations have been reported was from Difco. When needed, were added as (16). In a strictly analogous set of experiments using donor follows: ampicillin and kanamycin at 50 [kg/ml, tetracycline at phage grown on a Acystop strain (DM800) to transduce 10 ,tg/ml, and at 500 ,tg/ml. Antibiotics were from RFM431 to Trp+, Trp+Cys- transductants are only recovered Sigma and isopropyl ,B-D-thiogalactoside (IPTG) was from at 37°C. A Trp+Cys- transductant (RFM475) recovered at Boehringer Mannheim. 37°C displays the same Ts and cold-sensitive phenotypes shown Molecular Biology Techniques. Cloning, bacterial transfor- by the RFM480 strain. The Ts character of RFM480 and mations, PCRs, and other molecular biology techniques were RFM475 is not unexpected as they contain the Ts gyrase allele performed essentially as described (21). For CaCl2 transfor- gyrB203 present in RFM431. The cold-sensitive phenotype is mations, the bacterial strains carrying the gyrB temperature- novel. Presumably at 37°C the mutant gyrase allele is defective sensitive (Ts) mutation were heat-shocked for 6 min at 37°C, but retains enough activity to allow the strain to grow. This instead of 90 sec at 42°C. reduced activity at 37°C allows the inheritance of the topA defects. We postulate that by lowering the temperature to 300C the mutant gyrase allele regains a more wild-type level of RESULTS gyrase activity and is no longer sufficient to function as a mutation. We have Inactivation of a a compensatory extracted different plasmid topA Causes Strain with Ts DNA Gyrase DNAs [pBluescript (Stratagene) and pBR322APtetA (12)] to Become Cold-Sensitive. The for a requirement compensa- from a set of isogenic strains grown at 28 and 37°C: RFM443 tory mutation can be demonstrated in a marker exclusion (WT), RFM445 [gyrB(Ts)], RFM475 [Acys-topA, gyrB(Ts)], experiment where a neutral marker is selected and the coin- and RFM480 (topA::TnlO, gyrB(Ts)]. These particular plasmid heritance of a physically linked marker, under investigation, is DNAs were chosen for our studies because they do not carry scored. If a particular genetic background contains an appro- encoding membrane-bound proteins, such as TetA, priate compensatory mutation, then linkage between the which cause transcription-induced hypernegative supercoiling selected and unselected marker will be noted. We illustrate the of plasmid DNAs in topA mutants (22). For the wild-type use of marker exclusion in Table 2. We note that tetracycline strain, we didn't observe any significant temperature effects on resistance (from a topA::TnlO mutation) is inherited 40% of plasmid DNA supercoiling, as measured by electrophoresis in the time when prototrophic Trp+ transductants are selected in chloroquine-containing agarose gels. However, in both a genetic background containing the gyrB225 allele, an allele gyrB(Ts) and double gyrB(Ts)-topA mutants, we observed a previously shown to function as a compensatory mutation for significant increase in negative supercoiling when plasmid a cysB-topA deletion (4). When prototrophic Trp+ transduc- DNAs were extracted from cells exposed to 28°C for 2 h (data tants are selected in a genetic background with a wild-type not shown). The level of plasmid supercoiling was similar in gyrB+ allele, tetracycline resistance is not coinherited. The gyrB(Ts) and gyrB(Ts)-topA strains grown at 37°C, and the topA::TnlO mutation behaves as an excluded marker when a increase in negative supercoiling at 28°C was more profound compensatory mutation is not present. for the double mutants. Presumably, because of an active DNA Downloaded by guest on October 1, 2021 3528 Genetics: Drolet et at Proc. Natl. Acad. Sci. USA 92 (1995) gyrase at 28°C the absence of the topA gene is manifested by H may improve the growth of topA null mutants. The multi- a relative increase in negative supercoiling. These data suggest copy plasmid pSK760 when present in E. coli results in a 10- that during this short growth at 28°C, our double gyrB(Ts)- to 15-fold elevation in the levels of RNase H (23, 24). As a topA mutants behave as true topA mutants without compen- control, we used the plasmid pSK762c, a derivative of pSK760 satory mutations. in which the rnhA gene was inactivated by deletion of an Although linkage disruption of the unselected topA defects internal restriction site (25). A set of isogenic strains RFM445 is noted at 30°C with the gyrB221 gyrB203 combination, and [gyrB(Ts)], RFM475 [Acys-topA, gyrB(Ts)], and RFM480 RFM475 and RFM480 fail to yield single colonies after 18 h (topA::TnlO, gyrB(Ts)] were transformed with these plasmids. at 30°C on LB agar, the cold-sensitive nature of RFM475 and Transformants of the topA null mutants with the pSK760 RFM480 is not absolute. After several days at 30°C on LB agar, (rhnA+) plasmid grew much better than transformants with the single colonies begin to appear for both RFM475 and pSK762c (rhnA-) plasmid: with pSK760, medium-size colonies