Identification of Active Site Residues in the “GyrA” Half of Yeast DNA Topoisomerase II
1998; Elsevier BV; Volume: 273; Issue: 32 Linguagem: Inglês
10.1074/jbc.273.32.20252
ISSN1083-351X
AutoresQiyong Peter Liu, James C. Wang,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoSite-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate intrans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5′ and 3′ sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct. Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate intrans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5′ and 3′ sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct. Because of the double helix structure of DNA, its cellular transactions often require the passage of individual DNA strands or double helices through one another. The DNA topoisomerases are enzymes that have evolved to fulfill such requirements. The type I enzymes catalyze DNA strand passage by transiently breaking one DNA strand at a time, and the type II enzymes create transient breaks in both strands of a DNA segment for the enzyme-mediated passage of a second DNA segment (for reviews, see Refs. 1Reese R.J. Maxwell A. CRC Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (565) Google Scholar, 2Wigley D.B. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 185-208Crossref PubMed Scopus (88) Google Scholar, 3Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2093) Google Scholar and references therein). The type I enzymes are further divided into two subfamilies IA and IB that are distinct in terms of their catalytic characteristics and amino acid sequences (3Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2093) Google Scholar). Although the type II enzymes were thought to form a single subfamily, recent studies suggest that the enzyme DNA topoisomerase VI from archaeal hyperthermophiles may represent a separate subfamily (4Bergerat A. de Massy B. Gradelle D. Varoutas P.C. Nicolas A. Forterre P. Nature. 1997; 386: 414-417Crossref PubMed Scopus (719) Google Scholar). DNA topoisomerases catalyze the breakage of DNA strands via transesterification between an enzyme tyrosyl group and a DNA backbone phosphoryl group, forming a phosphotyrosine link between the two and leaving a deoxyribosyl hydroxyl group on the other end of the broken DNA strand (5Tse Y.-C. Kirkegaard K. Wang J.C. J. Biol. Chem. 1980; 255: 5560-5565Abstract Full Text PDF PubMed Google Scholar, 6Champoux J.J. J. Biol. Chem. 1981; 256: 4805-4809Abstract Full Text PDF PubMed Google Scholar, 7Rowe T.C. Tewey K.M. Liu L.F. J. Biol. Chem. 1984; 259: 9177-9181Abstract Full Text PDF PubMed Google Scholar, 8Horowitz D.S. Wang J.C. J. Biol. Chem. 1987; 262: 5339-5344Abstract Full Text PDF PubMed Google Scholar, 9Worland S.T. Wang J.C. J. Biol. Chem. 1989; 264: 4412-4416Abstract Full Text PDF PubMed Google Scholar, 10Lynn R.M. Bjornsti M.A. Caron P. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3559-3563Crossref PubMed Scopus (111) Google Scholar, 11Lynn R.M. Wang J.C. Proteins. 1989; 6: 231-239Crossref PubMed Scopus (59) Google Scholar). Rejoining of the strand occurs via an apparent reversal of the DNA cleavage reaction; the hydroxyl group formed in the cleavage reaction acts as the nucleophile in a second transesterification, breaking the phosphotyrosine link and reforming the DNA phosphodiester bond. The rejoining reaction could be the exact microscopic reversal of the breakage reaction; conformational changes in the enzyme between the DNA cleavage and rejoining steps are plausible, however, and thus the two reactions could involve transition states that are not identical (12Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 15449-15458Crossref PubMed Scopus (47) Google Scholar). For the type II DNA topoisomerases, sequence alignments of polypeptides other than those of the archaeal DNA topoisomerase VI class show a high degree of homology (see for example, Ref. 13Caron P.R. Wang J.C. Liu L. DNA Topoisomerase and Their Applications in Pharmacology. Academic Press, San Diego1994: 271-297Google Scholar). All type II DNA topoisomerases act as homodimers of 1–3 subunits (14Gellert M. Mizuuchi K. O'Dea M.H. Nash H.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3872-3876Crossref PubMed Scopus (878) Google Scholar, 15Liu L.F. Liu C.C. Alberts B.M. Nature. 