Direct Interaction between Escherichia coli RNA Polymerase and the Zinc Ribbon Domains of DNA Topoisomerase I
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m303403200
ISSN1083-351X
AutoresBokun Cheng, Chang-Xi Zhu, Chengling Ji, Adriana Ahumada, Yuk-Ching Tse-Dinh,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoEscherichia coli DNA topoisomerase I (encoded by the topA gene) is important for maintaining steady-state DNA supercoiling and has been shown to influence vital cellular processes including transcription. Topoisomerase I activity is also needed to remove hypernegative supercoiling generated on the DNA template by the progressing RNA polymerase complex during transcription elongation. The accumulation of hypernegative supercoiling in the absence of topoisomerase I can lead to R-loop formation by the nascent transcript and template strand, leading to suppression of transcription elongation. Here we show by affinity chromatography and overlay blotting that E. coli DNA topoisomerase I interacts directly with the RNA polymerase complex. The protein-protein interaction involves the β′ subunit of RNA polymerase and the C-terminal domains of E. coli DNA topoisomerase I, which are homologous to the zinc ribbon domains in a number of transcription factors. This direct interaction can bring the topoisomerase I relaxing activity to the site of transcription where its activity is needed. The zinc ribbon C-terminal domains of other type IA topoisomerases, including mammalian topoisomerase III, may also help link the enzyme activities to their physiological functions, potentially including replication, transcription, recombination, and repair. Escherichia coli DNA topoisomerase I (encoded by the topA gene) is important for maintaining steady-state DNA supercoiling and has been shown to influence vital cellular processes including transcription. Topoisomerase I activity is also needed to remove hypernegative supercoiling generated on the DNA template by the progressing RNA polymerase complex during transcription elongation. The accumulation of hypernegative supercoiling in the absence of topoisomerase I can lead to R-loop formation by the nascent transcript and template strand, leading to suppression of transcription elongation. Here we show by affinity chromatography and overlay blotting that E. coli DNA topoisomerase I interacts directly with the RNA polymerase complex. The protein-protein interaction involves the β′ subunit of RNA polymerase and the C-terminal domains of E. coli DNA topoisomerase I, which are homologous to the zinc ribbon domains in a number of transcription factors. This direct interaction can bring the topoisomerase I relaxing activity to the site of transcription where its activity is needed. The zinc ribbon C-terminal domains of other type IA topoisomerases, including mammalian topoisomerase III, may also help link the enzyme activities to their physiological functions, potentially including replication, transcription, recombination, and repair. DNA topoisomerases are ubiquitous enzymes that have functional roles in many vital cellular processes (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2188) Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2073) Google Scholar). Among different classes of topoisomerases, type IA topoisomerases found in archea, prokaryotes, and eukaryotes share the mechanistic feature of cutting and rejoining a single strand of DNA via a 5′-phosphotyrosine linkage and homologous amino acid sequences (3Tse-Dinh Y.-C. Biochim. Biophys. Acta. 1998; 1400: 19-27Crossref PubMed Scopus (51) Google Scholar). Escherichia coli DNA topoisomerase I (encoded by the topA gene) is the most extensively studied example of this class of enzyme. Its most apparent physiological role is the maintenance of steady-state DNA supercoiling (4Drlica K. Mol. Microbiol. 1992; 6: 415-433Crossref Scopus (286) Google Scholar, 5Zechiedrich E.L. Khodursky A.B. Bachellier S. Schneider R. Chen D. Lilley D.M. Cozzarelli N.R. J. Biol. Chem. 2000; 275: 8103-8113Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). During transcription, the movement of the RNA polymerase complex on the DNA template creates local transcription-driven supercoiling with negative supercoiling generated behind the RNA polymerase and positive supercoiling generated ahead of the RNA polymerase (6Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Crossref PubMed Scopus (1505) Google Scholar, 7Tsao Y.