RFM480. The number of single colonies appearing after 1 were visible after 16 h of incubation at 37°C, whereas with week at 30°C on LB agar approaches the number seen after 1 pSK762c, very small colonies began to appear after 24 h of day at 37°C. All such strains grow robustly when reisolated for incubation (data not shown). No differences in colony sizes single colonies after 18 h at 30°C on LB agar. These "rever- were observed for transformants of RFM445 with pSK760 or tants," however, are remarkably unstable when grown nonse- pSK762c. Upon restreaking the transformants, it is obvious lectively (37°C, LB liquid). We believe they represent dupli- that pSK760 increases the growth capability of the topA null cations of the tolC region (16). For selected cold-growing mutants (Fig. 1, compare pSK760 with pSK762c for RFM475 "revertants," we have shown by transduction that both tolC and and RFM480). In Table 3 we summarize the ability of various the tolC-linked metC genes are duplicated and that the insta- strains to form colonies at 28°C. The efficiency ofplating of the bility of these duplications is absolutely linked to the instability topA null mutants harboring pSK760 is at least four orders of of the 30°C growth phenotype (data not given). The fact that magnitude greater than these same topA null mutants carrying the unstable "revertants" arise with a frequency >90% of the pSK762c. plated cells suggests that RFM475 and RFM480 at 30°C do not To confirm that these results can be attributed to RNase H die but are rather growing very slowly under a constant activity, we measured the RNase H present in crude extracts selection for the frequent toiC region duplications. Careful as described (23). In the absence of pSK760, all strains have analysis of the efficiency of plating at more reduced temper- RNase H activity close to each other and similar to that seen atures shows that it is not until 18°C that the topA defects in a E. not RNase H does not manifest a truly lethal character (A. Segall, personal commu- wild-type coli (data shown). nication). It is unclear whether this is due to the fact that it is appear to be down regulated in response to a loss of topA. not until 18°C that the gyrB(Ts) defect is truly restored to a When pSK760 was present in our isogenic strains, we observed completely wild-type character or whethei it is not until 18°C an increase in RNase H activity (10- to 15-fold) similar to that that the requirement for topA activity becomes absolute. The previously reported for strains bearing pSK760 (data not fact that duplications (16) previously shown to compensate for shown). When the topA null mutants bearing pSK760 are a topA defect arise at the reduced temperature underscores the propagated at 28°C, they do not accumulate duplications of the fact that the cold-sensitive phenotype is related to the loss of tolC-metC region (data not shown), and we believe that the topA. Factors restoring or improving growth at reduced tem- overproduction of RNase H alone is sufficient to at least peratures in the RFM480 or RFM475 strains are expected to partially correct some of the growth problems resulting from involve the suppression of the problems arising from the loss the loss of topA. of the topA gene function. The de novo construction of If the positive effect of RNase H overexpression on the compensated topA defects in strains with the gyrB(Ts) allele growth of RFM475 and RFM480 is not due to the induction provides a carefully controlled situation in which to study the of some stable genetic changes, it should be possible to consequences of losing topA function. This, we believe, rep- demonstrate reversible suppression of the 28°C growth defect. resents an improvement in the genetic analysis oftopA function To demonstrate this, we constructed a plasmid (pPH310) in over the use of a strain, such as DM800, that contains unknown which the rnhA gene is under the control of the IPTG- compensatory mutations in addition to the characterized inducible tac promoter. This plasmid carries the lacIq gene, and gyrB225 allele (4). in the absence of IPTG, rnhA is tightly repressed. When Partial Complementation of the Cold-Sensitive Phenotype pPH310 is transformed into RFM475 and RFM480, these oftopA gyrB(Ts) Mutants by Overexpression of RNase H. Our strains are able to growth at 28°C but only when IPTG is previous biochemical studies have shown that DNA TOPO I included in the medium. Furthermore, we find that the growth was efficient at inhibiting hypernegative supercoiling and its rate can be adjusted by varying the IPTG concentration (Fig. associated R loops during transcription in the presence of 2). This IPTG-dependent suppression of the growth defect is DNA gyrase (12). If R-loop formation in the absence ofTOPO completely reversible, demonstrating that the suppression does I presents problems to E. coli, then overexpression of RNase not involve an induced stable genetic change.

