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Intradimerically Tethered DNA Topoisomerase II Is Catalytically Active in DNA Transport (Leucine Zipper/Cross-Linking/DNA Gyrase) JANET E

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Intradimerically Tethered DNA Topoisomerase II Is Catalytically Active in DNA Transport (Leucine Zipper/Cross-Linking/DNA Gyrase) JANET E Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2975-2980, April 1996 Biochemistry Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport (leucine zipper/cross-linking/DNA gyrase) JANET E. LINDSLEY Department of Biochemistry, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132 Communicated by Nicholas R. Cozzarelli, University of California, Berkeley, CA, December 18, 1995 (receivedfor review August 14, 1995) ABSTRACT A covalently cross-linked dimer ofyeast DNA Two general mechanisms, often termed the "one-gate" and topoisomerase II was created by fusing the enzyme with the "two-gate" models, have been proposed for the functioning of GCN4 leucine zipper followed by two glycines and a cysteine. topoisomerase II (3, 19-22). It is proposed in the one-gate Upon oxidation ofthe chimeric protein, a disulfide bond forms model that all DNA substrates and products enter and exit between the two carboxyl termini, covalently and intradimeri- from the same face of the enzyme. In the two-gate model, the cally cross-linking the two protomers. In addition, all nine of transported duplex enters one face of the enzyme, is passed the cysteines naturally occurring in topoisomerase II have through the cleaved DNA duplex, and exits from the opposite been changed to alanines in this construct. This cross-linked, face. Results of an elegant, although indirect, experiment by cysteine-less topoisomerase II is catalytically active in DNA Roca and Wang (20) support a two-gate mechanism. duplex passage as indicated by ATP-dependent DNA supercoil To further understand the interactions between the two relaxation and kinetoplast DNA decatenation assays. How- halves of topoisomerase II we designed a derivative of the yeast ever, these experiments do not directly distinguish between a enzyme in which the two protomers of the dimer can be "one-gate" and a "two-gate" mechanism for the enzyme. efficiently and covalently tethered. In addition, all of the cysteines in this topoisomerase II derivative have been changed Type II DNA topoisomerases are ubiquitous enzymes that to alanines. As described in this communication, purified type catalyze the ATP-dependent transport of one segment of II DNA topoisomerase does not require any of its cysteines for duplex DNA through an enzyme-mediated transient break in activity, and it remains catalytically active when its carboxyl a second DNA duplex (1-4). These enzymes participate in termini are covalently linked. many DNA metabolic pathways, including the segregation of newly replicated DNA molecules (5, 6) and the condensation MATERIALS AND METHODS of chromosomes in mitosis and meiosis (7, 8). They are also the targets of a diverse group of antibiotics and anticancer agents Materials. Standard reagents were purchased from com- (9, 10). mercial sources as described below: ATP, Pharmacia; dithio- Type II DNA topoisomerases from organisms as diverse as threitol, Boehringer Mannheim; leupeptin, pepstatin, benz- bacteriophages, prokaryotes, and eukaryotes are structurally amidine, and diamide, Sigma; pBluescript, Stratagene; goat and mechanistically related (11, 12). They are all dyadic, anti-mouse and goat anti-rabbit IgG-horseradish peroxidase existing in their active conformations as homodimers, A2B2 conjugates and SDS/PAGE molecular-weight standards, Bio- tetramers, or A2B2C2 hexamers. Each identical half possesses Rad. S. cerevisiae DNA topoisomerase II rabbit polyclonal two distinct catalytic active sites: one for ATP binding and antibodies and anti-Ha mouse monoclonal antibodies (12CA5) hydrolysis and the second for DNA cleavage and religation. were from James C. Wang (Harvard University). Kinetoplast The latter functions by forming a transient covalent interme- DNA was purified from Crithidia fasciculata as described (23). diate consisting of an active-site tyrosine and a 5' phosphoryl DNA oligonucleotides used for construction of topoisomerase end of the severed DNA. expression vectors, site-directed mutagenesis, and sequencing Purified topoisomerase II from the budding yeast Saccha- were made at the DNA Peptide Synthesis Facility, Huntsman romyces cerevisiae has been used to study how the ATPase and Cancer Institute, University of Utah (National Institutes of DNA cleavage/religation active sites from the two halves of Health grant CA 42014). the homodimer coordinate their activities to catalyze the Construction of Expression Vectors. All vectors used for the transport of one DNA duplex through another. The enzyme expression of altered S. cerevisiae DNA topoisomerase II are can hydrolyze ATP in the absence of DNA, and cleave/religate derivatives of the plasmid YEpTOP2-PGAL1 (24). The codons DNA in the absence of ATP (13, 14). However, the rate of for all nine of the naturally occurring cysteines were mutated to ATP hydrolysis is stimulated 20-fold