-P. Wu H.-Y. Liu L.F. Cell. 1989; 56: 111-118Abstract Full Text PDF PubMed Scopus (260) Google Scholar). DNA gyrase is needed for removing the positive supercoils, and topoisomerase I is responsible for removing the excess negative supercoils. In the absence of topoisomerase I function due to mutation in the topA gene, the accumulation of hypernegative supercoiling can lead to R-loop formation by nascent transcription and template stranding with the consequent suppression of transcription elongation (8Drolet 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 (194) Google Scholar, 9Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In previous studies, Tn5 transposase was found to copurify with E. coli DNA topoisomerase I and inhibit the topoisomerase I activity (10Yigit H. Reznikoff W.S. J. Bacteriol. 1999; 181: 3185-3192Crossref PubMed Google Scholar). RNA polymerase was also found to copurify with Tn5 transposase, but the copurification was reduced in extracts from a topA mutant strain, suggesting that the interaction between RNA polymerase and DNA topoisomerase I was responsible for the copurification of RNA polymerase with Tn5 transposase (10Yigit H. Reznikoff W.S. J. Bacteriol. 1999; 181: 3185-3192Crossref PubMed Google Scholar). The proposed function of topoisomerase I activity in removal of transcription-driven hypernegative supercoiling (8Drolet 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 (194) Google Scholar, 9Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) would be greatly facilitated by direct protein-protein interaction with RNA polymerase. Experiments described here provide direct evidence for such interaction as well as identifying the domain of topoisomerase I and the subunit in RNA polymerase that are responsible for this protein-protein interaction. Enzymes—E. coli DNA topoisomerase I and its subdomains were expressed and purified as described previously (11Zhu C.-X. Tse-Dinh Y.-C. Bjornsti M.-A. Osheroff N. Methods in Molecular Biology. Humana Press, Totowa, NJ1999: 145-151Google Scholar, 12Zhu C.-X. Samuel M. Pound A. Ahumada A. Tse-Dinh Y.-C. Biochem. Mol. Biol. Int. 1995; 35: 375-385PubMed Google Scholar, 13Ahumada A. Tse-Dinh Y.-C. Biochem. Biophys. Res. Commun. 1998; 251: 509-514Crossref PubMed Scopus (24) Google Scholar, 14Ahumada, A., and Tse-Dinh, Y.-C. (2002) BMC Biochemistry http://www.biomedcentral.com/1471-2091/3/13.Google Scholar). Purified E. coli RNA polymerase was purchased from Epicentre and USB Corp. Maltose-binding Protein Affinity Chromatography—A PCR fragment encoding the E. coli topA gene was generated by Pfu DNA polymerase (from Stratagene) and cloned into the XmnI site of pMal-c2X (New England BioLabs) to create a fusion protein with MBP 1The abbreviations used are: MBP, maltose-binding protein; DIG, digoxigenin; NEM, N-ethylmaleimide. linked to the N terminus of topoisomerase I. Expression of MBP-topoisomerase I and MBP in E. coli TB1 cells was induced with isopropyl-1-thio-β-d-galactopyranoside. Cells from a 250-ml culture were lysed by lysozyme treatment combined with freeze-thawing in phosphate-buffered saline. The soluble extract obtained after centrifugation was applied to 1 ml of amylose resin (New England BioLabs) equilibrated with column buffer (20 mm Tris, pH 7.5, 200 mm NaCl, 1 mm EDTA). After extensive washing, the proteins bound to the column were eluted with column buffer containing 10 mm maltose. RNA Polymerase Affinity Chromatography—Polyol-responsive monoclonal antibodies against E. coli RNA polymerase β′ subunit (NT73) was purchased from Neoclones and coupled to cyanogen bromide-activated Sepharose according to published procedures using 0.9 ml of the antibodies (15Thompson N.E. Hager D.A. Burgess R.R. Biochemistry. 1992; 31: 7003-7008Crossref PubMed Scopus (70) Google Scholar). The NT73 affinity matrix was mixed with 1 ml of extracts of E. coli BL21 cells prepared from 400 ml of culture expressing intact topoisomerase I (11Zhu C.-X. Tse-Dinh Y.-C. Bjornsti M.-A. Osheroff N. Methods in Molecular Biology. Humana Press, Totowa, NJ1999: 145-151Google Scholar) or its subdomains (12Zhu C.-X. Samuel M. Pound A. Ahumada A. Tse-Dinh Y.-C. Biochem. Mol. Biol. Int. 1995; 35: 375-385PubMed Google Scholar, 13Ahumada A. Tse-Dinh Y.-C. Biochem. Biophys. Res. Commun. 