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Mapping the Active-Site Tyrosine of Vaccinia Virus DNA Topoisomerase I

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Mapping the Active-Site Tyrosine of Vaccinia Virus DNA Topoisomerase I Proc. Natl. Acad. Sci. USA Vol. 86, pp. 9793-9797, December 1989 Biochemistry Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I (covalent catalysis/tyrosyl phosphodiester linkage/yeast topoisomerase) STEWART SHUMAN*, EILEEN M. KANE, AND SCOTT G. MORHAM Program in Molecular Biology, Sloan-Kettering Institute, New York, NY 10021 Communicated by Paul A. Marks, September 27, 1989 ABSTRACT Site-directed mutagenesis of the vaccinia vi- we have taken advantage of the ability to express active rus gene encoding a type I DNA topoisomerase implicates vaccinia topoisomerase in a heterologous system, Esche- Tyr-274 as the active-site residue that forms a covalent adduct richia coli (29). By studying the effects of a series of Tyr with DNA during cycles of DNA-strand breakage and reunion. Phe point mutations in the topoisomerase gene on the cata- Replacement of Tyr-274 by phenylalanine results in loss of the lytic activity of the resulting mutant proteins, we deduced ability of the enzyme to relax negatively supercoiled DNA as that Tyr-274 constitutes the active-site tyrosine of vaccinia well as to form the covalent DNA-protein intermediate. Sub- topoisomerase. While our work was in progress, the labora- stitution of phenylalanine for tyrosine at nine other sites in the tories of Wang and Sternglanz reported the mapping of the protein has no apparent effect on enzyme activity. Amino acid active-site tyrosines of Saccharomyces cerevisiae and Sac- sequence alignment reveals Tyr-274 to be homologous to Tyr- charomyces pombe type I topoisomerases to Tyr-727 and 727 and Tyr-771, respectively, of the type I topoisomerases Tyr-771, respectively (10, 11). Sequence alignment shows from Saccharomyces cerevisiae and Saccharomyces pombe; that the active-site tyrosine residues are conserved in all Tyr-727 and Tyr-771 have been shown to represent the active- known eukaryotic type I topoisomerases. site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr com- mon to the viral and cellular type I topoisomerases, including MATERIALS AND METHODS the human enzyme. Site-Directed Mutagenesis. A DNA fragment containing the vaccinia topoisomerase gene was excised from the plasmid A mechanistic feature common to all known DNA topoisom- pvtopS (29) by cleavage with enzymes BamHI and HindIll erases is the formation of a covalent protein-DNA interme- and then subcloned into M13mpl8 to generate M13topo. diate during each cycle of DNA-strand breakage and reunion Uracil-substituted single-stranded phage DNA was isolated (1). Type II topoisomerases, both prokaryotic and eukary- from M13topo that had been grown in E. coli CJ236 (dut-, otic, bind covalently to the 5'-phosphoryl end of the cleaved ung ). Mutagenesis was performed by hybridizing to the DNA strand (2-4). The same is true of the prokaryotic type uracit-substituted DNA various mutagenic oligonucleotides; I DNA topoisomerases (2). In contrast, eukaryotic type I these oligonucleotides, each 21 nucleotides long, contained at topoisomerases form a 3'-phosphoryl linkage to DNA (5). In position 11 a single base change (relative to the wild-type each instance, DNA is bound to protein by means of a topoisomerase gene) that converted a tyrosine codon (either phosphodiester bond to a tyrosyl residue (2-6). TAT or TAC) to a phenylalanine codon (either TTT or TTC). Interest in the structure of the enzyme-DNA intermediate The primed circles were then converted to replicative form has been fueled by the realization that numerous antimicro- by the action of the DNA polymerase III system and DNA bial and antitumor agents of clinical import act by stabilizing ligase purified from E. coli (provided by K. Marians and R. or trapping the enzyme in its DNA-bound state (the so-called Digate, Sloan-Kettering Institute). Phage plaques arising cleavable complex) (7). Initial studies have focused on the from transformation ofthe replicative form into E. coli JM105 identification of the tyrosine residue(s) to which DNA is were amplified. Single-strand DNA was isolated from phage attached (8-11). This identification has been facilitated by the supernatants, and the presence of the oligonucleotide- cloning, sequencing, and expression of the genes encoding directed mutation was assayed by dideoxynucleotide se- topoisomerases I and II from prokaryotic and eukaryotic quencing. Typically, 50-90% of phage isolates contained the sources (12-26). desired mutation. Replicative-form DNA isolated from Our report addresses the identity ofthe active-site tyrosine phage-infected cells was cleaved with nucleases BamHI and of the vaccinia virus DNA topoisomerase, an Mr 32,000 type HindIII, and the resulting topo-containing fragment was I enzyme that is encapsidated within the infectious poxvirus subcloned into pUC19 to generate a series of mutant con- particle (17, 27, 28). Vaccinia topoisomerase, like eukaryotic structs