A DNA nicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties.

Abstract

Vaccinia virus cores contain an activity which is able to relax both left-and right-handed superhelical DNA. This virus-specific nicking-closing enzyme has been highly purified and differs from the corresponding host enzyme in salt optimum, in sedimentation coefficient, and in polypeptide composition as determined on sodium dodecyl sulfate/polyacrylamide gels. The enzyme is probably newly synthesized after the cessation of host protein synthesis which follows virus infection. The most highly purified preparation contains two polypeptides, one of molecular weight 24,000 and the other 35,000. The former polypeptide is a major constituent of the virus (7% of total protein by weight), whereas the latter is present in a much smaller amount (0.2%). Chromatography with denatured DNA-cellulose reveals that the activity is predominately associated with those fractions enriched in the polypeptide of greater molecular weight. Many fundamental biological processes are associated with changes in the duplex winding of DNA. These include DNA replication; transcription; recombination; formation of nucleosomes; packaging of DNA in organelles, such as mitochondria or chloroplasts; formation of complex prokaryotic chromosomes (1); and encapsidation into viruses. Enzymes from several sources have recently been discovered which are able to relieve torsional stress on the DNA duplex. None of these appears to require a cofactor, and their primary function is to introduce a transient local swivel that allows the DNA to rotate in response to a torque.The prokaryotic enzymes, termed omega proteins (2), have been purified from Escherichia coli 1100 (2, 3) and from E. coli B (4) and are able to catalyze duplex rotation in the righthanded (overwinding) direction only. The eukaryotic enzymes are able to catalyze rotation in both directions and have so far been discovered in several animal cell nuclei (5-9). In the present report we show that such an activity is encapsidated in mature vaccinia virions and is active in viral cores, which are able to carry out transcription as intact complexes. The activity is not identical to the corresponding host protein and is newly synthesized following infection. This result, representing a virus-specific nicking-closing enzymet, confirms the widespread distribution of this activity.The only presently available assay for nicking-closing activity is based upon the ability of the enzyme to remove superhelical turns from closed circular duplex DNA. Such DNA molecules are subject to the topological constraint (11) that the net interstrand winding, a, is a constant and is the sum of the number MATERIALS AND METHODS Reagents. Yeast alcohol dehydrogenase and horse heart cytochrome c were obtained from Worthington and Sigma, respectively. Ethidium bromide (EtdBr) was obtained from Calbiochem, and [35S]methionine was purchased from New England Nuclear. All other chemicals were of reagent grade.Preparation of Virus. Vaccinia virus strain WR, grown in HeLa S cells, was purified by the method of Joklik (14). ...

DNA-relaxing enzyme from Micrococcus luteus

“…Taking into account that the relaxation process apparently follows a multihit mechanism and that one PM2 DNA molecule has 100 superhelical turns (20)(21)(22), one enzyme molecule removed approximately 150 superhelical turns under standard conditions. Therefore, we conclude that the DNA-relaxing enzyme from Micrococcus luteus acts in a catalytic way as do other DNA-relaxing enzymes (5,6,11,13,23).…”

“…9b). DISCUSSION DNA-relaxing enzymes were first found in E.coli (1), subsequently in various eukaryotic cells (2,4,6,12,13) and recently in vaccinia virus (11).…”

“…The enzymes may be involved in any process requiring winding or unwinding of the double helix such as replication (8) or transcription (9). DNA-relaxing activities have been purified from Escherichia coli (1,10), vaccinia virus (11), Drosophila melanogaster (3), yeast (Thielmann and Hess, in preparation), calf thymus (12), rat liver (13), mouse embryo cells (4), HeLa (4) and KB cells (6). The enzymes of eukaryotic origin are distinguished from the w protein of E.coli in that they relaxe both negatively and positively twisted DNAs and they do not require Mg++ for activity (1)(2)(3)(4)6,12,13).…”

Association of Type I DNA Topoisomerase with Herpes Simplex Virus

“…In both prokaryotes and eukaryotes these enzymes appear ubiquitous and have been isolated from a variety of sources (Gellert, 1981;Liu, 1983). Topoisomerases have also been identified in vaccinia virus cores (Bauer et al, 1977) and simian virus 40 (SV40) minichromosomes (Tsubota et al, 1978).…”

Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein

“…An insoluble core protein fraction prepared from disrupted, radiolabelled VV (Bauer et al, 1977) was fi'actionated by electrophoresis on SDS-containing Prosieve agarose (FMC). The corresponding Coomassiestained 4a band was excised from the gel, melted at 75 °C in 7 vols of extract buffer (50mM-Tris, pH 7.8, 10%, w/v, SDS and 1 mM-EDTA), frozen in dry ice, and thawed at room temperature.…”