1998; 251: 509-514Crossref PubMed Scopus (24) Google Scholar, 14Ahumada, A., and Tse-Dinh, Y.-C. (2002) BMC Biochemistry http://www.biomedcentral.com/1471-2091/3/13.Google Scholar) as described for affinity purification of RNA polymerase (15Thompson N.E. Hager D.A. Burgess R.R. Biochemistry. 1992; 31: 7003-7008Crossref PubMed Scopus (70) Google Scholar). After extensive washing, the bound proteins were eluted with 1 ml of 40% ethylene glycol with 0.75 m NaCl. Blotting of RNA Polymerase with Digoxigenin (DIG)-labeled Topoisomerase I and Its Subdomains—Purified RNA polymerase (0.8 μg) was electrophoresed in SDS-polyacrylamide gel to separate the subunits. The proteins were transferred onto either supported nitrocellulose membrane for chemiluminescence detection or polyvinylidene difluoride membrane for color detection. Topoisomerase I and its subdomains were labeled with DIG using the labeling kit from Roche Applied Science. Each DIG-labeled protein was incubated with the membrane at 25 °C for 1 h. Anti-DIG antibodies linked to peroxidase were used for detection by the ECL Plus system (Amersham Biosciences). Anti-DIG antibodies linked to alkaline phosphatase were used for color detection with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (Roche Applied Sciences). After color detection, the position of the topoisomerase I-binding signal was marked and photographed before staining of the nylon membrane by Coomassie Blue. In Vitro Transcription—In vitro transcription with E. coli RNA polymerase (3 units) was carried out with procedures similar to those described previously (16Leng F. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9139-9144Crossref PubMed Scopus (54) Google Scholar). The reaction (30 μl) contained 40 mm Hepes/KOH (pH 7.6), 11 mm magnesium acetate, 100 mm potassium glutamate, 1 mm dithiothreitol, 4 mm ATP, 0.5 mm each of GTP, CTP, and UTP, 0.5 μg of negatively supercoiled plasmid pBR322. Transcription was carried out at 37 °C for 10 min. After deproteinization by phenol extraction, the DNA samples were analyzed by electrophoresis in 1% agarose gel with 0.5× TPE (90 mm Tris, 90 mm phosphate, 2 mm EDTA, pH 8.0) buffer with the indicated chloroquine concentrations for detection of positively supercoiled DNA (17Drolet M. Bi X. Liu L.F. J. Biol. Chem. 1994; 269: 2068-2074Abstract Full Text PDF PubMed Google Scholar). One-dimensional agarose gels were stained with ethidium bromide and photographed. When analyzed by two-dimensional agarose gel electrophoresis, DNA was visualized by hybridization to 32P-labeled probes as described previously (17Drolet M. Bi X. Liu L.F. J. Biol. Chem. 1994; 269: 2068-2074Abstract Full Text PDF PubMed Google Scholar). Induction of Synthesis of the 14-kDa C-terminal Fragment in Vivo— DNA coding for the 14-kDa C-terminal fragment of E. coli DNA topoisomerase I was generated by PCR using the Pfu DNA polymerase and inserted into the pBADThio-TOPO expression vector (from Invitrogen). The resulting plasmid pBAD14K has the 14-kDa C-terminal fragment fused to the carboxyl end of thioredoxin and under the control of pBAD promoter. E. coli strain TOP10 with wild-type topoisomerase genotypes (from Invitrogen) was transformed with either pBAD14K or the control plasmid pBAD/Thio expressing thioredoxin. Cells were grown at 37 °C in LB containing 100 μg/ml ampicillin until A 600 reached 0.5, when transcription from the PBAD promoter was induced by addition of 0.005% arabinose. Cell growth was continued for 4 h at 37 °C before harvest of the cell pellets for plasmid preparation using the Qiagen kit. Plasmid DNA supercoiling was analyzed by two-dimensional gel electrophoresis using chloroquine concentrations specific for identification of hypernegatively supercoiled DNA (16Leng F. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9139-9144Crossref PubMed Scopus (54) Google Scholar, 17Drolet M. Bi X. Liu L.F. J. Biol. Chem. 1994; 269: 2068-2074Abstract Full Text PDF PubMed Google Scholar) and was visualized by hybridization to a 32P-labeled probe and by autoradiography. Survival Rates after N-Ethylmaleimide (NEM) Treatment—RFM445 (gyrB22(couR)gyrB203(Ts)), RFM475 (gyrB22(couR)gyrB203(Ts), and Δ(topAcysB)) and its transformants were cultured in LB broth at 37 °C to stationary phase (>18 h). Plasmid pJW312 (18Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar) was used for expression of full-length topoisomerase I, whereas its linker insertion derivative pJW2277ter (18Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar) was used for expressing Top85 lacking the 14-kDa C-terminal fragment (19Beran-Steed R.K. Tse-Dinh Y.