1979; 281: 456-461Crossref PubMed Scopus (164) Google Scholar, 16Stetler G.L. King G.J. Huang W.M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3737-3741Crossref PubMed Scopus (58) Google Scholar, 17Miller K.G. Liu L.F. Englund P.T. J. Biol. Chem. 1981; 256: 9334-9339Abstract Full Text PDF PubMed Google Scholar, 18Sander M. Hsieh T. J. Biol. Chem. 1983; 258: 8421-8428Abstract Full Text PDF PubMed Google Scholar, 19Goto T. Wang J.C. Cell. 1984; 36: 1073-1080Abstract Full Text PDF PubMed Scopus (119) Google Scholar), and they create transient openings or gates in a double-stranded DNA through transesterification between a pair of active site tyrosyl residues in each enzyme and a pair of phosphates 4 base pairs apart in the DNA (18Sander M. Hsieh T. J. Biol. Chem. 1983; 258: 8421-8428Abstract Full Text PDF PubMed Google Scholar,20Morrison A. Cozzarelli N.R. Cell. 1979; 17: 175-184Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 21Liu L.F. Rowe T.C. Yang L. Tewey K.M. Chen G.L. J. Biol. Chem. 1983; 258: 15365-15370Abstract Full Text PDF PubMed Google Scholar). These enzymes utilize ATP in their transport of a second DNA segment through such a transiently opened DNA gate (1Reese R.J. Maxwell A. CRC Crit. Rev. Biochem. Mol. Biol. 1991; 26: 335-375Crossref PubMed Scopus (565) Google Scholar, 2Wigley D.B. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 185-208Crossref PubMed Scopus (88) Google Scholar, 3Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2093) Google Scholar). According to the two-gate protein clamp model (22Roca J. Wang J.C. Cell. 1992; 71: 833-840Abstract Full Text PDF PubMed Scopus (297) Google Scholar, 23Roca J. Wang J.C. Cell. 1994; 77: 609-616Abstract Full Text PDF PubMed Scopus (255) Google Scholar), a homodimeric type II enzyme bound to a DNA segment (the gate or G segment) acts as an ATP-modulated protein clamp. A second DNA segment (the transported or T segment) can enter an open entrance gate of the protein clamp. The binding of ATP to the enzyme triggers the closure of the entrance gate, and this closure in turn causes a cascade of reactions: the entered DNA segment is forced through a transient opening in the DNA G segment created by the enzyme and expelled through the exit gate of the enzyme (22Roca J. Wang J.C. Cell. 1992; 71: 833-840Abstract Full Text PDF PubMed Scopus (297) Google Scholar, 23Roca J. Wang J.C. Cell. 1994; 77: 609-616Abstract Full Text PDF PubMed Scopus (255) Google Scholar). The crystal structure of a 92-kDa fragment of yeast DNA topoisomerase II spanning amino acid residues 409–1201 was reported in 1996 (24Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Crossref PubMed Scopus (751) Google Scholar). 1Numbering of amino acid residues was corrected for the presence of an extra asparagine at position 72 of the reported sequence. The region of the single polypeptide yeast enzyme from amino acid residues 409 to about 660 corresponds to the C-terminal half of the B subunit or GyrB 2The abbreviations used are: GyrA and GyrBthe A and B subunit of bacterial DNA gyrase, respectivelyADPPNP5′-adenylyl-β,γ-imidodiphosphateEenzymeWTwild typekbkilobase pairHMKheart muscle kinase. protein ofEscherichia coli DNA gyrase and has been termed the B′ subfragment; the region of the yeast enzyme extending from the end of the B′ subfragment to around residue 1200 corresponds to the N-terminal two-thirds of the A subunit or GyrA protein of E. coli DNA gyrase and has been termed the A′ subfragment. The 92-kDa yeast B′A′ fragment forms a homodimer in the crystal and in solution, and this dimeric protein has been shown to cleave double-stranded DNA to form a pair of protein-DNA covalent links (24Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Crossref PubMed Scopus (751) Google Scholar). The last result shows that all residues involved in DNA breakage and rejoining are located within the yeast (B′A′)2 protein. the A and B subunit of bacterial DNA gyrase, respectively 5′-adenylyl-β,γ-imidodiphosphate enzyme wild type kilobase pair heart muscle kinase. Examination of the 92-kDa structure led to the suggestion that the conformation of the polypeptide in this structure corresponds to that of the yeast enzyme after it has cleaved the DNA G segment and pulled the enzyme-linked DNA ends apart for the passage of the T segment through the widened opening (24Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Crossref PubMed Scopus (751) Google Scholar). A pair of short DNA helices, each with a four-nucleotide single-stranded extension at a 5′ end, has been modeled into a DNA-binding site in each half of the (B′A′)2protein (24Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Crossref PubMed Scopus (751) Google Scholar). This structural model is supported by recent protein footprinting experiments in which the effects of DNA binding on the citraconylation of individual lysyl side chains in the yeast enzyme were measured (25Li W. Wang J.C. J. Biol. Chem. 1997; 272: 31190-31195Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Recently, the crystal structure of a 59-kDa fragment of E. coli GyrA protein, corresponding to the A′ portion of the 92-kDa yeast fragment, was reported (26Morais Cabral J.H. Jackson A.P. Smith C.V. Shikotra N. Maxwell A. Liddington R.C. Nature. 