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• W2071796772 abstract "Alanine substitution mutagenesis of Escherichia coli DNA topoisomerase I, a member of the type IA subfamily of DNA topoisomerases, was carried out to identify amino acid side chains that are involved in transesterification between DNA and the active site tyrosine Tyr-319 of the enzyme. Twelve polar residues that are highly conserved among the type IA enzymes, Glu-9, His-33, Asp-111, Glu-115, Gln-309, Glu-313, Thr-318, Arg-321, Thr-322, Asp-323, His-365, and Thr-496, were selected for alanine substitution. Each of the mutant enzymes was overexpressed, purified, and characterized. Surprisingly, only substitution at Glu-9 and Arg-321 was found to reduce the DNA relaxation activity of the enzyme to an insignificant level. The R321A mutant enzyme, but not the E9A mutant enzyme, was found to retain a reduced level of DNA cleavage activity. Two additional mutant enzymes R321K and E9Q were also constructed and purified. Replacing Arg-321 by lysine has little effect on enzymatic activities; replacing Glu-9 by glutamine greatly reduces the supercoil removal activity but not the DNA cleavage and rejoining activities. From these results and the locations of the amino acids in the crystal structure of the enzyme, it appears that Glu-9 has a critical role in DNA breakage and rejoining, probably through its interaction with the 3′ deoxyribosyl oxygen. The positively charged Arg-321 may also participate in these reactions by interacting with the scissile DNA phosphate as a monodentate. Because of the strict conservation of these residues, the findings for the E. coli enzyme are likely to apply to all type IA DNA topoisomerases. Alanine substitution mutagenesis of Escherichia coli DNA topoisomerase I, a member of the type IA subfamily of DNA topoisomerases, was carried out to identify amino acid side chains that are involved in transesterification between DNA and the active site tyrosine Tyr-319 of the enzyme. Twelve polar residues that are highly conserved among the type IA enzymes, Glu-9, His-33, Asp-111, Glu-115, Gln-309, Glu-313, Thr-318, Arg-321, Thr-322, Asp-323, His-365, and Thr-496, were selected for alanine substitution. Each of the mutant enzymes was overexpressed, purified, and characterized. Surprisingly, only substitution at Glu-9 and Arg-321 was found to reduce the DNA relaxation activity of the enzyme to an insignificant level. The R321A mutant enzyme, but not the E9A mutant enzyme, was found to retain a reduced level of DNA cleavage activity. Two additional mutant enzymes R321K and E9Q were also constructed and purified. Replacing Arg-321 by lysine has little effect on enzymatic activities; replacing Glu-9 by glutamine greatly reduces the supercoil removal activity but not the DNA cleavage and rejoining activities. From these results and the locations of the amino acids in the crystal structure of the enzyme, it appears that Glu-9 has a critical role in DNA breakage and rejoining, probably through its interaction with the 3′ deoxyribosyl oxygen. The positively charged Arg-321 may also participate in these reactions by interacting with the scissile DNA phosphate as a monodentate. Because of the strict conservation of these residues, the findings for the E. coli enzyme are likely to apply to all type IA DNA topoisomerases. DNA topoisomerases are enzymes that participate in nearly all cellular transactions of DNA, including replication, transcription, and chromosome condensation (for reviews see Ref. 1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar and references therein). There are two types of DNA topoisomerases: the type I enzymes catalyze the transport of individual DNA strands through one another and the type II enzymes catalyze the interpenetration of double-stranded DNA segments. By doing so, the DNA topoisomerases alleviate the topological problems encountered by intracellular DNA. The transport of DNA strands through one another requires the transient breakage of one of the encountering pair, and all DNA topoisomerases catalyze this reaction through the formation of covalent enzyme-DNA intermediates; the phenolic oxygen of an enzyme tyrosyl residue undergoes nucleophilic attack of a DNA phosphorous to break a DNA phosphodiester bond and form a phosphotyrosine link (2Tse Y.