-C. Proteins Struct. Funct. Genet. 1989; 6: 249-258Crossref PubMed Scopus (46) Google Scholar). Plasmid for expression of Top67 lacking the 30-kDa terminal fragment was constructed by introduction of a termination codon in pJW312 via site-directed mutagenesis (14Ahumada, A., and Tse-Dinh, Y.-C. (2002) BMC Biochemistry http://www.biomedcentral.com/1471-2091/3/13.Google Scholar). The cells were washed and resuspended in M9 medium as described (20Tse-Dinh Y.-C. J. Bacteriol. 2000; 182: 829-832Crossref PubMed Scopus (27) Google Scholar) before treatment for 1 h with 8 mm NEM at 37 °C. The rate of survival was determined by serial dilutions and plating of treated cells as well as control cells not treated with NEM. Colonies on LB plates were counted after 36 h of incubation at 37 °C. Direct Interaction between E. coli DNA Topoisomerase I and RNA Polymerase—Extract of E. coli cells expressing recombinant E. coli DNA topoisomerase I with MBP fused to its N-terminal end was applied to amylose resin. The bound topoisomerase I and any associated cellular proteins were then eluted with buffer containing maltose. Identical procedures were carried out with elution of recombinant MBP from amylose resin. When the maltose eluate fractions were analyzed by Western blotting, RNA polymerase α and β′ subunits were detected in the eluate fractions from cells expressing MBP-topoisomerase I but not in the eluate fractions prepared from cells expressing MBP (Fig. 1). This demonstrates that the linkage of DNA topoisomerase I to MBP is required for binding of RNA polymerase to the amylose resin. The Zinc Ribbon Domains of Topoisomerase I Are Responsible for the Interaction with RNA Polymerase—An RNA polymerase affinity column was prepared using polyol-responsive monoclonal antibodies against the β′ subunit of E. coli RNA polymerase (15Thompson N.E. Hager D.A. Burgess R.R. Biochemistry. 1992; 31: 7003-7008Crossref PubMed Scopus (70) Google Scholar). Soluble extracts of E. coli BL21 cells expressing DNA topoisomerase I (11Zhu C.-X. Tse-Dinh Y.-C. Bjornsti M.-A. Osheroff N. Methods in Molecular Biology. Humana Press, Totowa, NJ1999: 145-151Google Scholar) or its subdomains (12Zhu C.-X. Samuel M. Pound A. Ahumada A. Tse-Dinh Y.-C. Biochem. Mol. Biol. Int. 1995; 35: 375-385PubMed Google Scholar, 13Ahumada A. Tse-Dinh Y.-C. Biochem. Biophys. Res. Commun. 1998; 251: 509-514Crossref PubMed Scopus (24) Google Scholar, 14Ahumada, A., and Tse-Dinh, Y.-C. (2002) BMC Biochemistry http://www.biomedcentral.com/1471-2091/3/13.Google Scholar) were applied individually to the affinity column in separate experiments carried out with identical procedures (Fig. 2). Topoisomerase I could be detected in the bound proteins eluted from this affinity column with buffer containing 40% ethylene glycol and 0.75 m NaCl. In similar experiments carried out separately with lysates expressing an individual topoisomerase I subdomain, it was determined that the 67-kDa N-terminal transesterification domain (Top67) did not bind to the affinity column but the 20-kDa (ZD2) and 14-kDa C-terminal fragments could bind to the RNA polymerase affinity column. These two fragments contain three and two copies of zinc ribbon domains, respectively, and are homologous to the zinc ribbon domains in transcription regulators including RPB9, TFIIS, and TFIIB (21Grishin N.V. J. Mol. Biol. 2000; 299: 1165-1177Crossref PubMed Scopus (47) Google Scholar). However, only the three zinc ribbon domains on ZD2 have Zn(II) bound to tetracysteine motifs (13Ahumada A. Tse-Dinh Y.-C. Biochem. Biophys. Res. Commun. 