On the Molecular Basis of the Thermal Sensitivity of an Escherichia coli topA Mutant
2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês
10.1074/jbc.m109436200
ISSN1083-351X
AutoresYong Wang, A. Simon Lynch, Sue-Jane Chen, James C. Wang,
Tópico(s)DNA Repair Mechanisms
ResumoStudies of two temperature-sensitiveEscherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 °C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 °C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity. Studies of two temperature-sensitiveEscherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 °C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 °C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity. temperature-sensitive Bacterial DNA topoisomerase I is a member of the type IA subfamily of DNA topoisomerases found in all living organisms (reviewed in Refs.1Drlica K. Mol. Microbiol. 1992; 6: 425-433Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar, 3Tse-Dinh Y.C. Biochim. Biophys. Acta. 1998; 1400: 19-27Google Scholar). The enzyme was first identified in Escherichia colithree decades ago as the "omega protein" (4Wang J.C. J. Mol. Biol. 1971; 55: 523-533Google Scholar), and its structural gene topA was mapped and sequenced in the 1980s (5Sternglanz R. Dinardo S. Voelkel K.A. Nishimura Y. Hirota Y. Becherer K. Zumstein L. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2747-2751Google Scholar, 6Trucksis M. Depew R.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2164-2168Google Scholar, 7Tse-Dinh Y.C. Wang J.C. J. Mol. Biol. 1986; 191: 321-331Google Scholar). Extensive mechanistic studies of the enzyme and its mutants have been carried out (1Drlica K. Mol. Microbiol. 1992; 6: 425-433Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar, 3Tse-Dinh Y.C. Biochim. Biophys. Acta. 1998; 1400: 19-27Google Scholar), and the crystal structure of a 67-kDa fragment of it was reported in 1994 (8Lima C.D. Wang J.C. Mondragon A. Nature. 1994; 367: 138-146Google Scholar). In the three-dimensional structure, the polypeptide chain folds into a torus, with four distinct domains enclosing a large central hole (8Lima C.D. Wang J.C. Mondragon A. Nature. 1994; 367: 138-146Google Scholar). The same architecture was seen in the crystal structure of E. coli DNA topoisomerase III (9Mondragon A. DiGate R. Struct. Fold Des. 1999; 7: 1373-1383Google Scholar), another member of the type IA subfamily. E. coli DNA topoisomerase I specifically relaxes negatively supercoiled DNA (4Wang J.C. J. Mol. Biol. 1971; 55: 523-533Google Scholar) and has a key role in the modulation of intracellular DNA supercoiling (1Drlica K. Mol. Microbiol. 1992; 6: 425-433Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar, 3Tse-Dinh Y.C. Biochim. Biophys. Acta. 1998; 1400: 19-27Google Scholar). Inactivation of the enzyme is detrimental to cell viability (10DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Google Scholar, 11Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Google Scholar, 12Raji A. Zabel D.J. Laufer C.S. Depew R.E. J. Bacteriol. 1985; 162: 1173-1179Google Scholar). Excessive negative supercoiling of intracellular DNA, particularly in regions behind the transcribing RNA polymerases (13Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Google Scholar), appears to be a major cause of lethality ofE. coli topA null mutants (1Drlica K. Mol. Microbiol. 1992; 6: 425-433Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar, 3Tse-Dinh Y.C. Biochim. Biophys. Acta. 1998; 1400: 19-27Google Scholar). In support of this notion, secondary mutations that reduce the cellular level of gyrase (DNA topoisomerase II), an activity that catalyzes DNA negative supercoiling or the removal of positive supercoils, was found to suppresstopA lethality (10DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Google Scholar, 11Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Google Scholar). Furthermore, expression of eukaryotic DNA topoisomerase I (14Bjornsti M.A. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8971-8975Google Scholar) or vaccinia virus topoisomerase (15Fernandez-Beros M.E. Tse-Dinh Y.C. J. Bacteriol. 1992; 174: 7059-7062Google Scholar), as well as overexpression of E. coli DNA topoisomerase III (16Broccoli S. Phoenix P. Drolet M. Mol. Microbiol. 2000; 35: 58-68Google Scholar) or IV (17Kato J. Nishimura Y. Imamura R. Niki H. Hiraga S. Suzuki H. Cell. 1990; 63: 393-404Google Scholar, 18McNairn E. Bhriain N.N. Dorman C.J. Mol. Microbiol. 1995; 15: 507-517Google Scholar), was also found to compensate fortopA inactivation. One consequence of excessive negative supercoiling of intracellular DNA appears to be hybrid formation between nascent RNA and the DNA template ("R-looping"), as suggested by the partial suppression of topA lethality by overexpressing RNaseH (19Drolet M. Phoenix P. Menzel R. Masse E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Google Scholar, 20Masse E. Drolet M. J. Biol. Chem. 1999; 274: 16654-16658Google Scholar). Unlike E. coli,Salmonella typhimurium and Shigella flexneri topAnulls appear to survive in the absence of a compensatory mutation (21Richardson S.M. Higgins C.F. Lilley D.M. EMBO J. 1984; 3: 1745-1752Google Scholar,22Bhriain N.N. Dorman C.J. Mol. Microbiol. 1993; 7: 351-358Google Scholar). In addition to its role in the regulation of DNA supercoiling, bacterial DNA topoisomerase I is likely to participate in other processes that require the passage of one DNA single strand through an enzyme-mediated transient break in another. E. coli topA topB double mutants lacking both DNA topoisomerase I and III are nonviable even in the presence of a mutation that compensates fortopA inactivation (23Li Z.Y. Hiasa H. Kumar U. DiGate R.J. J. Biol. Chem. 1997; 272: 19582-19587Google Scholar, 24Zhu Q. Pongpech P. DiGate R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9766-9771Google Scholar). The involvement of bacterial DNA topoisomerase I in multiple cellular transactions of DNA is manifested by a pleiotropic phenotype oftopA mutants. In addition to their effects on E. coli cell lethality (10DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Google Scholar, 11Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Google Scholar, 12Raji A. Zabel D.J. Laufer C.S. Depew R.E. J. Bacteriol. 1985; 162: 1173-1179Google Scholar), mutations in topA were also reported to affect adaptive responses to changes in environmental conditions (reviewed in Refs. 25Dorman C.J. Trends Microbiol. 1996; 4: 214-216Google Scholar, 26Tse-Dinh Y.C. Qi H. Menzel R. Trends Microbiol. 1997; 5: 323-326Google Scholar; see also Refs. 27Higgins C.F. Dorman C.J. Stirling D.A. Waddell L. Booth I.R. May G. Bremer E. Cell. 1988; 52: 569-584Google Scholar, 28Graeme-Cook K.A. May G. Bremer E. Higgins C.F. Mol. Microbiol. 1989; 3: 1287-1294Google Scholar, 29Tse-Dinh Y.C. J. Bacteriol. 2000; 182: 829-832Google Scholar, 30Weinstein-Fischer D. Elgrably-Weiss M. Altuvia S. Mol. Microbiol. 2000; 35: 1413-1420Google Scholar), plasmid partition (31Miller C.A. Beaucage S.L. Cohen S.N. Cell. 1990; 62: 127-133Google Scholar, 32Austin S.J. Eichorn B.G. J. Bacteriol. 1992; 174: 5190-5195Google Scholar), chromosome segregation in E. coli mukmutants (33Sawitzke J.A. Austin S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1671-1676Google Scholar), the development of genetic competence in Hemophilus influenza (34Chandler M.S. Smith R.A. Gene. 1996; 169: 25-31Google Scholar), sensitivity of Salmonella typhimuriumto ultraviolet irradiation (35Overbye K.M. Basu S.K. Margolin P. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 785-791Google Scholar, 36Smith C.M. Arany Z. Orrego C. Eisenstadt E. Environ. Mol. Mutagen. 1992; 19: 185-194Google Scholar), and recA-independent recombination (37Reddy M. Gowrishankar J. J. Bacteriol. 2000; 182: 1978-1986Google Scholar). Because of the multiple cellular roles of bacterial DNA topoisomerase I, conditional topA mutants were constructed to facilitate functional studies of this enzyme. In one strain, the coding sequence of topA was placed under the control of a lac promoter, so that expression of the gene could be tightly regulated (38Wang J.C. J. Cell Sci. (Suppl.). 