Relaxation of Transcription-induced Negative Supercoiling Is an Essential Function of Escherichia coli DNA Topoisomerase I
1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês
10.1074/jbc.274.23.16654
ISSN1083-351X
Autores Tópico(s)DNA Repair Mechanisms
ResumoIt has been suggested that the essential function of DNA topoisomerase I in Escherichia coli is to prevent chromosomal DNA from reaching an unacceptably high level of global negative supercoiling. However, other in vivo studies have shown that DNA topoisomerase I is very effective in removing local negative supercoiling generated during transcription elongation. To determine whether topoisomerase I is essential for controlling global or local DNA supercoiling, we have prepared a set of topAnull mutant strains in combination with different plasmid DNAs. Although we found a correlation between the severity of the growth defect with both transcription-induced and global supercoiling, near to complete growth inhibition correlated only with transcription-induced supercoiling. This result strongly suggests that the major function of DNA topoisomerase I is to relax local negative supercoiling generated during transcription elongation. It has been suggested that the essential function of DNA topoisomerase I in Escherichia coli is to prevent chromosomal DNA from reaching an unacceptably high level of global negative supercoiling. However, other in vivo studies have shown that DNA topoisomerase I is very effective in removing local negative supercoiling generated during transcription elongation. To determine whether topoisomerase I is essential for controlling global or local DNA supercoiling, we have prepared a set of topAnull mutant strains in combination with different plasmid DNAs. Although we found a correlation between the severity of the growth defect with both transcription-induced and global supercoiling, near to complete growth inhibition correlated only with transcription-induced supercoiling. This result strongly suggests that the major function of DNA topoisomerase I is to relax local negative supercoiling generated during transcription elongation. Based on genetic evidence, it has been postulated that the maintenance of a global level of chromosomal negative supercoiling within a ±15% range is required for good growth of Escherichia coli cells (1Drlica K. Mol. Microbiol. 1992; 6: 425-433Crossref PubMed Scopus (289) Google Scholar). The global level of DNA supercoiling reflects the average superhelical density of all supercoiling domains. In this context, the essential function of DNA topoisomerase I is to prevent chromosomal DNA from reaching an inappropriate level of negative supercoiling. This model stems from the observation thattopA null mutants are viable only if they accumulate compensatory mutations that are very often found in one of the genes encoding a subunit of DNA gyrase. As a result, global negative supercoiling of both chromosomal and plasmid DNA were decreased below the normal level (2Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 3DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google Scholar). Therefore, this global level of negative supercoiling is believed to be regulated by the opposing enzymatic activities of DNA topoisomerase I, encoded by the topA gene, which specifically relaxes negative supercoiling, and DNA gyrase, with two different subunits encoded by gyrA and gyrBthat introduces negative supercoiling. However, exactly how a high level of global negative supercoiling could be detrimental to cell growth is not known. In vivo and in vitro studies have shown that DNA topoisomerase I is highly efficient in removing negative supercoils produced in the wake of moving transcription complexes (4Tsao Y.P. Wu H.