-C. Kirkegaard K. Wang J.C. J. Biol. Chem. 1980; 255: 5560-5565Abstract Full Text PDF PubMed Google Scholar, 3Champoux J.J. J. Biol. Chem. 1981; 256: 4805-4809Abstract Full Text PDF PubMed Google Scholar). Following DNA strand breakage and passage, the deoxyribosyl hydroxyl formed during DNA breakage acts as the nucleophile to break the phosphotyrosine link and rejoin the DNA strand. By analogy to the cleavage of DNA by nucleases (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar) and from studies of the pH dependences of the reaction steps catalyzed by vaccinia virus topoisomerase (5Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar, 6Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 15449-15458Crossref PubMed Scopus (46) Google Scholar), it was suggested that transesterification mediated by the DNA topoisomerases might involve general acid-base catalysis. In the formation of the enzyme-DNA covalent adduct, a general base in the enzyme might assist in the removal of the hydroxyl proton of the active site tyrosine, and a separate general acid in the enzyme might assist in the protonation of the departing deoxyribosyl oxygen. The DNA rejoining reaction after strand passage could be the exact microscopic reversal of the DNA breakage reaction (5Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar, 6Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 15449-15458Crossref PubMed Scopus (46) Google Scholar, 7Wang J.C. J. Mol. Biol. 1971; 55: 523-533Crossref PubMed Scopus (512) Google Scholar), but there has been no definitive experimental test of this conjecture. In this work, we report a mutational analysis of Escherichia coli DNA topoisomerase I for the identification of amino acid side chains that might be directly involved in transesterification between Tyr-319 of the enzyme and the DNA scissile phosphorus (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar). The E. coli enzyme is representative of a subfamily of type I DNA topoisomerases, the type IA enzymes, from a diverse collection of organisms including bacteria, eukarea, and archaea (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar, 8Tse-Dinh Y.-C. Adv. Pharmacol. 1994; 29A: 21-27Crossref PubMed Scopus (16) Google Scholar). Alignment of amino acid sequences of these enzymes showed a large number of highly conserved amino acid residues (9Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (98) Google Scholar). This subfamily of enzymes are very different, however, from the type IB DNA topoisomerases, whose members include eukaryotic DNA topoisomerase I and vaccinia virus topoisomerase, both in amino acid sequences and reaction mechanisms (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar). Several studies on the identification of active site residues in vaccinia virus DNA topoisomerase have been reported recently (10Peterson B.O. Shuman S. J. Biol. Chem. 1997; 272: 3891-3896Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Cheng C. Wang L.K. Sekiguchi J. Shuman S. J. Biol. Chem. 1997; 272: 8263-8269Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12Wang L.K. Wittschieben J. Shuman S. Biochemistry. 1997; 36: 7944-7950Crossref PubMed Scopus (10) Google Scholar, 13Wittschieben J. Shuman S. Nucleic Acids Res. 1997; 25: 3001-3008Crossref PubMed Scopus (69) Google Scholar). E. coli DNA topoisomerase I is encoded by the topA gene comprised of 865 codons (14Tse-Dinh Y.-C. Wang J.C. J. Mol. Biol. 1986; 191: 321-331Crossref PubMed Scopus (73) Google Scholar). For convenience, the positions of all amino acid residues in the 97-kDa single polypeptide protein are referred to by their codon numbers, even though the N-terminal methionine is removed post-translation (14Tse-Dinh Y.-C. Wang J.C. J. Mol. Biol. 1986; 191: 321-331Crossref PubMed Scopus (73) Google Scholar). The three-dimensional structure of a 67-kDa fragment of the enzyme has been determined by x-ray crystallography (15Lima C.D. Wang J.C. Mondragón A. Nature. 1994; 367: 138-146Crossref PubMed Scopus (262) Google Scholar), and that of a C-terminal fragment comprised of amino acids 745–865 has been determined by nuclear magnetic resonance (16Yu L. Zhu C.X. Tse-Dinh Y.