37°C 280C I RFM445 RFM475 RFM480 I I RFM445 RFM475 RFM480 (gyrBts) (gyrBts (gyrBts (gyrBts) (gyrBts (g yrBts AtopA) topA::Tn IO) AtopA) topA::Tn 1O) V _.27~ ;: ~_~2_f_ = FIG. 1. Effects of RNase H overexpression on the growth of pSK762c I (rn hA-) 3 topA null mutants on LB plates. Cells were grown to saturation in I VB minimal medium with required I amino acids and ampicillin at 50 ,ug/ml and streaked for single col- onies on LB plates with the same pSK760 (rnhA+) amount of ampicillin. Incubations at 37°C and 28°C were, respec- tively, for 22 h and 40 h. Downloaded by guest on October 1, 2021 Genetics: Drolet et at Proc Natl. Acad. Sci. USA 92 (1995) 3529

Table 3. Plating efficiencies of RFM445 [gyrB(Ts)] derivatives a loss of RNase H activity could, in part, be compensated for with various topA mutations in combination with pSK760 or by a decrease in DNA gyrase activity, if a balance in the extent pSK762C plasmid DNAs of in vivo R-loop structures were an important parameter for Ratio of colonies at 28°C to colonies at 37°C E. coli. It is known that strain AQ666 with the rnhA224 mutation Strains and pSK762C pSK760 grows very poorly in Luria plates (0.05% NaCl; ref. 27) topA genotypes BHI TB BHI TB compared to the isogenic wild-type AQ634 (rnhA+). We RFM445 0.94 0.95 0.97 0.92 observed that the introduction of the gyrB203 gyrB221 (topA +) [gyrB(Ts)] combination into AQ666 is able to partially reverse RFM475 4.3 x 10-4 5 x 10-4 0.43 0.90 the poor growth phenotype at 34°C, but not at 28°C (data not (AtopA) shown). We also observed that the rnhA224 mutation confers RFM480 6.6 x 10-5 2 x 10-4 0.36 0.70 a poor growth phenotype on MacConkey plates in all genetic (topA::TnlO) background tested. It can be seen in Fig. 3 that the presence of the gyrB(Ts) allele corrects this phenotype at 37°C but not Cells were grown to saturation in VB minimal medium at 37°C with the required amino acids and ampicillin, diluted, and plated. Incuba- at 280C, as expected if compensation is due to a defect in DNA tions at 37°C were for 20 h and for 40 h with topA mutant strains gyrase activity. Another rnhA224 phenotype that is abolished containing pSK762C. Incubations at 28°C were for 44 h for the topA by the gyrB(Ts) mutation at 37°C is the high rate of plasmid mutants and 24 h for the topA+ strains. multimerization (ref. 28 and data not given). It is clear that a defect in DNA gyrase can suppress several phenotypes caused RNase H overexpression will correct the cold-sensitive by the rnhA224 mutation. growth defects of RFM475 and RFM480 down to 27°C. RNase H overproduction, apparently, does not fully suppress a topA null mutant. To confirm this conclusion, we trans- DISCUSSION formed the Atrp strain RFM430 (gyrB+) with pSK760 and then Growth defects noted in our topA null mutants can be reversed transduced the resulting transformant to prototrophy with by overproducing RNase H. These in vivo observations are in Plvir phage grown on RFM480 (trpI topA::TnlO). Of 200 Trp+ agreement with previous in vitro transcription experiments transductants tested, none was tetracycline-resistant, indicat- (12). The observation that the overproduction of RNase H ing that overexpression of RNase H (at these levels) is not does not correct all the phenotypes associated with a topA null sufficient to fully suppress a topA defect. mutant suggests that topA activity may have functions in If RNase H overexpression helps the growth of topA mu- addition to controlling R-loop formation. However, the dra- tants, the presence of an active rnhA gene might be essential matic suppression observed after the overproduction of RNase for the viability of topA mutants. To test this hypothesis, we H suggests an important