-Y. Liu L.F. Cell. 1989; 56: 111-118Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 5Wu H.Y. Shyy S.H. Wang J.C. Liu L.F. Cell. 1988; 53: 433-440Abstract Full Text PDF PubMed Scopus (546) Google Scholar, 6Drolet M. Bi X. Liu L.F. J. Biol. Chem. 1994; 269: 2068-2074Abstract Full Text PDF PubMed Google Scholar). Such supercoiling can be generated during transcription elongation because of the difficulty for a moving transcription complex to rotate around the double helix (the twin-domain model for transcription; see Ref. 7Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Crossref PubMed Scopus (1518) Google Scholar). In this situation, domains of negative and positive supercoiling are transiently generated, respectively, behind and ahead of the moving transcription complex. In the absence of DNA topoisomerase I, the local negative supercoiled domain can build up, whereas the positive one can be removed by DNA gyrase. In some cases, especially when the transcribed genes encode membrane bound proteins, extreme negative supercoiling is generated by transcription (8Cook D.N. Ma D. Pon N.G. Hearst J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10603-10607Crossref PubMed Scopus (58) Google Scholar, 9Lynch A.S. Wang J.C. J. Bacteriol. 1993; 175: 1645-1655Crossref PubMed Scopus (130) Google Scholar). When such genes are present on a plasmid DNA, transcription in the absence of DNA topoisomerase I has been shown to generate hypernegatively supercoiled DNA (5Wu H.Y. Shyy S.H. Wang J.C. Liu L.F. Cell. 1988; 53: 433-440Abstract Full Text PDF PubMed Scopus (546) Google Scholar, 8Cook D.N. Ma D. Pon N.G. Hearst J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10603-10607Crossref PubMed Scopus (58) Google Scholar, 9Lynch A.S. Wang J.C. J. Bacteriol. 1993; 175: 1645-1655Crossref PubMed Scopus (130) Google Scholar, 10Pruss G.J. J. Mol. Biol. 1985; 185: 51-63Crossref PubMed Scopus (95) Google Scholar). Therefore, in the context of transcription elongation, the major role of DNA topoisomerase I is to control important local fluctuations of negative supercoiling, as opposed to simply maintaining global chromosome supercoiling at a constant level. Given the fact that in all these studies, the experiments were performed withtopA null mutants with compensatory gyrase mutations, one may conclude that the removal of transcription-induced negative supercoiling by DNA topoisomerase I is not essential for cell growth. In the work reported here, we present data suggesting that the essential function of DNA topoisomerase I is linked to transcription elongation and not to the control of the global level of negative supercoiling. These results were obtained by measuring the linking number deficit of pBR322 derivatives extracted from varioustopA mutants having different growth capacities. On these pBR322 derivatives, the effect of transcription of the tetAgene that encodes a membrane bound protein is either kept at its minimum or totally abolished. These plasmid DNAs allowed us to measure both global negative supercoiling and transcription-induced negative supercoiling and to make a correlation between these parameters and the growth of the various topA mutants. Our results indicate that the essential function of DNA topoisomerase I is linked to transcription-induced negative supercoiling. Our results presented in Ref. 11Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar further suggest that one detrimental consequence of the failure to relax transcription-induced supercoiling is R-loop formation (11Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The E. coli strains used are listed and described in Table I. DM800 derivatives were constructed by P1vir transduction (12Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). The RFM475 cold-sensitive strain is well described by Drolet et al. (13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar).Table IE. coli strains used in this studyStrainsGenotypeReference/constructionRFM475rpsL galK2, Δlac74, gyrB221(couR), gyrB203(Ts) Δ(topA cysB)20413Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google ScholarDM800Δ(topA cysB)204 gyrB225, acrA132Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 3DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google ScholarCAG18592zie-3163::Tn10kan14Singer M. Baker T.A. Schnitzler G. Deischel S.M. Goel M. Dove W. Jaacks K.J. Grossman A.D. Erickson J.W. Gross C.A. Microbiol. Rev. 1989; 53: 1-24Crossref PubMed Google ScholarRFM445rpsL galK2, Δlac74, gyrB221(couR),gyrB203(Ts)13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google ScholarPH326RFM445zie-3163::Tn10kanRFM445XP1.CAG18592, select Kmr, Cour and TsEM169DM800gyrB221(couR),gyrB203(Ts)DM800XP1.PH326, select Kmr, Cour and TsEM176aThe gyrB + selection is based on the observation that gyrB + cells are Bgl−, whereas gyrB225 cells are Bgl+ due to the activation of the cryptic bgl operon (3). Bgl+ cells will be able to