-C. Fesik S.W. Biochemistry. 1995; 34: 7622-7628Crossref PubMed Scopus (46) Google Scholar). The latter fragment is dispensable for catalytic activity but appears to participate in substrate binding (17Beran-Steed R.K. Tse-Dinh Y.-C. Proteins Struct. Funct. Genet. 1989; 6: 249-258Crossref PubMed Scopus (46) Google Scholar,18Zhu C.-X. Samuel M. Pound A. Ahumada A. Tse-Dinh Y.-C. Biochem. Mol. Biol. Int. 1995; 35: 375-385PubMed Google Scholar). The polypeptide bridging these two fragments contains three motifs with four cysteines in each. These tetracysteine motifs are most likely the binding sites of three Zn(II) ions (19Zhu C.-X. Qi H.Y. Tse-Dinh Y.-C. J. Mol. Biol. 1995; 250: 609-616Crossref PubMed Scopus (21) Google Scholar). The 67-kDa N-terminal fragment is capable of covalent adduct formation with single-stranded DNA (15Lima C.D. Wang J.C. Mondragón A. Nature. 1994; 367: 138-146Crossref PubMed Scopus (262) Google Scholar). Therefore, it is most likely to contain all residues that are essential for covalent catalysis. In the crystal structure of this fragment, the polypeptide is folded into four distinct domains (15Lima C.D. Wang J.C. Mondragón A. Nature. 1994; 367: 138-146Crossref PubMed Scopus (262) Google Scholar). The fragment can be viewed to comprise a “base” formed by domains I and IV and a “lid” formed by domains II and III (see Fig. 1 A). In the crystal, the base and the lid are touching on one side through contacts between domain III in the lid and domains I and IV in the base and are linked on the other side by a pair of long strands between domains II and IV. The four domains and the pair of connecting strands enclose a 28 Å hole. The active site tyrosine has been identified to be Tyr-319 (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar). It is located in domain III at the interface between this domain and domains I and IV. This strategic position suggests that during the catalysis of DNA breakage, passage, and rejoining, domain III on one side and domains I and IV on the other are likely to undergo large relative movements (15Lima C.D. Wang J.C. Mondragón A. Nature. 1994; 367: 138-146Crossref PubMed Scopus (262) Google Scholar). Following the formation of the covalent adduct, for example, the lid is probably lifted away from the base to allow the passage of a second strand (15Lima C.D. Wang J.C. Mondragón A. Nature. 1994; 367: 138-146Crossref PubMed Scopus (262) Google Scholar). The structural features of the 67-kDa fragment and the homology alignment of the amino acid sequences of the type IA enzymes provided a useful backdrop for the studies described below. Alanine substitution mutants were constructed by site-directed mutagenesis, using a commercial kit and following the protocol of the supplier (CLONTECH). The E. coli DNA topoisomerase I overexpression plasmid pJW312 (16Yu L. Zhu C.X. Tse-Dinh Y.-C. Fesik S.W. Biochemistry. 1995; 34: 7622-7628Crossref PubMed Scopus (46) Google Scholar) was used in these constructions. A selection primer, which changes a unique ScaI site within the β-lactamase gene of the plasmid to a SalI site, and mutagenesis primers for the intended alanine codon substitutions, were purchased from commercial suppliers. The nucleotide sequences of the mutagenesis primers used in the construction of the E9A, H33A, D111A, E115A, Q309A, E313A, T318A, Y319A, R321A, T322A, D323A, H365A, and T496A were, respectively, 5′-CTT-GTC-ATC-GTT-GCT-AGC-CCG-GCA-AAA-GCC, 5′-TCC-AGC-GTC-GGC-GCC-ATC-CGC-GAT-TTG-C, 5′-C-ATC-TAT-CTC-GCG-ACC-GCC-CTT-GAC-CGC-G, 5′-C-CTT-GAC-CGC-GCC-GGC-GAA-GCC-ATT-GCA-TG, 5′-CC-ATG-ATG-ATG-GCG-GCG-CGC-TTG-TAT-GAA-GCA-GGC, 5′-CAG-CGT-TTG-TAT-GCA-GCC-GGC-TAT-ATC-ACT-TAC, 5′-GCA-GGC-TAT-ATC-GCT-TAC-ATG-CGC-ACC-GAC-TCC-AC, 5′-CA-GGC-TAT-ATC-ACT-GCC-ATG-CGC-ACC-GAC-TCC-AC, 5′-C-TAT-ATC-ACT-TAC-ATG-GCC-ACC-GAC-TCC-AC, 5′-C-ACT-TAC-ATG-CGC-GCC-GAC-TCC-ACT-AAC, 5′-CT-TAC-ATG-CGT-ACG-GCC-TCC-ACT-AAC-CTG, 5′-C-TCA-CAG-GCA-GCG-GCC-GAA-GCG-ATT-CGC, 5′-CA-GAT-GCG-CAG-AAG-CTA-GCC-CAG-TTA-ATC-TGG-C, and 5′-GT-CGT-CCG-TCT-GCA-TAT-GCG-TCG-ATC (for clarity, hyphens are inserted in between codons). The oligonucleotides used in the construction of E9Q and R321K were 5′-CTT-GTC-ATA-GTT-CAG-TCG-CCG-GCA-AAA-3′ and 5′-ATC-ACT-TAC-ATG-AAG-ACC-GAC-TCC-ACT-3′, respectively. In