1998; 251: 509-514Crossref PubMed Scopus (24) Google Scholar). Binding of the RNA polymerase subunits to this affinity column was confirmed by both Coomassie Blue staining of the eluted proteins and Western blot analysis using antibodies against the α, β, and β′ subunits (Fig. 2c). The β′ Subunit of RNA Polymerase Interacts with Topoisomerase I—E. coli RNA polymerase was expected to bind to the affinity matrix as a multisubunit complex. To determine which subunit was responsible for the interaction with DNA topoisomerase I, the RNA polymerase subunits were electrophoresed in a 7% SDS-polyacrylamide gel and transferred onto membrane. Overlay blotting was carried out using DIG-labeled topoisomerase I, DIG-labeled Top67, DIG-labeled ZD2, and DIG-labeled 14-kDa fragment. All of these DIG-labeled proteins, except DIG-labeled Top67, gave a positive chemiluminescence signal with peroxidase-linked anti-DIG antibody at a position where RNA polymerase β and β′ subunits would migrate in the 7% SDS-polyacrylamide gel (Fig. 3a). This result was in agreement with the data in Fig. 2b and showed that DNA that might have been present in the E. coli protein extract during binding to the affinity columns was not required for interaction between DNA topoisomerase I and RNA polymerase. There was also no signal when the RNA polymerase subunits on the membrane were blotted with DIG-labeled bovine serum albumen. To determine whether the β or β′ subunit was interacting with topoisomerase I, a 5% polyacrylamide gel was used for SDS-gel electrophoresis of the RNA polymerase subunits to better separate these two high molecular weight subunits. After transfer, the membrane was incubated with DIG-labeled ZD2 followed by alkaline-phosphatase linked anti-DIG antibodies. The signal of the DIG-topoisomerase I fragment bound to the nylon membrane was developed with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color reagent. The position of the signal was marked on the membrane and photographed alongside a ruler. The nylon membrane was then stained with Coomassie Blue to visualize both the β and β′ subunits in the gel lane analyzed previously with the alkaline-phosphatase linked anti-DIG antibodies. A comparison with the previously marked and photographed signal from alkaline phosphatase indicated that the topoisomerase I subdomain was interacting with the β′ subunit (Fig. 3b). The experiment was repeated using DIG-topoisomerase I or DIG 14-kDa fragment, and both of these also interacted with the β′ subunit (Fig. 3b and data not shown). Direct Interaction between E. coli RNA Polymerase and Topoisomerase I Is Important for Removal of Transcription-driven Negative Supercoils—It has been proposed that topoisomerase I plays an important physiological role in the removal of negative supercoils formed during transcription because of the movement of the RNA polymerase complex (6Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Crossref PubMed Scopus (1505) Google Scholar, 7Tsao Y.-P. Wu H.-Y. Liu L.F. Cell. 1989; 56: 111-118Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 8Drolet 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 (194) Google Scholar, 9Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In an in vitro transcription reaction in the presence of DNA topoisomerase I, positive supercoiling of the DNA template results from the removal of transcription-driven negative supercoils by topoisomerase I (7Tsao Y.-P. Wu H.-Y. Liu L.F. Cell. 1989; 56: 111-118Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 16Leng F. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9139-9144Crossref PubMed Scopus (54) Google Scholar). To demonstrate that the direct interaction between E. coli RNA polymerase and topoisomerase I is important for the removal of transcription-driven negative supercoils, a recombinant 14-kDa C-terminal fragment (purified from an overexpression system (12Zhu C.-X. Samuel M. Pound A. Ahumada A. Tse-Dinh Y.-C. Biochem. Mol. Biol. Int. 1995; 35: 375-385PubMed Google Scholar) at >99% purity as determined by Coomassie Blue staining of SDS gel (data not shown)) was also added to the in vitro transcription reaction. The 14-kDa C-terminal fragment could compete with topoisomerase I for interaction with E. coli RNA polymerase, and the formation of positively supercoiled DNA template was found to be inhibited (Fig. 4). In a relaxation reaction in the absence of RNA polymerase, the 14-kDa fragment has no significant effect on the relaxation of negatively supercoiled DNA by DNA topoisomerase I; thus so the 14-kDa fragment does not inhibit the catalytic activity of DNA topoisomerase I (Fig. 4c). The significance of the RNA polymerase-topoisomerase I interaction for removal of transcription driven supercoils was also demonstrated in vivo. The 14-kDa C-terminal domain was expressed in E. coli as a thioredoxin fusion protein via the tightly regulated PBAD promoter (22Schleif R. Annu. Rev. Biochem. 