use the β-glucoside analog 5-bromo-4-chloro-3-indolyl β-d-glucoside and form blue colonies, whereas Bgl− cells will not use this analog and will form white colonies.DM800gyrB +DM800XP1.CAG18592, select Kmr, small colonies and white colonies in the presence of 5-bromo-4-chloro-3-indolyl β-d-glucosidea The gyrB + selection is based on the observation that gyrB + cells are Bgl−, whereas gyrB225 cells are Bgl+ due to the activation of the cryptic bgl operon (3DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google Scholar). Bgl+ cells will be able to use the β-glucoside analog 5-bromo-4-chloro-3-indolyl β-d-glucoside and form blue colonies, whereas Bgl− cells will not use this analog and will form white colonies. Open table in a new tab pBR322ΔPtet is a pBR322 derivative with a small deletion within the tetA promoter region (5Wu H.Y. Shyy S.H. Wang J.C. Liu L.F. Cell. 1988; 53: 433-440Abstract Full Text PDF PubMed Scopus (546) Google Scholar). This deletion was further characterized in the present study by sequencing the appropriate region of the plasmid (Fig. 1). pBR322Δtet5′ was obtained by deleting theHindIII-EcoRV DNA fragment from pBR322. TheHindIII-EcoRV-digested pBR322 vector was treated with the Klenow enzyme to fill in the HindIII site, before being treated with DNA ligase. Transformants were selected on LB medium with ampicillin and were screened for sensitivity to tetracycline. The plasmid DNA of some Tets transformants was analyzed by using appropriate restriction enzymes to confirm theHindIII-EcoRV deletion. Unless otherwise indicated, bacteria were grown in VB Casa or LB medium (13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar) supplemented with cysteine (50 μg/ml) at the temperature indicated in the table and figure legends. When needed, antibiotics were added as follows: ampicillin at 50 μg/ml, and chloramphenicol at 30 μg/ml. Because of the acrA13 mutation in the DM800 derivatives, which renders these cells more permeable to many antibiotics (3DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google Scholar), chloramphenicol was used at 10 μg/ml for these cells. Isopropyl-β-d-thiogalactoside and 5-bromo-4-chloro-3-indolyl β-d-glucoside were purchased from Sigma. For the extraction of plasmid DNAs for supercoiling analysis, bacterial cells carrying various pBR322 derivatives were grown overnight in VB Casa medium at 37 °C and then diluted 1/75 in prewarmed LB medium. The cells were grown to an A 600 of 0.4 at 37 °C at which time they were transferred to the desired temperature. The plasmid DNAs were extracted when theA 600 reached about 0.7 or after an exposition of about 2 h at the respective temperature when anA 600 of 0.7 could not be reached. We found that when hypernegatively supercoiled plasmid DNAs were produced at 21 °C, the proportion of such topoisomers reached a maximum after about 1 h at this temperature and did not change for at least another hour. Growth was stopped by transferring the cells in a tube filled with ice. By this procedure, the temperature of the cultures immediately dropped to 0 °C. Plasmid DNAs were extracted by an alkaline lysis procedure (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The current molecular biology techniques were performed essentially as described (16Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). CaCl2 transformations were carried out as described by Drolet et al. (13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar). One-dimensional and two-dimensional agarose gel electrophoresis in the presence of chloroquine in 0.5 × TBE were performed essentially as described (16Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Chloroquine was used at the concentration indicated in the figure legends. After electrophoresis, agarose gels were dried and prepared for in situhybridization as described (16Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Most of the previous studies related to the effects of transcription on DNA supercoiling were designed to support the twin-domain model for transcription (7Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Crossref PubMed Scopus (1518) Google Scholar) but not to test if DNA topoisomerase I activity during transcription is crucial for cell growth. In these studies, transcription of genes encoding membrane bound proteins allowed the extraction of hypernegatively supercoiled plasmid DNAs from topA null mutants (5Wu H.Y. Shyy S.H. Wang J.C. Liu L.F. Cell. 1988; 53: 433-440Abstract Full Text PDF PubMed Scopus (546) Google Scholar, 8Cook D.N. Ma D. Pon N.G. Hearst J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10603-10607Crossref PubMed Scopus (58) Google Scholar, 9Lynch A.S. Wang J.C. J. Bacteriol. 