the design of each mutagenesis primer, silent mutations were often included to introduce a restriction site (underlined hexameric sequences in the mutagenesis primers specified above), so that the presence of the intended mutation could be checked by digestion of the mutated plasmid with the particular restriction enzyme. In the construction of R321K, aRsaI site in the wild-type topA gene was eliminated for the same purpose. Further confirmation of the presence of the intended mutation in each of the mutant plasmid was carried out by DNA sequencing. pJW312 or its mutated derivative, which expresses wild-type or mutant E. coli DNA topoisomerase I from an inducible lac promoter (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar), was transformed into E. coli BL21 topA +or DM800 ΔtopA cells bearing a pACYC184-basedlacI clone. Induction of cells for overexpression of the topoisomerase by the addition of isopropyl-1-thio-β-d-galactoside and lysis of the cells with lysozyme and the nonionic detergent Brij-58 were carried out as described previously for the preparation of wild-type E. coli DNA topoisomerase I (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar). Following the removal of cell debris by centrifugation, the lysate was directly loaded on a phosphocellulose column and eluted as described (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar). Peak fractions were pooled and further purified by high pressure liquid chromatography, using an SP column (Bio-Rad). Purity of each protein was examined by SDS-polyacrylamide gel electrophoresis. Expression levels of all mutants were found to be comparable with that of wild-type E. coli DNA topoisomerase I. Higher yields were generally achieved in preparations from BL21 cells than DM800 cells, but preparations from DM800 cells were used in the relaxation assays owing to the possibility of contaminating wild-type DNA topoisomerase I in preparations from BL21 cells. Cleavage of uniquely end-labeled DNA was carried out in a buffer containing 40 mm Tris·HCl, pH 7.5, and 10 mm KCl. In some experiments, EDTA was also present at 0.1 mm. A 388-base pair longEcoRI-NcoI restriction fragment was used in the cleavage assays. The plasmid pJW312 containing the fragment was first cut with EcoRI, and the 5′ ends were 32P-labeled by a cycle of dephosphorylation with calf intestine alkaline phosphatase and phosphorylation with T4 polynucleotide kinase in the presence of [γ-32P]ATP. After phenol extraction and ethanol precipitation in the presence of ammonium acetate to remove the unincorporated triphosphate, the labeled DNA was resuspended and digested with NcoI, and the DNA fragment labeled only at itsEcoRI end was purified by electrophoresis in a 1.5% agarose gel. This uniquely end-labeled DNA was heat denatured and used in the cleavage assays. Further details of the DNA relaxation and cleavage assays are described in the legends to Figs. 2 and 3.Figure 3DNA cleavage by wild-type E. coliDNA topoisomerase I and several of its alanine substitution derivatives. Lane 1, wild type; lane 2, E9A;lane 3, E9Q; lane 4, Y319A; lane 5, R321A; lane 6, control without enzyme. Each cleavage assay mixture of 10 μl contained 40 mm Tris·HCl, pH 7.5, 10 mm KCl, 100 ng of E. coli DNA topoisomerase I, and approximately 20 ng of a denatured 388-base pair restriction fragment 32P-labeled at a unique 5′ end. The mixture was incubated at 37 °C for 30 min, and SDS was added to a final concentration of 1% to reveal the topoisomerase-mediated DNA cleavage. All reaction mixtures were desalted by ethanol precipitation, and the pellets were dissolved in 10 μl of water for electrophoresis in a 6% polyacrylamide DNA sequence gel.View Large Image Figure ViewerDownload (PPT) Reversal of DNA cleavage by the addition of NaCl to 0.8 m or NaCl and MgCl2 to 0.8 m and 10 mm, respectively, was carried out to test whether a mutant enzyme capable of cleaving single-stranded DNA could rejoin the cleaved DNA (21Depew R.E. Liu L.F. Wang J.C. J. Biol. Chem. 1978; 253: 511-518Abstract Full Text PDF PubMed Google Scholar, 22Liu L.F. Wang J.C. J. Biol. Chem. 1979; 254: 11082-11088Abstract Full Text PDF PubMed Google Scholar). Following incubation of the enzyme and the 5′ end-labeled DNA fragment, each sample was split into three equal volume portions. One of each triplicate was used for measuring DNA cleavage by the addition of SDS to 1%. For the remaining two, MgCl2 was added to one to a final concentration of 10 mm, and NaCl was added to both to a final concentration of 0.8 m. The pair of samples were incubated at 37 °C for an additional 30 min before the addition of SDS. Following the removal of salt by ethanol precipitation, the samples were resuspended in water for electrophoresis in a 6% polyacrylamide DNA sequencing gel. A gel-purified nonamer 5′-CAATGCGCT-3′, 32P-labeled at its 5′ end, was used as the substrate. The concentrations of E. coli DNA topoisomerase I and the oligonucleotide were approximately 0.4 and 1 μm, respectively. Ethanol precipitation of the radiolabeled oligomer was done in the presence of glycogen (Boehringer Mannheim). To identify amino acid side chains of E. coli DNA topoisomerase I that might be directly involved in the catalysis of DNA breakage and rejoining, 12 point mutants, E9A, H33A, D111A, E115A, Q309A, E313A, T318A, R321A, T322A, D323A, H365A, and T496A, each designated by the particular amino acid residue replaced by alanine, were constructed by site-directed mutagenesis. An additional mutant Y319A was also constructed for comparison with the others, because inactivation of the enzyme by this mutation was anticipated from the known function of Tyr-319 in catalysis (4Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar), as well as from previous studies of the Y319F and Y319S mutant enzymes (23Wilkinson A.J. Wang J.C. Wu F.Y.-H. Wu C.-W. Structure and Function of Nucleic Acids and Proteins. Raven Press, New York1990: 61-75Google Scholar). In selecting the residues for alanine substitution, it was assumed that a particular catalytic residue must be present at corresponding positions in all members of this subfamily and that it must possess a polar side chain, which is likely to be involved in the catalysis of DNA breakage and rejoining. Variability in residues at a conserved position was deemed acceptable only if all residues at the position contained a similar chemical group: interchanges among aspartate, glutamate, asparagine, and glutamine at a particular position in the homology alignment, for example, were considered acceptable because of the presence of a carbonyl group in each of the residues; on the other hand, a highly conserved amino acid residue such as Tyr-312 was considered to be an unlikely candidate because of the presence of a phenylalanine at this position in Sulfolobus acidocaldariusreverse gyrase (9Caron P.R. Wang J.C. Adv. Pharmacol. 1994; 29B: 271-297Crossref PubMed Scopus (98) Google Scholar). Several amino acid residues, including Lys-90, Arg-209, Tyr-391, and Glu-547, which fulfill the criterion of being functionally conserved, were not selected for mutagenesis because of their relatively distal locations from the active site tyrosine Tyr-319 (15Lima C.D. Wang J.C. Mondragón A. Nature. 1994; 367: 138-146Crossref PubMed Scopus (262) Google Scholar). The locations of Tyr-319 and the other 12 amino acid residues selected for alanine substitution mutagenesis are shown in Fig. 1 B. Wild-type E. coli DNA topoisomerase I and the 13 alanine substitution mutant proteins were individually overexpressed in ΔtopA cells harboring plasmids encoding the proteins. All mutant proteins were found to be expressed to a high level comparable with that of the wild-type enzyme. Each of the overexpressed proteins was purified to homogeneity and assayed for its ability to relax negatively supercoiled DNA. In Fig. 2, each set of four lanes represents assays in which the concentration of the topoisomerase was successively diluted 5-fold each time from left to right. Although all mutants were constructed by substituting a highly conserved polar residue by alanine, the majority of these were found to be catalytically active. In addition to Y319A, in which the active site tyrosine was replaced by alanine, only E9A and R321A were found to have no detectable DNA relaxation activity. The above findings led to the construction of two additional mutants: E9Q, in which Glu-9 is replaced by a glutamine, and R321K, in which