1992; 61: 199-223Crossref PubMed Scopus (387) Google Scholar). Accumulation of hypernegative supercoils in the plasmid DNA was observed when synthesis of the 14-kDa C-terminal fragment by pBAD14K was induced by addition of 0.005% arabinose to the culture (Fig. 5a). The induced 14-kDa C-terminal domain was expected to compete with topoisomerase I for interaction with RNA polymerase, impeding the removal of transcription-driven negative supercoils by DNA topoisomerase I. The level of 14-kDa C-terminal domain synthesis and resulting accumulation of hypernegatively supercoiled DNA did not vary significantly when the arabinose concentration was varied between 0.001 and 0.1% (data not shown). Induction of expression of thioredoxin in the control pBAD/THIO plasmid under the same experimental conditions did not result in accumulation of hypernegatively supercoiled DNA (Fig. 5b). Zinc Ribbons Are Important for in Vivo Function of Topoisomerase I during Stress Response—An 85-kDa truncated topoisomerase I lacking the 14-kDa C-terminal fragment (Top85) has been shown previously to be active in relaxation of supercoiled DNA (18Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar, 19Beran-Steed R.K. Tse-Dinh Y.-C. Proteins Struct. Funct. Genet. 1989; 6: 249-258Crossref PubMed Scopus (46) Google Scholar). The in vitro relaxing activity of Top85 was >75% of that of the full-length enzyme, albeit with lower processivity in high salt (19Beran-Steed R.K. Tse-Dinh Y.-C. Proteins Struct. Funct. Genet. 1989; 6: 249-258Crossref PubMed Scopus (46) Google Scholar). In vivo, Top85 could fully complement the viability of the E. coli strain AS17 that has a temperature-sensitive topoisomerase I (18Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar, 23Wang Y. Lynch A.S. Chen S.-J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Top85 has three of the five zinc ribbon domains present in topoisomerase I. We hypothesize that during stress response, when rapid transcription of induced genes takes place, direct interaction between topoisomerase I and RNA polymerase via the zinc ribbon domains may be important for survival. We have previously shown that the E. coli strain RFM475 with the topA gene deleted is much more sensitive to killing by the toxic electrophile NEM than the isogenic topA + strain RFM445 (20Tse-Dinh Y.-C. J. Bacteriol. 2000; 182: 829-832Crossref PubMed Scopus (27) Google Scholar). The increased sensitivity of RFM475 to NEM can be reversed by the presence of a plasmid expressing DNA topoisomerase I (20Tse-Dinh Y.-C. J. Bacteriol. 2000; 182: 829-832Crossref PubMed Scopus (27) Google Scholar). Strain RFM475 was transformed with plasmids expressing full-length topoisomerase I or its truncated forms lacking the C-terminal fragments. Comparison of the survival rates of the RFM475 transformants after NEM treatment (Fig. 6) showed that the survival rate of the transformant expressing Top85 was higher than the transformant expressing Top67 but still about 100-fold lower than that of the transformant expressing the full-length topoisomerase I. Although not required for viability of E. coli under optimal laboratory growth conditions, the absence of the 14-kDa fragment in topoisomerase I thus affected the function of topoisomerase I in stress response significantly. The results presented here support a role of the C-terminal zinc ribbon domains in E. coli DNA topoisomerase I for interacting with the β′ subunit of RNA polymerase, so that negative DNA supercoiling formed during transcription can be removed immediately. The rapid removal of transcription-driven negative supercoils would prevent the formation of R-loops (8Drolet 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 (194) Google Scholar, 9Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This suppression of R-loops during transcription has been proposed to be an essential function for E. coli DNA topoisomerase I (24Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16654-16658Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Sequence analysis shows that the number of zinc ribbon domains in type IA DNA topoisomerases found in different organisms varies from zero to five (20Tse-Dinh Y.-C. J. Bacteriol. 2000; 182: 829-832Crossref PubMed Scopus (27) Google Scholar). If present, these zinc ribbon domains can potentially interact with RNA polymerase in these organisms during transcription. Other DNA topoisomerases have also been shown previously to be involved directly in transcription. Human topoisomerase I, a type IB topoisomerase, has been shown to be a cofactor of RNA polymerase II transcription (25Merino A. Madden K.R. Lane W.S. Champoux J.J. Reinberg D. Nature. 