1993; 175: 1645-1655Crossref PubMed Scopus (130) Google Scholar). Although the results of these studies supported very well the twin-domain model for transcription, the question of DNA topoisomerase I activity being essential during transcription was not addressed or could not even be addressed, because the topA null mutants used in these studies grew very well. Indeed, the fact that these strains grew very well may suggest that DNA topoisomerase I activity during transcription is not normally essential. Therefore, to better address the role of DNA topoisomerase I in transcription elongation, we had to design a genetic system in which at least two requirements needed to be met: 1) the plasmid DNAs used should not carry genes encoding for membrane bound products; 2) thetopA mutants used should not benefit from compensatory mutations in order for the topA phenotypes to be fully expressed. Because most of the results supporting the twin-domain model for transcription were obtained by using pBR322 DNA, we decided to use several of its derivatives in our studies. In the early studies, the extraction of hypernegatively supercoiled pBR322 DNA fromtopA null mutants carrying compensatory mutations, was clearly shown to be dependent on tetA gene expression, which encodes a membrane-bound protein (9Lynch A.S. Wang J.C. J. Bacteriol. 1993; 175: 1645-1655Crossref PubMed Scopus (130) Google Scholar). One derivative used in one study, pBR322ΔPtet, has a deletion within the promoter responsible for tetA expression (Fig.1 A), which was shown to abolish the production of hypernegatively supercoiled pBR322 DNA (5Wu H.Y. Shyy S.H. Wang J.C. Liu L.F. Cell. 1988; 53: 433-440Abstract Full Text PDF PubMed Scopus (546) Google Scholar). We therefore decided to use this pBR322 derivative in our studies. However, as can be seen in Table II, this plasmid confers residual tetracycline resistance, because bacterial cells carrying it can grow in the presence of 4 μg/ml tetracycline, whereas cells carrying no plasmid DNAs can only grow when tetracycline concentrations do not exceed 1 μg/ml. This residual tetAexpression can be explained by the fact that a weak −10 promoter region, according to the consensus, was created during plasmid construction (Fig. 1 B). We constructed an additional pBR322 derivative, pBR322Δtet5′ (Fig. 1 A), from whichtetA gene expression is not detectable (Table II). This plasmid has an HindIII-EcoRV deletion, removing the 5′ part of the gene including the original −10 promoter region, the Shine-Dalgarno sequence, the ATG initiator codon, and one transmembrane domain responsible for the anchorage of the TetA protein to the membrane (Fig. 1 A). Interestingly, by performing such a deletion, a −10 region that restored a promoter sequence was produced (Fig. 1 B). Indeed, promoter activity was detected when the lacZ gene was cloned downstream in the appropriate orientation (Fig. 1 B).Table IIGrowth of RFM475 carrying pBR322 derivatives on LB plates with various amounts of tetracyclinePlasmidsTetracycline01246μg/mlNo plasmid+++−−−pBR322++++++++++pBR322ΔPtet+++++++−pBR322Δtet5′+++−−−RFM475 cells carrying the indicated plasmid were grown overnight in liquid VB Casa medium with cysteine and ampicillin (except for the strain carrying no plasmid) at 37 °C, and 2 μl were streaked on LB plates with the indicated concentration of tetracycline. The plates were incubated for 20 hrs at 37 °C. +, reflects colony size; −, absence of growth. Open table in a new tab RFM475 cells carrying the indicated plasmid were grown overnight in liquid VB Casa medium with cysteine and ampicillin (except for the strain carrying no plasmid) at 37 °C, and 2 μl were streaked on LB plates with the indicated concentration of tetracycline. The plates were incubated for 20 hrs at 37 °C. +, reflects colony size; −, absence of growth. The relative growth capacity of the various topA null strains used in our studies is shown in TableIII. It can be seen that the strain showing the most