1997; 388: 903-906Crossref PubMed Scopus (407) Google Scholar). This structure is believed to resemble closely the conformation of the polypeptide in a DNA-bound enzyme before the DNA is cleaved by the enzyme (26Morais Cabral J.H. Jackson A.P. Smith C.V. Shikotra N. Maxwell A. Liddington R.C. Nature. 1997; 388: 903-906Crossref PubMed Scopus (407) Google Scholar). Based on the distribution of surface charges and the positions of the active site tyrosines in the crystal structure, a curved DNA segment has been modeled into the structure (26Morais Cabral J.H. Jackson A.P. Smith C.V. Shikotra N. Maxwell A. Liddington R.C. Nature. 1997; 388: 903-906Crossref PubMed Scopus (407) Google Scholar). Whereas there are significant differences in the spatial arrangements of the various domains within the A′ subfragment in the yeast (B′A′)2 crystal structure and their counterparts in the 59-kDa E. coli GyrA fragment crystal structure, the remarkable conservation of the overall architecture of the polypeptide in the two structures provides strong support of the notion that these enzymes act similarly in their ATP-dependent transport of one duplex DNA segment through another. The crystal structures of the yeast DNA topoisomerase II 92-kDa fragment and the E. coli DNA GyrA 59-kDa fragment have provided much needed structural information for understanding the mechanism of DNA breakage/rejoining by the type II DNA topoisomerases. To gain further mechanistic insight on the reactions they catalyze, we have applied site-directed mutagenesis in the search of residues that are involved in the catalysis of DNA cleavage and rejoining. In the present communication, we report mutagenesis analysis of the A′ subfragment of yeast DNA topoisomerase II. A 6.4-kb plasmid pSW201 bearing the yeast TOP2 gene encoding DNA topoisomerase II was used in the construction of the alanine substitution mutants, using a commercial kit and following the experimental protocol of the supplier (CLONTECH). The sequences of the mutagenic oligodeoxyribonucleotides used in the various constructions are listed in Table I. To facilitate the identification of the desired mutants, each mutagenic oligomer was designed to alter the restriction pattern of the parental plasmid in addition to introducing the desired codon change. The oligomer 5′-GAAAAGTGCCACCTGAGCTCTAAGAAACCATTATTATC-3′, which was previously designed to change a unique AatII site in pSW201 to the underlined SacI site (27Roca J. Berger J.M. Harrison S.C. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4057-4062Crossref PubMed Scopus (167) Google Scholar), was used as the selection primer in the construction of the mutants. Mutant plasmids were screened by digestion with appropriate restriction enzymes that cut at the altered sites, and final confirmation of the mutations was done by direct nucleotide sequencing of the altered regions.Table ISequences of mutagenic oligodeoxynucleotidesMutantOligonucleotides (5′–3′)Restriction site alterationR690AG GCC GAT AAT ATA GC A TCG ATT CCC AAT G+ClaID697ACCC AAT GTT TTA GCC GGA TTT AAA CCT GGC+MspIK700AGAT GGA TTT GCA CCC GGG CAA AGA AAA GTT C+SmaIR704AGAT GGA TTT AAA CCG GGC CAA GCA AAA GTT CTT TAT GGT TGT+MspIH735ATGT ACG GCA TAT GCG CAT GGT GAG CAG+FspI, −NcoIH736ACG GCA TAT CAC GCT GGT GAG CAG TC−NcoIE738AT CAC CAT GGT GCG CAG TCA TTG G+FspIQ750AATT ATT GGG CTG GCC GCA AAC TTT GTT GG−NheIR781AGAT GCA GCT GCC GCG GCA TAT ATC TAC ACA G+NotIN828ACT ATG ATT CTT GTT GCC GGT GCT GAG−HpaIAdjacent codon triplets are separated by a space for clarity, and differences in nucleotides from the original sequence are indicated by boldface letters. The restriction site added or removed by the introduction of a particular mutagenic oligonucleotide is specified in the right-hand column (plus for addition and minus for removal), and the restriction site added or removed is underlined in each sequence. Open table in a new tab Adjacent codon triplets are separated by a space for clarity, and differences in nucleotides from the original sequence are indicated by boldface