Arg-321 is replaced by a lysine. The lysine substitution mutant R321K was found to be as active as the wild-type enzyme in the removal of DNA negative supercoils, but the E9Q mutant enzyme showed little activity in comparison with the wild-type enzyme (results not shown). Fig. 3illustrates the results of a typical experiment in which covalent adduct formation between DNA and wild type (lane 1), E9A (lane 2), E9Q (lane 3), Y319A (lane 4), and R321A mutant enzyme (lane 5) was examined. A 388-base pair-long DNA fragment uniquely labeled at a 5′ end was used in this experiment. The fragment was heat denatured and incubated with wild-type or mutant E. coli DNA topoisomerase I in 40 mm Tris·HCl, pH 7.5, 10 mm KCl, and SDS was then added to 1%. As shown previously, protein-DNA covalent adduct formation is accompanied by cleavage of single-stranded DNA at sites that are determined by both structural and sequence features (24Kirkegaard K. Pflugfelder G. Wang J.C. Cold Spring Harbor Symp. Quant. Biol. 1984; 49: 411-419Crossref PubMed Scopus (47) Google Scholar, 25Kirkegaard K. Wang J.C. J. Mol. Biol. 1985; 185: 625-637Crossref PubMed Scopus (128) Google Scholar). There is a strong preference of a cytosine at position −4, that is, four nucleotides upstream of the cleavage site (2Tse Y.-C. Kirkegaard K. Wang J.C. J. Biol. Chem. 1980; 255: 5560-5565Abstract Full Text PDF PubMed Google Scholar). In the case of wild-type E. coli DNA topoisomerase I (lane 1 of Fig. 3), the specificity of the cleavage reaction resulted in a distinctive distribution of cleavage products (compare the pattern of the lane 1 sample with that of the untreated control run in lane 6). For the two mutant proteins E9A and R321A that showed little DNA relaxation activity (Fig. 2), the former showed no cleavage activity (lane 2 of Fig. 3), whereas the latter showed reduced but significant level of DNA cleavage (lane 5of Fig. 3). In contrast to the E9A mutant, the E9Q mutant protein showed full cleavage activity (lane 3 of Fig. 3). In some cleavage assays, the reaction mixtures also contained 0.1 mm EDTA; no difference in the cleavage patterns was observed by the inclusion of this metal chelating agent. As expected, the active site tyrosine mutant Y319A showed little DNA cleavage activity (lane 4 of Fig. 3). Careful inspection of the autoradiogram revealed, however, the presence of faint bands corresponding to cleavages at a subset of the cleavage sites of the wild-type enzyme. The significance of these bands will be discussed in a later section. Experiments similar to the one shown in Fig. 3 were carried out for the other mutant enzymes. As expected, mutant proteins that showed DNA relaxation activity comparable with that of the wild-type enzyme were found to cleave DNA with efficiencies and site preferences similar to those of the wild-type enzyme (data not shown). For the complex between single-stranded or negatively supercoiled DNA and bacterial DNA topoisomerase I, it is known that addition of excess salt leads to the dissociation of the complex to give DNA with intact strands (21Depew R.E. Liu L.F. Wang J.C. J. Biol. Chem. 1978; 253: 511-518Abstract Full Text PDF PubMed Google Scholar, 22Liu L.F. Wang J.C. J. Biol. Chem. 1979; 254: 11082-11088Abstract Full Text PDF PubMed Google Scholar). Prior to salt addition, a fraction of the enzyme-DNA complex is presumably in the form of the covalent intermediate that can be revealed by the addition of a protein denaturant. The addition of salt to the enzyme-DNA complex therefore appears to drive the dissociation of the enzyme and the rejoining of the DNA. In the absence of added Mg(II) and in the presence of excess EDTA, however, a significant fraction of the enzyme, termed the “salt-stable complex,” was found to remain bound to single-stranded DNA upon addition of molar amounts of salt (21Depew R.E. Liu L.F. Wang J.C. J. Biol. Chem. 1978; 253: 511-518Abstract Full Text PDF PubMed Google Scholar, 22Liu L.F. Wang J.C. J. Biol. Chem. 1979; 254: 11082-11088Abstract Full Text PDF PubMed Google Scholar). The salt-induced reversal of DNA cleavage was exploited to test whether a mutant enzyme that showed DNA cleavage activity might be deficient in rejoining the broken DNA; following the DNA