severe growth defect and therefore, in our view, the strain that most closely resembles a true topA null mutant without compensatory mutations is RFM475. Low temperatures are more restrictive for this strain because of the gyrB(Ts) allele that regains a more wild-type level of activity at these temperatures (13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar). The other strain that grows poorly is a gyrB+ derivative of the widely used DM800 strain (ΔtopA andgyrB225). The fact that it is possible to introduce a wild-type gyrB allele in DM800 indicates a possible presence of additional compensatory mutation(s) as previously considered when a similar transduction experiment was performed (3DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google Scholar). Moreover agyrB(Ts) derivative of DM800, a strain that should almost be identical to RFM475, grows significantly better than RFM475 (TableIII), again suggesting that additional compensatory mutation(s) might be present. It is also important to note that all the ΔtopA strains used in our studies carry the sametopA deletion [Δ(topA cysB)204].Table IIIRelative colony size on LB medium of the various ΔtopA strains used in this studyBacterial strainsTemperature37 °C28 °C21 °CDM800 [ΔtopA, gyrB225]++++++++++++EM176 [DM800 gyrB +]++1/2EM169 [DM800gyrB(Ts)]++++++1/2++RFM475 [ΔtopA, gyrB(Ts)]++−aThe few colonies that appeared are made of bacterial cells with tolC region duplications known to compensate for the absence of topA, as previously described for this bacterial strain (13).−Bacterial cells were grown overnight in liquid VB Casa medium with cysteine at 37 °C, and 2 μl were streaked on LB plates that were incubated at 37, 28, and 21 °C, respectively, for 20, 42, and 72 h (EM176 and RFM475 were incubated for 6 days at 21 °C). +, reflects colony size; −, absence of growth.a The few colonies that appeared are made of bacterial cells with tolC region duplications known to compensate for the absence of topA, as previously described for this bacterial strain (13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar). Open table in a new tab Bacterial cells were grown overnight in liquid VB Casa medium with cysteine at 37 °C, and 2 μl were streaked on LB plates that were incubated at 37, 28, and 21 °C, respectively, for 20, 42, and 72 h (EM176 and RFM475 were incubated for 6 days at 21 °C). +, reflects colony size; −, absence of growth. The various ΔtopAmutants carrying pBR322ΔPtet were grown in LB medium at 37 °C and exposed to the indicated temperatures as described under "Experimental Procedures." All the strains were exposed to various temperatures to discriminate between true temperature effects and allele specific effects [gyrB(Ts)] on DNA supercoiling. The extracted plasmid DNAs were subjected to electrophoresis in agarose gel in the presence of chloroquine at 7.5 μg/ml, as described under "Experimental Procedures." Under these conditions the more negatively supercoiled topoisomers migrate slowly except for the fastest migrating band pointed to by an arrow (Fig.2 A, lane 12 [- -]), which represents hypernegatively supercoiled plasmid DNAs. It can be seen that the global DNA supercoiling level in the various strains, represented by the topoisomers distributions of pBR322ΔPtet DNA without considering hypernegatively supercoiled DNA, reflects very well the level of gyrase activity within these strains (Fig. 2 A). Indeed, plasmid DNAs extracted from the DM800 strain that carries the gyrB225 mutation are less negatively supercoiled than plasmid DNAs extracted from the DM800gyrB+ derivative (EM176) or the gyrB(Ts) strains (EM169 and RFM475) exposed to temperatures of 28 °C and below (Fig.2 A). It is also obvious that the global negative supercoiling level correlates with the growth defects (Table III). However, this correlation is not complete because the global negative supercoiling level eventually reaches a maximum value even though some bacterial strains are still growing, albeit slowly (EM169 at 28 °C and 21 °C, EM176 at 28 °C, and very slowly at 21 °C), whereas others are not (RFM475 at 28 °C (almost undetectable growth) and 21 °C). Under more restrictive conditions, when the ΔtopA mutant completely fails to grow (RFM475 at 21 °C), hypernegatively supercoiled pBR322ΔPtet DNA is found (Fig. 2, A, lane 12; B,bottom right panel). The