and central in vivo role for topA in transduced the Atrp strain MD317 [rnhA224 gyrB(Ts)], to blocking transcription-associated R-loop formation. prototrophy with Plvir phage grown on RFM480 (trp+ The finding in the present study that a defect in DNA gyrase topA::TnlO). The rnhA224 mutation is a nonsense allele lead- function can correct some of the phenotypes associated with an ing to a 45% truncation of the mature RNase H enzyme with rnhA mutation is consistent with the in vitro experiments <0.8% wild-type activity (26). Of 300 Trp+ transductants showing that DNA gyrase activity is an important factor tested, no tetracycline-resistant clones were found. This result promoting R-loop formation during transcription elongation shows that a strain missing both TopA and RNase H is not (12). It also suggests that even in the presence of a functional viable. TOPO I, R-loop formation linked to DNA supercoiling has Correction of Some Phenotypes Associated with an rnhA important consequences. Other phenotypes common to both Mutation by a Ts DNA Gyrase. DNA gyrase and RNase H rnhA and topA mutants include sensitivity to nutritional shifts have opposite and competing effects on the formation of R (ref. 29 and our unpublished results) and poor growth on loops. The supercoiling activity of DNA gyrase in an in vitro MacConkey medium (unpublished results). These phenotypes transcription system provides the required driving force for are also corrected by RNase H overexpression in our topA R-loop formation (12). RNase H has the enzymatic activity mutants. The effects of various rpoB alleles on some pheno- appropriate for removal of such R loops. It seems possible that types associated with an rnhA null mutant have been studied

FIG. 2. Effects of RNase H overex- pression on the growth of topA null X mutants in liquid BHI at 29°C. Cells $* 100 were grown in liquid BHI (with ampicil- at a -~~---01000 0 '1 lin) 29°C after 1:100 dilution from oh overnight growth in VB minimal me- 0 Q *-'10000 dium (with required amino acids and 0 - pSK760 ampicillin). OD600 measurements were co taken by using a Shimadzu spectropho- --- pSK762cj tometer (model UV160U). The pPH310 plasmid was constructed by ligating an -4---0 EcoRI-HindIII fragment containing the E 1 0000 X 1n E. coli rnhA gene with its Shine- 9 pSK760 Dalgarno sequence (but no promoter) C obtained by PCR, into the EcoRI- A pSK762 HindIII-digested pTRC99A plasmid (an inducible expression vector from Phar- macia). Numbers, from 0 to 10,000,,rep- 0 2 4 6 8 10 12 14 resent IPTG micromolar concentrations that were added to induce rnhA gene Time, h transcription from pPH310. Downloaded by guest on October 1, 2021 3530 Genetics: Drolet et aL Proc. Natl. Acad ScL USA 92 (1995)

3 70C 2 8°C tive supercoils, will suppress it. It is also possible that the R-loop structure, with its single-stranded DNA region, con- stitutes a hot spot for DNA TOPO 1. The action of DNA TOPO I as soon as R-loop formation is initiated may result in gyrBts the destabilization of the short RNADNA hybrid, leading to rhA gyrBts its dissociation. Since our present study did not address this rnhA question, it is subject to speculation. The current study implies that in the absence of TOPO I, R loops accumulate and that some of the problems associated with a topA mutant are related to R-loop formation. The FIG. 3. Effect of a gyrBts mutation on the growth of an rnhA nature of the problems presented by R loops is also open to mutant on MacConkey medium. Cells were grown to saturation in VB R Do R loops minimal medium with required amino acids and streaked for single speculation. Are loops a block to transcription? colonies on MacConkey plate with 0.2% glucose. Incubations