letters. The restriction site added or removed by the introduction of a particular mutagenic oligonucleotide is specified in the right-hand column (plus for addition and minus for removal), and the restriction site added or removed is underlined in each sequence. Two sets of expression plasmids were constructed from the mutagenized pSW201 derivatives. For genetic complementation tests of the mutants, the KpnI-AvrII fragment from each of the mutated derivative of pSW201 was used to replace the corresponding wild-type TOP2 fragment in YEpTOP2-PGAL1, a multicopy plasmid used previously in the overexpression of full-length yeast DNA topoisomerase II from an inducible yeast GAL1 gene promoter (9Worland S.T. Wang J.C. J. Biol. Chem. 1989; 264: 4412-4416Abstract Full Text PDF PubMed Google Scholar). For overexpression of the mutant enzymes for biochemical characterization, theKpnI-AvrII fragment from each of the mutated pSW201 was used to replace the corresponding fragment in pGAL1Top2-(1–1196)-HMK-(His)6, in which the first 1196 codons of the wild-type yeast TOP2 gene are fused to two short runs of codons, one encodes a phosphorylation site of heart muscle kinase and the other a stretch of six histidines (25Li W. Wang J.C. J. Biol. Chem. 1997; 272: 31190-31195Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The tagged enzyme expressed by pGAL1Top2-(1–1196)-HMK-(His)6has previously been shown to be fully active in vitro (25Li W. Wang J.C. J. Biol. Chem. 1997; 272: 31190-31195Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,28Caron P.R. Watt P. Wang J.C. Mol. Cell. Biol. 1994; 14: 3197-3207Crossref PubMed Scopus (83) Google Scholar), and the presence of the six histidines at the C terminus facilitates the purification of the enzyme on Ni-resin (Novagen). YEpTOP2-PGAL1 and its mutated derivatives were individually transformed into yeast strain JCW26 carrying the temperature-sensitive allele top2-4 (29Holm C. Goto T. Wang J.C. Botstein D. Cell. 1985; 41: 553-563Abstract Full Text PDF PubMed Scopus (574) Google Scholar) or strain CH834 carrying the cold-sensitive top2-13 allele (30Thomas W. Spell R.M. Ming M.E. Holm C. Genetics. 1991; 128: 703-716Crossref PubMed Google Scholar). One single colony of a strain JCW26 transformant was picked and suspended in 1 ml of water. Following serial dilution of the cell suspension, equal volume aliquots of the final dilutions were plated in duplicates. One set of plates was incubated at 25 °C and the other set at 35 °C for 72 h. Complementation test with strain CH834top2-13 (30Thomas W. Spell R.M. Ming M.E. Holm C. Genetics. 1991; 128: 703-716Crossref PubMed Google Scholar) was done similarly, but the two sets of plates were incubated at 25 and 13 °C. Overexpression of the plasmid-borne Top2-(1–1196)-HMK-(His)6 and its various alanine substitution derivatives was carried out in a protease-deficient yeast strain BCY123 (originally obtained from the laboratory of R. Kornberg, Stanford University) or JEL1 (31Lindsley J.E. Wang J.C. J. Biol. Chem. 1993; 268: 8096-8104Abstract Full Text PDF PubMed Google Scholar) according to the procedures previously described for overexpression of the full-length yeast enzyme (9Worland S.T. Wang J.C. J. Biol. Chem. 1989; 264: 4412-4416Abstract Full Text PDF PubMed Google Scholar). Single colonies of transformants were picked and inoculated in minimal medium lacking uracil and supplemented with 3% (v/v) glycerol, 2% (v/v) lactic acid, and 2% (w/v) glucose. Cultures were grown at 30 °C for 24 h to reach a cell density of 0.4–1.5 × 108. These were diluted by 100-fold into YPD medium (32Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 145Google Scholar) in 2-liter flasks, and growth of cells was resumed by shaking the flasks on an orbital shaker platform in a 30 °C incubator. When the cell density of a culture climbed back to 0.4–1.5 × 108(in about 24 h), galactose was added to a final concentration of 2% (w/v) to induce the expression of the plasmid-borne top2gene from the GAL1 promoter. Cells were harvested by centrifugation 8–9 h post-induction. The pelleted cells were used immediately for the next step or were flash-frozen with liquid N2 and stored at −70 °C until use. Freshly pelleted cells or cells thawed from the frozen pellets were resuspended in an equal volume of 1× Ni-resin binding buffer (20 mm Tris·HCl, pH 7.9, 500 mm NaCl, 5 mm imidazole, 1 mmphenymethylsulfonyl fluoride, and 1 μg/ml each of leupeptin, pepstatin A, and aprotinin) and vortexed vigorously with glass beads in 50-ml conical plastic tubes (9Worland