formation of such topoisomers is completely dependent on transcription, because it is abolished by rifampicin treatment (data not shown). Such topoisomers are not found when pBR322ΔPtet DNA is either extracted from DM800 carrying the gyrB+ allele (Fig. 2, A,lanes 4-6; B, top middle panel), or DM800 carrying the gyrB(Ts) allele and grown at 21 °C (Fig. 2, A, lane 9;B, top right panel), again supporting our conclusion about the presence of additional compensatory mutation(s) within DM800. Similar results are also obtained when the same set of experiments are performed with pBR322Δtet5′ (Fig.3). A larger proportion of hypernegatively supercoiled topoisomers was detected when pBR322Δtet5′ DNA was extracted from RFM480 grown at 21 °C (and a small amount from cells grown at 28 °C), a strain identical to RFM475, but carrying atopA::Tn10 allele instead of the [Δ(topA cysB)204] found in RFM475 (11Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This is possibly due to the fact that RFM480 is genetically more stable than RFM475.Figure 3Transcription-induced hypernegative supercoiling of pBR322Δtet5′ DNA in various topA null mutant. The varioustopA null mutants carrying pBR322Δtet5′ were grown, and the plasmid DNAs were extracted and analyzed as described in the legend to Fig. 2.View Large Image Figure ViewerDownload (PPT) Our results show that severe growth inhibition of Escherchia coli in the absence of DNA topoisomerase I correlates with transcription-induced negative supercoiling, but not with global negative supercoiling. Therefore, it suggests that topAmutants fail to grow when DNA gyrase is too active during the process of transcription elongation. Under these conditions, DNA gyrase activity is efficient whether or not genes encode for membrane bound proteins. In fact, under these conditions, we have also detected hypernegatively supercoiled topoisomers for several others plasmid DNAs that do not carry genes encoding for membrane bound proteins. For the moment, we cannot exclude the possibility that the apparent increase in DNA gyrase activity at low temperatures is not, for unknown reasons, only linked to the gyrB(Ts) allele, but to the low temperature itself. Indeed, our preliminary data with DM800 derivatives suggest that this might be the case. 1E. Massé and M. Drolet, unpublished results. Either way, the result of this activity, if not counteracted by DNA topoisomerase I, is inhibitory to cell growth. The results presented in the accompanying manuscript (11Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) show that extensive R-loop formation can occur under these conditions (11Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This is most likely inhibitory to cell growth, because the cellular level of RNase H activity must be properly increased to support growth under conditions where topAmutations are fully expressed (13Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar). Our results also strongly suggest that gyr mutations arose in topA mutants to reduce gyrase activity during transcription elongation. We can also conclude that the increase in the level of global negative supercoiling in some topA mutants (see Ref. 1Drlica K. Mol. Microbiol. 1992; 6: 425-433Crossref PubMed Scopus (289) Google Scholar and this work), is only a secondary consequence of the absence of DNA topoisomerase I and is not linked to the essential function of this enzyme. This notion is further supported by our recent observations that the overproduction of both RNase H (11Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and topoisomerase III (TopB) 2S. Broccoli, P. Phoenix, E. Massé, and M. Drolet, manuscript in preparation. can very well correct the growth defect of topA null mutants without, however, altering the global supercoiling level. In conclusion, although the previous studies with varioustopA null mutants have been very useful to reveal the regulatory potential of negative supercoiling on DNA functions, they did not reveal the essential function of DNA topoisomerase I. In our view, this enzyme should be considered, at least in part, as a transcription factor and not as a regulator of the global supercoiling level. We thank Pauline Phoenix for technical assistance and Sonia Broccoli for careful reading of the manuscript.
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