Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion.Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe;Tyr-727 and Tyr-771 have been shown to represent the activesite tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.A mechanistic feature common to all known DNA topoisomerases is the formation of a covalent protein-DNA intermediate during each cycle of DNA-strand breakage and reunion (1). Type II topoisomerases, both prokaryotic and eukaryotic, bind covalently to the 5'-phosphoryl end of the cleaved DNA strand (2-4). The same is true of the prokaryotic type I DNA topoisomerases (2). In contrast, eukaryotic type I topoisomerases form a 3'-phosphoryl linkage to DNA (5). In each instance, DNA is bound to protein by means of a phosphodiester bond to a tyrosyl residue (2-6).Interest in the structure of the enzyme-DNA intermediate has been fueled by the realization that numerous antimicrobial and antitumor agents of clinical import act by stabilizing or trapping the enzyme in its DNA-bound state (the so-called cleavable complex) (7). Initial studies have focused on the identification of the tyrosine residue(s) to which DNA is attached (8-11). This identification has been facilitated by the cloning, sequencing, and expression of the genes encoding topoisomerases I and II from prokaryotic and eukaryotic sources (12-26).Our report addresses the identity of the active-site tyrosine of the vaccinia virus DNA topoisomerase, an Mr 32,000 type I enzyme that is encapsidated within the infectious poxvirus particle (17,27,28). Vaccinia topoisomerase, like eukaryotic cellular type I enzymes, forms a covalent bond to the 3'-phosphoryl group of a cleaved DNA strand (28,29). The linkage of DNA to vaccinia topoisomerase is stable to acid, alkali, and hydroxylamine (S.S., unpublished observations), properties consistent with the formation of a phosphotyrosyl bond. The amino acid sequence of the vaccinia enzyme, deduced from the nucleotide sequence of the vaccinia gene that encodes topoisomerase, includes 14 tyrosine residues (out of 314 amino acids). To map the active-site tyrosine(s), we have taken advantage of the ability to express active vaccinia topoisomerase in a heterologous system, Escherichia coli (29). By studying the effects of a series of Tyr Phe point mutations in the topoisomerase...
1983) has been mapped by marker rescue to the 17L open reading frame located within the genomic HindIll I DNA fragment. The 17 gene encodes a 423-amino-acid polypeptide. Thermolabile growth was attributed to an amino acid substitution, Pro-344-*Leu, in the predicted 17 protein. A normal temporal pattern of viral protein synthesis was elicited in cells infected with tsl6 at the nonpermissive temperature (40°C). Electron microscopy revealed a defect in virion assembly at 40°C. Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles. Western immunoblot analysis with antiserum directed against the 17 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection. Immunoblotting of extracts of wild-type virions showed that the 17 protein is encapsiWated within the virus core. The 17 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae.
Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (tsl, ts3l, ts55, and ts58) comprising a single complementation group (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) have been mapped by marker rescue to the H4L open reading frame located within the genomic HindIH-H DNA fragment. The H4 gene is predicted to encode a 93.6-kDa polypeptide expressed at late times during infection. Nucleotide sequence alterations responsible for thermolabile growth lead to amino acid
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