1984; 1: 21-29Google Scholar). Temperature-dependent expression of E. coli topA was also accomplished in strains AS17 and BR83, which were constructed by the introduction of anamber mutation in topA and the expression of a plasmid-borne or chromosomally located temperature-sensitive (ts)1amber-suppressor (R. E. Depew, cited in Refs. 39Zumstein L. Wang J.C. J. Mol. Biol. 1985; 191: 33-340Google Scholar, 40Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Google Scholar). The isolation of ts alleles within topA has been unsuccessful; introducing a plasmid-borne topA tsmutation C662H (41Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Google Scholar) into the chromosomal topA gene, for example, yielded a mutant that grew well at 30 or 42 °C. 2V. Stupina, Y. Wang, and J. C. Wang, unpublished data. Because of the lack of known ts alleles within topA, the thermal-sensitive topA strains AS17 and BR83 were used in a number of previous studies (see for examples, Ref. 14Bjornsti M.A. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8971-8975Google Scholar, 39Zumstein L. Wang J.C. J. Mol. Biol. 1985; 191: 33-340Google Scholar, 40Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Google Scholar, 41Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Google Scholar, 42Lynch A.S. Wang J.C. J. Bacteriol. 1993; 175: 1645-1655Google Scholar, 43Zhu C.X. Roche C.J. Papanicolaou N. DiPietrantonio A. Tse-Dinh Y.C. J. Biol. Chem. 1998; 273: 8783-8789Google Scholar). In the course of working with these strains, however, we encountered several observations that could not be explained by a common molecular basis of the temperature sensitivity of these strains, namely the temperature-dependent synthesis of a functional DNA topoisomerase I. We report here that the plasmid-borne supD amber suppressor in strain AS17 had apparently lost its temperature sensitivity. Both in vivo and in vitro experiments provided strong evidence that the ts phenotype of strain AS17 resulted from the synthesis of a temperature-sensitive DNA topoisomerase I in the presence of a functional supD, and not from the temperature-dependent suppression of anamber codon in the topA allele of the strain. Our results also indicate that the growth of E. coli cells is critically dependent on the presence of a functional DNA topoisomerase I in media of any osmolarity. Previously, studies of BR83 cells grown in different media had led to the suggestion that the enzyme might be dispensable in growth media of low osmolarity (40Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Google Scholar). The topA region of DNA from strain AS17 (F−topA17(am) pLL1(Tet®supD43,74)) cells was amplified by the polymerase chain reaction (PCR), using a pair of primers 5′-AAT-CCG-CTC-GAG-CTC-GTT-GCC-AGT-GGA-AGG-TTT-3′ and 5′-GGC-TAG-TCT-AGA-CCA-CTA-TAT-CAT-TTA-TAG-CCT-3′. Each of the primers incorporated a unique restriction site (underlined) to facilitate subsequent cloning of the PCR product. The 2.8-kbXhoI-XbaI fragment containing the entire coding region of topA was purified by gel electrophoresis from the restriction endonuclease-treated PCR products and subcloned. ThetopA insert in the subclone was then sequenced (carried out by the Molecular Biology Core Facility of the Medical College of Georgia, Augusta, Georgia). Site-directed mutagenesis using a commercial kit (Stratagene) was done to introduce specific mutations into the topA region of pJW312, a plasmid previously constructed for the overexpression of wild-type topA from alac promoter (39Zumstein L. Wang J.C. J. Mol. Biol. 1985; 191: 33-340Google Scholar). Two pairs of mutagenic oligonucleotides, 5′-CT-AAA-AAG-GAT-GAA-CGT-AAC-GCG-TTA-GTC-AAC-CGT-ATG-3′ and 5′-GGG-GTT-GAC-CCA-TGG-CAC-AAT-TCG-GAG-GCG-CAC-TAT-G-3′, and their complements were used to introduce the G65N or