by DNA, the DNA alanine codons byoligonucleotide-directed mutagenesisusing the cleavage/religation equilibrium is perturbed by the presence of Altered Sites II kit (Promega); construction of this plasmid ATP and, most importantly, the passage of one DNA through (pDAT10) will be described in detail elsewhere. Derivatives of a transient break in another only occurs in the presence of ATP pDAT10 in which the last 95 codons of the TOP2 gene were (15, 16). When the topoisomerase binds ATP, it undergoes a replaced with codons for either the Ha epitope from the influenza major conformational change such that the enzyme topolog- hemagglutinin protein (25) (pJEL205) or the yeast GCN4 leucine ically clamps down around DNA (17). By studying a prepara- zipper (26) (pJEL203), were constructed by standard recombi- tion of topoisomerase II in which one subunit was immuno- nant DNA methodology. The exact peptide sequences following tagged and defective in ATP binding and the other was wild Ala-1334 of topoisomerase II for the expressed proteins are as type, it was shown that ATP binding to one protomer can follows: Ala-Arg-Gly-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala for induce a concerted conformational change in the entire en- topoII(C->A)Ha, and Ala-Arg-Gly-Gly-Gly-Arg-Met-Lys-Gln- zyme (18). Leu-Glu-Asp-Lys-Val-Glu-Glu-Leu-Leu-Ser-Lys-Asn-Tyr-His- Leu-Glu-Asn-Glu-Val-Ala-Arg-Leu-Lys-Lys-Leu-Val-Gly-Glu- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: G segment, gated segment; T segment, transported accordance with 18 U.S.C. §1734 solely to indicate this fact. segment. Downloaded by guest on September 24, 2021 2975 2976 Biochemistry: Lindsley Proc. Natl. Acad. Sci. USA 93 (1996) Arg-Gly-Gly-Cys for topoII(C->A)zipGGC; the boldface letters RESULTS represent the amino acids making up the Ha epitope and the the GCN4 leucine Design of the Tether. Determining the effect of linking zipper, respectively. two protomers of a topoisomerase II dimer together will help Expression, Purification, and Oxidative Cross-Linking of An effective cross-link Proteins. The vectors pJEL203 and pJEL205 were trans- define the mechanism of this enzyme. strain BCY123 would (i) be specific, (ii) form efficiently and (iii) be flexible formed into the protease-deficient yeast (27) enough for topoisomerase II to undergo its normal cycle of and expressed as described (24). Using these expression con- conformational changes. To achieve these goals, a chimeric ditions, <1% of the topoisomerase II purified comes from the protein consisting of the first 1334 amino acids of the yeast chromosomal copy of the wild-type TOP2 gene; the remainder topoisomerase II and the last 33 amino acids of the yeast of protein is from the plasmid-borne gene (16, 27). The with two and a were as described transcription factor GCN4, ending glycines topoisomerase derivatives purified (24), cysteine was designed (see Fig. 1A). The carboxyl-terminal 33 except that: (i) the yeast cells were cracked in the presence of amino acids of GCN4 dimerize byforming a leucine zipper; the leupeptin and pepstatin at 1 gtg/ml, 10 mM dithiothreitol, 1 dimerization of this peptide has been extensively studied (for mM benzamidine and the tubes of cells were first flushed with review, see ref. 32). Rapid formation of intradimeric disulfide nitrogen gas, and (ii) all buffers were thoroughly degassed and, bonds occurs between these peptides engineered with Gly- except for the final dialysis buffer, contained 3 mM dithio- Gly-Cys at the carboxyl termini (26). Therefore, if the two threitol. The purified protein (1 ml) was dialyzed against 2 carboxyl terminal GCN4 sequences of the dimeric topoisomer- liters of dialysis buffer (50 mM Tris-HCl, pH 8.0/200 mM ase fusion protein form a leucine zipper, oxidation should NaCl/10% glycerol) by stirring on ice in a nitrogen-filled glove produce a specific, disulfide-bonded cross-link. bag. Tubes containing aliquots of the protein were flushed For a separate series of experiments to be described else- with nitrogen gas before storage at -70°C. Protein concen- where, all nine cysteines in the yeast topoisomerase II were trations were determined with Coomassie Plus protein assay changed to alanines [(topoII(C->A)] as shown in Fig. 1A. reagent (Bio-Rad) and are reported as dimer concentrations. Although four of the cysteines are conserved among several Oxidative cross-linking of topoII(C->A)zipGGC by disulfide eukaryotic type II DNA topoisomerases, none are completely bond formation between the terminal cysteines was induced by conserved between the prokaryotic DNA gyrases and yeast the addition of diamide (1 mM final) (28). topoisomerase II (11, 12). Therefore it was reasoned that they Topoisomerase Activity Assays. Topoisomerase II deriva- may not be essential for protein stability or catalytic activity. tives were diluted to twice their final reaction concentration in In fact, topoII(C-*A) is at least half as active as the wild-type dialysis buffer. An equal volume of 100 nM pBluescript KS+ enzyme in supercoil relaxation and kinetoplast DNA decat- (plasmid concentration) or kinetoplast DNA at 66 ,tg/ml in enation (R.
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