S.T. Wang J.C. J. Biol. Chem. 1989; 264: 4412-4416Abstract Full Text PDF PubMed Google Scholar, 33Berger J.M. Structural Determination of a 92-kDa Fragment of Yeast Topoisomerase II by X-ray Crystallography at 2.7 Å ResolutionPh.D. thesis. Harvard University, Cambridge1995Google Scholar). Cell lysate was clarified by centrifugation and loaded on a Ni-resin column (Novagen, His·Bind Resin), following the manufacturer's protocol with minor modifications. The column was thoroughly washed with 1× binding buffer and then 1× binding buffer + 25 mm imidazole, and the hexahistidine-tagged protein was eluted by increasing the imidazole concentration to 1 m. The salt concentration in the protein eluate was reduced to 100 mm by the addition of 5 mm Tris·HCl, pH 7.7, 10 mm 2-mercaptoethanol, 10% glycerol (v/v), and 1 μg/ml each of leupeptin, pepstatin A, and aprotinin. The diluted protein was then concentrated by loading on a small heparin column (about 200 μl for 1 mg of protein) and eluting with the dilution buffer plus 45 mm Tris·HCl, pH 7.7, and 650 mm KCl. Drops were collected, and the peak fractions were pooled and mixed with equal volume of 80% (v/v) glycerol for storage at −70 °C. During the concentration step, some contaminant proteins were also removed. The yeast strain JEL1(Δtop1), a derivative of JEL1 constructed by targeted disruption of the TOP1 gene encoding DNA topoisomerase I, was transformed with both the R690A derivative of pTop2-(1–1196)-HMK-(His)6 and a second plasmid pJEL131-Y782F, which expresses full-length yeast DNA topoisomerase II with a Y782F mutation. The Y782F mutation in pJEL131-Y782F was constructed by site-directed mutagenesis of pJEL131, which was derived from YEpTOP2-PGAL1 by replacing the URA3 marker in it with aLEU2 marker (34Lindsley J.E. Wang J.C. Nature. 1993; 361: 749-750Crossref PubMed Scopus (57) Google Scholar). Induction of the GAL1 promoter-linked genes and the preparation of cell extracts were carried out as described above. Because of weaker binding of the heterodimeric protein to the Ni-resin, presumably owing to the presence of only one hexahistidine tag in a heterodimeric molecule, the procedure used in the purification of the other hexahistidine-tagged derivatives of yeast DNA topoisomerase II was slightly modified. After loading the protein on the Ni-resin, the column was thoroughly washed with only the 1× binding buffer before eluting the protein with the same buffer plus 1m imidazole. There are a large number of highly conserved amino acid residues in the type II DNA topoisomerases from a diverse collection of organisms ranging from T-even phage to human. In order to focus on residues that are likely to participate in the catalysis of DNA breakage and rejoining, we applied the same criteria used in a recent study of E. coli DNA topoisomerase I (35Chen S.-J. Wang J.C. J. Biol. Chem. 1998; 273: 6050-6056Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It is assumed that these residues are likely to possess polar side chains and that variations at a conserved position in any member of a particular subfamily of enzymes must preserve a common chemical group at that position (35Chen S.-J. Wang J.C. J. Biol. Chem. 1998; 273: 6050-6056Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The above considerations led to the construction of 10 mutants within the C-terminal or "GyrA" half of yeast DNA topoisomerase II: R690A, D697A, K700A, R704A, H735A, H736A, E738A, Q750A, R781A, and N828A. The positions of these residues in the 92-kDa yeast fragment structure are shown in Fig. 1 A, and the region containing these residues is shown in stereo in Fig.1 B. In both figures, the location of the known active site tyrosyl residue Tyr-782 is also shown, and a short double-stranded DNA with a four-nucleotide 5′-overhang is modeled into the putative DNA-binding site of the enzyme as described in Berger et al. (24Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Crossref PubMed Scopus (751) Google Scholar). As mentioned earlier, the binding of DNA to the site inferred from the crystal structure of the protein is supported by recent protein footprinting experiments (25Li W. Wang J.C. J. Biol. Chem. 1997; 272: 31190-31195Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To test for functionality of each mutant enzyme, each alanine substitution mutant of pTOP2-PGAL1, which expresses wild-type yeast DNA topoisomerase II from aGAL1 gene promoter, was used to transform a yeast strain with a temperature-sensitive mutation top2-4 in the chromosomal gene encoding DNA