the W79S mutation. In the oligonucleotides specified above, the triplets in boldface fonts indicate the Gly (GGC) to Asn (AAC) and Trp (TGG) to Ser (TCG) codon replacements. Several other modifications (single boldface letters) were also made to introduce restriction sites (underlined) without codon changes so that the presence of the intended mutations could be readily checked. The double mutant harboring both G65N and W79S mutations was constructed by two successive rounds of site-directed mutagenesis. Further confirmation of the presence of the intended mutation or mutations was carried out by nucleotide sequencing. Plasmids pJW312 and its derivatives bearing the specified mutations, pJW312(G65N), pJW312(W79S), pJW312(G65N/W79S), and pJW312(Y319A), were first digested with BglII and HindIII. The 2.9-kb fragment containing the lac promoter-linked wild-type or mutated topA gene was then inserted in between theBamHI and HindIII sites of a single-copy plasmid pBeloBAC11 (purchased from New England Biolabs). The resulting constructs were individually transformed into AS17 cells, and the transformants were checked for growth at different temperatures on Luria broth agar plates containing tetracycline and chloramphenicol. Plasmids expressing wild-type E. coli DNA topoisomerase I and the active site tyrosine mutant (Y319A) protein were included in this experiment as the positive and negative control, respectively. pJW312 and pJW312(G65N/W79S) were individually transformed into a ΔtopA E. coli strain DM800 bearing a pACYC184-based plasmid overexpressing thelac repressor (44Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Google Scholar). The use of a ΔtopAstrain as the host eliminates the possible contamination of wild-type DNA topoisomerase I in the preparation of the mutant protein. Induction of cells for overexpression of the wild-type and mutant enzymes was performed at 30 °C by the addition of isopropyl-1-thio-β-d-galactoside to 1 mm. Cell lysis and initial purification by phosphocellulose column-chromatography were carried out as described previously for purification of the wild-type enzyme (44Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Google Scholar). The pooled peak fractions from each preparation was further purified by the use of a 1 ml HiTrap-heparin column (Amersham Biosciences, Inc.). The peak fractions were collected and were flash frozen in liquid nitrogen for storage at −80 °C. Purity of the preparations was examined by SDS-polyacrylamide gel electrophoresis, and protein concentrations of the fractions were estimated from spectrophotometric readings in the presence of Coomassie Blue (Pierce) using bovine serum albumin as a standard. Relaxation of negatively supercoiled pBluescript KS (Stratagene) was carried out in a buffer containing 20 mm Tris-HCl, pH 7.5, 2.5 mm MgCl2, 0.1 mm EDTA, 100 μg/ml bovine serum albumin, and 10–180 mm KCl as specified elsewhere. For each reaction, 10 μl of the above buffer containing different amounts of the wild-type or mutant enzyme were incubated for 5 min at either 30 or 42 °C. Ten microliters of a solution containing 30 ng/μl plasmid, in the same buffer and preincubated at the same temperature, were rapidly pipetted and mixed into the enzyme solution. Following incubation for 20 min, the reaction was terminated by the addition of EDTA (pH 8) to a final concentration of 50 mm. The quenched reactions were analyzed by electrophoresis in a 0.9% agarose gel slab in 50 mm Tris-borate and 1 mm EDTA. The gel slab was stained for 1 h in 1 μg/ml ethidium bromide and photographed with a Kodak D120 camera over a ultraviolet light source. A 388-base pair-longNcoI-EcoRI restriction fragment from pJW312,32P-labeled at the NcoI 5′-end by successive phosphatase and polynucleotide kinase treatment, was heat denatured in 1 mm Tris-HCl (pH 7.5) and immediately used in the cleavage assays. For each cleavage reaction, 1 μl of 400 mmTris-HCl (pH 7.5), varying amounts of 1 m KCl and water, and 20 ng of the