1993; 365: 227-232Crossref PubMed Scopus (323) Google Scholar, 26Kretzachmar M. Meisterernst M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11508-11512Crossref PubMed Scopus (162) Google Scholar), whereas topoisomerase IIα is required for RNA polymerase II transcription on chromatin templates (27Mondal N. Parvin J.D. Nature. 2001; 413: 435-438Crossref PubMed Scopus (104) Google Scholar). We have observed previously that a recombinant plasmid expressing the 14-kDa C-terminal domain of E. coli DNA topoisomerase I under the T7 promoter was unstable in E. coli BL21DE3 (12Zhu C.-X. Samuel M. Pound A. Ahumada A. Tse-Dinh Y.-C. Biochem. Mol. Biol. Int. 1995; 35: 375-385PubMed Google Scholar). Attempts to construct a recombinant plasmid expressing the 14-kDa C-terminal domain as a MBP-fusion protein in pMal-c2X were unsuccessful. The results showing the interference of E. coli DNA topoisomerase I function during transcription by the 14-kDa C-terminal domain would account for the instability of these recombinant plasmids. It may be possible for topoisomerase I function or DNA supercoiling to be modulated via targeting of the protein-protein interactions between topoisomerase I and its partners in E. coli. Besides Tn5 transposase and RNA polymerase in E. coli, cellular proteins in other organisms may also interact with type IA topoisomerases. This is particularly intriguing for mammalian type IA topoisomerases. There are two type IA topoisomerases (TOP3α and TOP3β) present in both human and mouse (28Hanai R. Caron P.R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3653-3657Crossref PubMed Scopus (123) Google Scholar, 29Li W. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1010-1013Crossref PubMed Scopus (147) Google Scholar, 30Kawasaki K. Minoshima S. Nakato E. Shibuya K. Shintani A. Schmeits J.L. Wang J. Shimizu N. Genome Res. 1997; 7: 250-261Crossref PubMed Scopus (173) Google Scholar, 31Ng S.-W. Liu Y. Hasselblatt K.T. Mok S.C. Berkowitz R.S. Nucleic Acids Res. 1999; 27: 993-1000Crossref PubMed Scopus (44) Google Scholar, 32Seki T. Seki M. Onodera R. Katada T. Enomoto T. J. Biol. Chem. 1998; 273: 28553-28556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) with four zinc ribbon domains present in each of these enzymes (21Grishin N.V. J. Mol. Biol. 2000; 299: 1165-1177Crossref PubMed Scopus (47) Google Scholar). Human TOP3α has been shown to interact with the Bloom Syndrome helicase, BLM (33Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.-F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9644Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 34Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.-A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar), whereas both TOP3α and TOP3β interact with human RecQ5β helicase (35Shimamoto A. Nishikawa K. Kitao S. Furuichi Y. Nucleic Acids Res. 2000; 28: 1647-1655Crossref PubMed Google Scholar). There is evidence that interaction between Bloom Syndrome helicase and human TOP3α is important for genomic stability (36Hu P. Beresten S.F. van Brabant A.J. Ye T.Z. Pandolfi P.P. Johnson F.B. Guarente L. Ellis N.A. Hum. Mol. Genet. 2001; 10: 1287-1298Crossref PubMed Google Scholar). The domains in TOP3α and TOP3β responsible for the interactions with the RecQ family of helicases have not been identified experimentally. Multiple transcripts from alternative splicing with tissue specific expression pattern give rise to variant forms of TOP3β that have different numbers of zinc ribbon domains (31Ng S.-W. Liu Y. Hasselblatt K.T. Mok S.C. Berkowitz R.S. Nucleic Acids Res. 1999; 27: 993-1000Crossref PubMed Scopus (44) Google Scholar). This can affect the interaction of TOP3β with other cellular proteins. The potential involvement of these zinc ribbon domains in protein-protein interactions may play the important role of directing different forms of mammalian type IA topoisomerases to complexes involved in replication, transcription, recombination, or DNA repair. We thank Richard Burgess for suggestions on the RNA polymerase affinity chromatography, Victor Fried for suggestions on the β′ subunit identification, and Marc Drolet for helpful discussions.
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