cleavage reaction by a mutant enzyme blocked in its DNA rejoining activity, the addition of salt would not be expected to rejoin the cleaved DNA. Lanes 1–3 of Fig. 4 depict the results of such a salt-reversal experiment with wild-type E. coli DNA topoisomerase I. The lane 1 sample in Fig. 4 was treated in the same way as that analyzed in lane 1 of Fig. 3. A DNA fragment 32P-labeled at a unique 5′ end was denatured and incubated with the E. coli enzyme. SDS was then added to a final concentration of 1% to denature the enzyme and to reveal the formation of the covalent complex. For the sample run in lanes 2 and 3, incubation of the denatured DNA and the E. coli enzyme was carried out in the usual manner. Before the addition of SDS, however, NaCl was added to the lane 2sample to a final concentration of 0.8 m, and NaCl and MgCl2 were added to the lane 3 sample, to 0.8 and 10 mm, respectively. The pattern shown in lane 3 was expected from results of the earlier studies; exposure of the enzyme-DNA complex to high salt would lead to rejoining of any cleaved DNA and dissociation of the enzyme from the DNA. Thus in contrast to the sample run in lane 1, which showed substantial amounts of cleaved DNA, the bulk of the DNA in the lane 3 sample appeared uncleaved. The high salt-induced rejoining of DNA was also observed in the absence of added Mg(II) (lane 2). When EDTA was added to 10 mm before the addition of NaCl, however, rejoining was largely abolished unless Mg(II) was added to a concentration of 1 mm or higher (data not shown) as observed previously (21Depew R.E. Liu L.F. Wang J.C. J. Biol. Chem. 1978; 253: 511-518Abstract Full Text PDF PubMed Google Scholar, 22Liu L.F. Wang J.C. J. Biol. Chem. 1979; 254: 11082-11088Abstract Full Text PDF PubMed Google Scholar). Salt-induced reversal experiments with the mutant enzymes E9Q and R321A are shown in lanes 4–6 and 7–9 of Fig. 4, respectively. Similar to the case with the wild-type enzyme, the addition of salt to 0.8 m in either the presence or the absence of 10 mm Mg(II) was found to induce the rejoining of DNA by the mutant enzymes. The DNA cleavage activities of the E9Q and R321A mutant proteins were also tested with a short DNA oligomer 5′-CAAT*GCGCT-3′ known to be cleaved by E. coli DNA topoisomerase I at the position marked by an asterisk in the nonamer sequence (24Kirkegaard K. Pflugfelder G. Wang J.C. Cold Spring Harbor Symp. Quant. Biol. 1984; 49: 411-419Crossref PubMed Scopus (47) Google Scholar). 1Y.-C. Tse-Dinh, personal communication. Similar to the results shown in Fig. 4 for the longer DNA substrate, the wild-type and the E9Q mutant protein were observed to cleave the DNA nonamer with comparable efficiency (lanes 2 and 5 of Fig. 5, respectively), and the R321A mutant protein showed a reduced level of cleavage activity (lane 8of Fig. 5). In all cases the intensity of the labeled cleavage product remained constant, however, upon the addition of NaCl to 0.8m (Fig. 5, lanes 3, 6, and 9) or the addition of both NaCl and MgCl2 to 0.8m and 10 mm, respectively (lanes 4, 7, and 10). These results were expected; the noncovalently bound cleavage product 5′-CAAT-3′ would diffuse away from the catalytic pocket of the enzyme, and thus no significant rejoining with enzyme-linked DNA could occur following salt addition. We have applied alanine substitution mutagenesis in assessing the plausible roles of 12 residues, in addition to the nucleophile Tyr-319, in the catalysis of DNA breakage and rejoining by E. coliDNA topoisomerase I. Each of the residues was selected for site-directed mutagenesis based on the strict conservation of a polar group at that position in all homologues of phylogenetically diverse organisms including bacteria, eukarea, and archaea. It is therefore surprising that only substitution at Glu-9 or Arg-321 was found to affect transesterifica" @default.
• W2071796772 created "2016-06-24" @default.
• W2071796772 creator A5011866089 @default.
• W2071796772 creator A5072019761 @default.
• W2071796772 date "1998-03-01" @default.
• W2071796772 modified "2023-10-10" @default.
• W2071796772 title "Identification of Active Site Residues in Escherichia coli DNA Topoisomerase I" @default.
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