wild-type or 100 ng of the mutant enzyme were mixed in a total volume of 7 μl. The mixture was preincubated at either 30 or 42 °C for 5 min, and ∼2 ng of the labeled DNA in a volume of 3 μl were added. Incubation was continued for another 15 min, and SDS was then added to a final concentration of 1% to reveal the topoisomerase-mediated cleavage of DNA. Samples were subsequently mixed with equal volume of a loading buffer containing 50% formamide, 0.05% bromphenol blue, 0.03% xylene cyanol, and 5 mm EDTA (pH 8) and subjected to electrophoresis in a 6% denaturing polyacrylamide gel. Following electrophoresis, the gel was dried over a filter paper backing and autoradiographed in a PhosphoImager (Fuji). E. coli DM800ΔtopA cells were sequentially transformed with pBR322 and a pBeloBAC11 derivative expressing wild-type or the G65N/W79S or Y319A mutant DNA topoisomerase I from a lac promoter. Transformants bearing different pairs of plasmids were each grown in Luria broth containing tetracycline and chloramphenicol in a flask placed in a 30 °C gyratory shaker. When the cultures reached an apparent optical density of 0.4–0.6 at 595 nm, 5-ml portions of each were placed in two sets of 50-ml tubes, one kept in the 30 °C shaker and the other placed in a 42 °C shaker water-bath. After another 10 min, rapid lysis of cells was performed by pouring into each culture an equal volume of preheated lysis buffer (80 °C) containing 3% SDS and 0.2 m NaOH (14Bjornsti M.A. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8971-8975Google Scholar). Following neutralization and precipitation of the detergent with 0.75 volume of 3 mpotassium acetate (pH 4.8), the supernatant was cleared by centrifugation and passage through several layers of cheesecloth. The DNA samples were isolated from the filtered supernatants by alcohol precipitation, and the linking number distribution of the test plasmid pBR322 in each sample was analyzed by two-dimensional gel electrophoresis as described previously (45Peck L.J. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6206-6210Google Scholar). Following electrophoresis, DNA was transferred from the gel slab to a nylon membrane (Bio-Rad). A 32P-labeled DNA probe, prepared by random priming of an EcoRI-EagI fragment from thetet region of pBR322, was used to selectively detect pBR322 by Southern hybridization. Fig. 1 depicts the plating efficiency of E. coli strain AS17topA17(am) pLL1(supD43,74) cells on agar plates containing Luria broth and different concentrations of added NaCl. At either 30 or 42 °C, there was a gradual drop in plating efficiency with increasing salt concentration. Significantly, at all salt concentrations a large drop in plating efficiency, ranging from several thousand-fold in a low salt medium to about 105-fold in a high salt medium, was observed when the temperature was increased from 30 to 42 °C. Essentially the same results were obtained when sucrose instead of salt was added to the media to cover a similar range of osmolarity (data not shown). When the cells were transformed with pJW249 carrying a wild-type topAgene (46Wang J.C. Becherer K. Nucleic Acids Res. 1983; 11: 1773-1790Google Scholar), the plating efficiency was no longer ts at any osmolarity, confirming that the observed changes reflected a topAphenotype. The above results were surprising, however, when compared with similar data previously reported for strain BR83topA57(am) supD43,74 (40Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Google Scholar). When the temperature was increased from 30 to 42 °C, the plating efficiency of BR83 cells was shown to decrease sharply in media of high osmolarity, but remain unchanged in broth containing no added osmolyte (40Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Google Scholar). Because both AS17 and BR83 were supposed to express an inactive DNA topoisomerase I unless the amber mutation intopA was suppressed by the same ts suppressor,supD43,74, the results shown in Fig. 1 apparently contradicted