designated pUCtopo-phe. This series of plasmids was cellular type I enzymes, forms a covalent bond to the digested with nucleases Nde I and Bgl II; the DNA fragments 3'-phosphoryl group of a cleaved DNA strand (28, 29). The containing the topoisomerase gene were cloned into the linkage of DNA to vaccinia topoisomerase is stable to acid, T7-based expression plasmid pET3c (30) that had been alkali, and hydroxylamine (S.S., unpublished observations), cleaved with Nde I and BamHI, thereby generating the series properties consistent with the formation of a phosphotyrosyl of mutant plasmids, pETtopo-phe. A plasmid containing the bond. The amino acid sequence of the vaccinia enzyme, wild-type topoisomerase gene (designated pETtopo-wt) was deduced from the nucleotide sequence of the vaccinia gene constructed by cloning an Nde I/Bgl II fragment of pvtopS that encodes topoisomerase, includes 14 tyrosine residues into pET3c. Each pETtopo plasmid was used to transform E. (out of 314 amino acids). To map the active-site tyrosine(s), coli BL21. Topoisomerase Expression. E. coli BL21 transformants vaccinia The publication costs of this article were defrayed in part by page charge carrying pETtopo plasmids were induced to express payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9793 Downloaded by guest on September 27, 2021 9794 Biochemistry: Shuman et al. Proc. Natl. Acad. Sci. USA 86 (1989) topoisomerase by infection with bacteriophage ACE6 (con- tide shown previously to be vaccinia topoisomerase (29). A taining the T7 RNA polymerase gene) as described (29). polypeptide with identical mobility was evident in cells Bacteria (from 50 ml of culture) were harvested by centrif- bearing the pETtopo-phe plasmids (Fig. 1). The appearance ugation 4 hr postinfection, then stored frozen at -80'C. of the topoisomerase polypeptide was absolutely dependent Soluble cell lysates were prepared as described (29) and in each case on infection with ACE6 (data not shown). stored at -80'C. Wild-type and mutant proteins accumulated to similar levels Assay of Vaccinia Topoisomerase. Soluble bacterial lysates in both the soluble fraction (Fig. 1) and in whole cell lysates were screened for EDTA-resistant DNA relaxing activity as (data not shown). No polypeptides of Mr < 33,000 were described (29). Formation of a covalent complex (cleavable induced by bacteriophage ACE6 infection. Thus, the intro- complex) of vaccinia topoisomerase with radiolabeled nick- duction of Tyr -* Phe point mutations had no significant translated A DNA was assayed as described (28). deleterious effect on the expression, solubility, or stability of Materials. Sequenase and other DNA sequencing reagents the vaccinia topoisomerase in E. coli. were obtained from United States Biochemical. Reagents for Effect of Tyr -* Phe Mutations on Topoisomerase Activity. nick translation were purchased from Bethesda Research Topoisomerase activity in E. coli lysates was assayed by the Laboratories. Radionucleotides were from Amersham. relaxation of negatively supercoiled plasmid DNA in the presence of EDTA-i.e., under conditions that limit DNA RESULTS relaxation to that catalyzed by the vaccinia enzyme. As shown in Fig. 2, the soluble protein fraction from cells Expression of Mutated Vaccinia Topoisomerase Genes. A expressing wild-type topoisomerase relaxed the supercoiled series of 10 mutant alleles ofthe vaccinia topoisomerase gene DNA to completion. Extracts of cells expressing topoisom- (each resulting in a specific Tyr -* Phe change at the protein erases that contained phenylalanine in lieu of tyrosine at level) were generated by oligonucleotide-directed mutagen- positions 6, 28, 46, 70, 72, 209, 225, 229, or 233 also relaxed esis and then cloned into the T7-based expression vector the DNA fully. In contrast, an equivalent amount of extract pET3c (described in Materials and Methods). E. coli bearing from cells expressing topoisomerase mutated to phenylala- either wild-type or mutant pETtopo plasmids were induced to nine at position 274 showed no appreciable topoisomerase express the cloned gene by infection of the bacteria with activity. A more thorough analysis of topoisomerase activity bacteriophage ACE6 (29, 31). Lysates of ACE6-infected cells is shown in Fig. 3. Wild-type extract catalyzed extensive were prepared, and soluble polypeptides were analyzed by DNA relaxation, even when diluted 1000-fold, whereas the SDS/PAGE. As shown in Fig. 1, lane WT, cells bearing Phe-274 mutant extract failed to relax the plasmid DNA at pETtopo-wt accumulated high levels of a Mr 33,000 polypep- any level of dilution. Thus, within the limitations of this assay, it is concluded that the effect of the amino acid uLt) au coC- N a cm co co cO N C) N substitution at position 274 is to reduce the specific activity CvJ N N N co 'zt NN N N by 1000-fold or more, if not to wholly inactivate the enzyme. Similar analyses of the remaining nine mutant topoisomerase .4- extracts revealed little difference in activity of the mutants 97 compared to wild-type extract (data not shown).
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