at 37°C lead to inappropriate attempts at DNA replication? Do R and 28°C were, respectively, for 17 h and 22 h. Bacterial strains used loops represent hot spots for both homologous and nonho- are derivatives of RFM430 (WT) and RFM431 [gyrB(Ts)] and were mologous recombination? With our set of isogenic strains and constructed as follows: A phage lysate prepared by infecting CAG- the means to carefully control RNase H production, it should 18633 (zag-3198::TnlOkan) with Plvir was used to transduce AQ666 be possible to answer some of these questions about the (rnhA224) to kanamycin resistance. Recombinants that kept the formation, dynamics, and consequences of R-loop structures. rnhA224 allele were selected by screening for a growth defect on Luria and MacConkey plates at 28°C and high rate of plasmid DNA We thank T. Kogoma and C. Gross for the generous gift of bacterial multimerization. One such recombinant (MD310) was used to prepare strains and L. Gold for excellent technical assistance. This work was a Plvir lysate that was subsequently used to transduce RFM430 and supported by grants from the Medical Research Council of Canada RFM431 to kanamycin resistance. rnhA + and rnhA224 derivatives of (MT-12667), the FCAR (Fonds pour la Formation de Chercheurs et each strain (MD315, MD316, MD317, and MD318), selected as l'Aide a la Recherche), and the CAFIR (Comite d'Attribution des described above, were kept and used in the present experiment. The Fonds Internes de Recherche) to M.D. presence of the rhnA224 allele in MD315 and MD317 was further confirmed by nucleotide sequencing. 1. Wang, J. C. (1971) J. Mol. Bio. 55, 523-533. 2. Trucksis, M. & Depew, R. E. (1981) Proc. Natl. Acad. Sci. USA 78, (30). A strain with the rpoB8 allele, which produces a slow 2164-2168. moving and termination-proficient RNA (31), 3. Stemglanz, R., DiNardo, S., Voelkel, K. A., Nishimura, Y., Hirota, Y., Becherer, K., Zumstein, L. & Wang, J. C. (1981) Proc. Natl. Acad. Sci. provided some relief, while a strain with the rpoB3595 allele USA 78, 2747-2751. producing an RNA polymerase, which is fast moving and 4. DiNardo, S., Voelkel, K. A. & Sternglanz, R. (1982) Cell 31, 43-51. termination-deficient (31), exacerbated some growth defects. 5. Pruss, G. J., Manes, S. H. & Drlica, K. (1982) Cell 31, 35-42. Interestingly, we observe that the rpoB8 allele also helps the 6. Kirkegaard, K. & Wang, J. C. (1985) J. Mol. Biol. 185, 625-637. 7. Pruss, G. J., Franco, R. J., Chevalier, S. G., Manes, S. H. & Drlica, K. growth of our topA defective strains, but the rpoB3595 allele is (1986) J. Bacteriol. 168, 276-282. detrimental for the growth of these strains (unpublished 8. Bliska, J. & Cozzarelli, N. (1987) J. Mol. Bio. 194, 205-218. results). Moreover, we found that the growth problem of an 9. Pruss, G. J. & Drlica, K. (1986) Proc. Natl. Acad. Sci. USA 83, rnhA224 rpoB3595 mutant can be partially corrected at 37°C by 8952-8956. the introduction of the gyrB(Ts) mutation (unpublished re- 10. Wu, H.-Y., Shyy, S. H., Wang, J. C. & Liu, L. F. (1988) Cell 53, DNA mu- 433-440. sult). Thus, these observations suggest that gyrase 11. Liu, L. F., & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, tations may exert their topA compensating effects, in part, by 7024-7027. reducing the extent and/or amount of R loops formed. We 12. Drolet, M, Bi, X. & Liu, L. F. (1994) J. Bio. Chem. 269, 2068-2074. believe that many of the phenotypes associated with topA 13. Durrenberger, M., Bjornsti, M.-A., Uetz, T., Hobot, J. A. & Kellen- berger, E. (1988) J. Bacteriol. 170, 4757-4768. mutations may be linked to R-loop formation. 14. Drolet, M., Wu, H.-Y. & Liu, L. F. 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