those previously reported for BR83 (40Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Google Scholar). A clue to the molecular basis of this discrepancy came when the plasmid-borne suppressor in strain AS17 was introduced into BR83. When pLL1(supD43,74) isolated from AS17 cells was introduced into BR83 cells, the plating efficiencies of individual transformants were found to be temperature-independent at any osmolarity (data not shown). This result indicated that the suppressor on pLL1, as isolated from strain AS17 cells, was no longer ts. Thus upon introducing the plasmid into BR83, the product of the plasmid-borne suppressor would allow the synthesis of a full-length product of thetopA57(am) mutant gene, at either 30 or 42 °C, with an amber to Ser substitution dictated by the particular suppressor (47Oeschger M.P. Woods S.L. Cell. 1976; 7: 205-212Google Scholar, 48Zengel J.M. Lindahl L. J. Bacteriol. 1981; 145: 459-465Google Scholar). The temperature-sensitive phenotype of AS17topA17(am) cells bearing pLL1 would then suggest that the full-length DNA topoisomerase I produced in these cells, with a Ser substitution at the amber codon, must be itself ts. To test the above interpretation, the topA coding region of DNA isolated from AS17 cells was amplified by PCR and cloned into a plasmid vector for sequencing. Two and only two codon alterations were identified. The first changes Gly-65 (GGC) of wild-type topA to an Asn codon (AAC), and the second changes Trp-79 (TGG) of wild-type topAto an amber stop codon (TAG). The topA17(am) product in the presence of a functional supD suppressor is therefore expected to be a mutant DNA topoisomerase I with two amino acid changes, G65N and W79S. To express this mutant protein, to be referred to as TopA(G65N/W79S), site-directed mutagenesis was employed to introduce the desired mutations, either singly or together, into thetopA gene on a multicopy plasmid pJW312 previously constructed for overexpression of topA from a lacpromoter (39Zumstein L. Wang J.C. J. Mol. Biol. 1985; 191: 33-340Google Scholar). For complementation assays of the mutant proteins (see the section below), the lac promoter-linked topAcassettes from these constructs, as well as from pJW312 itself, were also individually cloned into a single-copy plasmid pBeloBAC11. In addition, a lac promoter-linked topA cassette with a Y319A mutation (49Chen S.J. Wang J.C. J. Biol. Chem. 1998; 274: 6050-6056Google Scholar), in which the active-site tyrosyl residue had been replaced by an alanine, was also moved into pBeloBAC11 to provide a topA null control in some of the experiments. The various constructs in the pBeloBAC11 vector were transformed individually into strain AS17 cells, and transformants picked from plates incubated at 30 °C were examined for growth at various temperatures. The results are tabulated in Table I. At a permissive temperature of 30 °C for strain AS17, all transformants were viable. At 42 °C, expression of wild-type topA or the mutanttopA(G65N) fully complemented the inviability of AS17 cells; AS17 cells expressing topA(Y319A), topA(W79S), or the double mutant topA(G65N/W79S) showed a plating efficiency of less than 0.001, however. Thus in agreement with the postulate of a temperature-sensitive enzyme, the anticipated product of the topA17(am) allele in the presence of a functional supD suppressor is apparently inactive at 42 °C.Table IViability of AS17 cells transformed with plasmids expressing wild-type or mutant E. coli DNA topoisomerase IDNA topoisomerase ICell viability37 °C42 °CWild-type1.01.0G65N1.01.0W79S0.14<0.001G65N/W79S0.04<0.001Y319A<0.001<0.001Plasmid-expressing wild-type or mutant DNA topoisomerase I as indicated in the leftmost column was transformed into AS17 cells. The 30 °C cultures of the transformants were plated on LB plates containing tetracycline and chloramphenicol, and incubated overnight at 30, 37, or 42 °C. Cell viability was expressed as the ratio of the number of colonies grown at 37 or 42 °C to the number of colonies grown at 30 °C. Open table in a new tab Plasmid-expressing wild-type or mutant DNA topoisomerase I as indicated in the leftmost column was transformed into AS17 cells. The 30 °C cultures of the transformants were plated on LB plates containing tetracycline and chloramphenicol, and incubated overnight at 30, 37, or 42 °C. Cell viability was expressed as the ratio of the number of colonies grown at 37 or 42 °C to the number of colonies grown at 30 °C. At an intermediate temperature of 37 °C, AS17 cells expressingtopA(Y319A) showed a plating efficiency of less than 0.001, but the same cells expressing topA(W79S) andtopA(G65N/W79S) showed significantly higher plating efficiencies of 0.14 and 0.04, respectively (Table I). Because thetopA(G65N/W79S) double mutant appeared to be more stringent in its temperature sensitivity than the topA(W79S) single mutant, it was chosen for in vitro characterization of the mutant enzyme. Wild-type E. coli DNA topoisomerase I and its mutated derivative TopA(G65N/W79S) were purified from strain DM800 ΔtopAcells harboring plasmid pJW312 or its mutated derivative. Both proteins were expressed to a comparable level upon induction of thelac promoter and were purified to apparently homogeneity. Relaxation of negatively supercoiled plasmid DNA by the wild-type and mutant enzyme was first examined at 30 °C, in an assay mixture containing 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 2.5 mm MgCl2, 0.1 mm EDTA, and 100 μg/ml bovine serum albumin. As shown in Fig.2, both the wild-type and mutant DNA topoisomerase I were capable of relaxing the negatively supercoiled DNA, but the latter was much less active. In 20 μl of the assay mixture containing about 300 ng of DNA, the bulk of the input negatively supercoiled DNA was relaxed after 20 min in the presence of 30–60 ng of the wild-type enzyme (Fig. 2, upper panel). Under the same conditions, the presence of 50 ng of the mutant enzyme converted only a minor fraction of the input DNA to topoisomers that migrated with significantly reduced mobilities (Fig.2, lower panel). Even at the highest concentration of the mutant enzyme used in this experiment, the topoisomer products retained a significant number of negative supercoils (see the patterns of topoisomers in the lower panel of Fig. 2). In Fig. 3, results of additional assays carried out in a buffer containing varying concentrations of KCl are depicted. The reduced activity of the mutant enzyme at 30 °C was again evident. Although 7.5 times more of the mutant enzyme was used relative to the wild-type enzyme in the two sets of reaction mixtures, relaxation of the negatively supercoiled plasmid DNA was generally less complete in the case of the mutant enzyme (compare the corresponding lanes in the upper left and upper right panels shown in Fig. 3). A shift of the assay temperature from 30 to 42 °C further reduced the relaxation activity of TopA(G65N/W79S) relative to that of the wild-type enzyme (compare theupper and lower right panels in Fig. 3for the mutant enzyme and the upper and lower left panels in Fig. 3 for the wild-type enzyme). It was clear, however, that the mutant enzyme retained some activity at 42 °C; relaxation of negatively supercoiled plasmid DNA by the mutant enzyme was readily detectable, especially in assay mixtures containing lower amounts of KCl (see Fig. 3, lower right panel). Cleavage of single-stranded DNA by the wild-type and TopA(G65N/W79S) was also examined. A 388-base pair-long DNA fragment uniquely32P-labeled at a 5′-end was denatured and used in this experiment. Cleavage of the denatured DNA by 100 ng of the mutant enzyme or 20 ng of the wild-type enzyme in assay mixtures containing different amounts of KCl was performed at 30 °C (Fig.4A). The mutant and wild-type enzyme appeared to cleave the DNA strand with the same sequence specificity, as similar ladders of labeled cleavage products were observed. Even though 5 times more of the mutant than the
Referência(s)