topoisomerase II (29Holm C. Goto T. Wang J.C. Botstein D. Cell. 1985; 41: 553-563Abstract Full Text PDF PubMed Scopus (574) Google Scholar). Transformants selected at 25 °C, a permissive temperature for thetop2-4 mutation, were tested for viability at 35 °C, a nonpermissive temperature for top2-4. Among the mutant plasmids constructed, those expressing R690A, D697A, K700A, R704A, R781A, and N828A failed to complement thetop2-4 temperature sensitivity, and those expressing H735A, H736A, E738A, and Q750A did. Residues that cannot be replaced by alanine without affecting the functionality of the enzyme are coloredred in Fig. 1, A and B, and those that can are colored blue in the same figures. These results indicate that the highly conserved polar side chains His-735, His-736, Glu-738, and Gln-750 are not essential in the reactions catalyzed by yeast DNA topoisomerase II. To test the possibility that a mutant might have failed the above complementation test because the mutation itself might have led to thermal sensitivity, the complementation test was repeated in a yeast strain carrying a cold-sensitive top2 mutationtop2-13 (30Thomas W. Spell R.M. Ming M.E. Holm C. Genetics. 1991; 128: 703-716Crossref PubMed Google Scholar). All mutants that were found to complement the thermal sensitive top2-4 mutation were also found to complement the cold-sensitive top2-13 mutation. Among those that failed to complement top2-4, N828A was found to complement top2-13, indicating that Asn-828 probably also lacks an essential role in catalysis. To characterize further mutant enzymes that failed one or both complementation tests, overexpression and purification of the mutant enzymes were carried out for biochemical studies. To facilitate the purification of the mutant enzymes, alanine substitutions were introduced into pGAL1Top2-(1–1196)-HMK-(His)6, a plasmid constructed for the overexpression of a C-terminal truncation of yeast DNA topoisomerase II with a hexahistidine tag (25Li W. Wang J.C. J. Biol. Chem. 1997; 272: 31190-31195Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In this plasmid, codons 1–1196 of yeast DNA topoisomerase II is fused to two short sequence motifs HMK and (His)6; the former encodes a heart muscle kinase phosphorylation site and the latter a string of six histidines. The entire open reading frame was placed under the control of the inducible promoter of the yeast GAL1 gene, and the presence of the hexahistidine tag at the C terminus of the desired polypeptide facilitates its purification upon induction. The HMK tag was previously introduced to permit radiolabeling of the polypeptide at its C terminus for protein footprinting and was of no consequence in the work reported here. In the absence of any mutation in the Top2-(1–1196) region, the fusion protein expressed by the plasmid-borne gene is known to be fully active in vitro (25Li W. Wang J.C. J. Biol. Chem. 1997; 272: 31190-31195Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,28Caron P.R. Watt P. Wang J.C. Mol. Cell. Biol. 1994; 14: 3197-3207Crossref PubMed Scopus (83) Google Scholar). To avoid cumbersome notations, in the biochemical experiments reported below the C-terminally tagged protein with amino acid residues 1–1196 of the wild-type yeast DNA topoisomerase II is simply referred to as the "wild-type" enzyme, and the same fusion protein with a mutation within residues 1–1196 of the yeast enzyme is referred to as a mutant enzyme. Fig. 2 depicts the results of a set of experiments designed and carried out based on preliminary assays of the various enzyme preparations. In each assay, a negatively supercoiled 3-kb plasmid was incubated with the wild-type enzyme or one of the mutant enzymes under standard assay conditions, and the reaction mixture was deproteinized for analysis by agarose gel electrophoresis. In the top margin above each lane of the gel slab, the molar ratio of enzyme to DNA (E/DNA) in the assay mixture analyzed is specified, and the presence or absence of ATP in the assay mixture is indicated by a plus or minus sign. In the incubation mixture without enzyme (leftmost lane), the majority of the DNA migrated at the position of the supercoiled form (bottom band), and a smaller amount of the DNA migrated as nicked DNA rings (upper band). For the set of four samples incubated with the wild-type enzyme (lanes below WT in Fig. 2), in the absence of ATP the electrophoretic pattern of the DNA remained unchanged from that of the no-enzyme control at a high E/DNA
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