Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences

Abstract

Type II DNA topoisomerase has been isolated from inflorescences of cauliflower (Brassica oleracea var. botrytis) through a sequence of polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex, hydroxyapatite and phosphocellulose. The molecular weight of the native enzyme, based on sedimentation coefficient (9S) and gel filtration analysis (Stokes radius, 60 Å), was estimated to be 223 000. This enzyme was able to catalyze fully the relaxation of supercoiled DNA by breaking and then rejoining the double-stranded DNA. The breaking reaction was reversible by a change in salt concentrations. When an antitumor drug, 4'-(9-acridinylamino)-methanesulfon-m-anisidide, was added to the topoisomerase reaction, DNA cleavage fragments were accumulated; and this suggested that the drug interfered with the reaction at the rejoining step. This enzyme also catalyzed the formation of DNA catenanes in the presence of 8% polyethylene glycol or histone H1, while few catenanes were formed in the presence of spermidine, which was highly effective on a bacterial enzyme.

Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays

“…As mentioned above, VM-26 induces the appearance of the linear form of pBR322 in the relaxation of supercoiled DNA reaction in the presence of ATP suggesting its capacity to interfere with the breakage-reunion reaction of topoisomerase II in embryonic nuclear extracts, but it does not interfere with the relaxing topoisomerase I activity. A similar result has been obtained with the intercalative antitumor drug acridine m-AM SA with purified topoisomerase II from cauliflower [ 13]. When VM-26 was tested in the P4 DNA unknotting assay by embryonic nuclear extracts, inhibition was also obtained.…”

“…The catalytic activity of topoisomerase I has been reported in different plant systems such as wheat embryo mitochondria [11], carrot and spinach cells [30], pea chloroplast [19,24] and the enzyme has been isolated from wheat germ [ 10 ]. Topoisomerase II activity has been reported in cauliflower inflorescences [13] and gyrase activity has been found in extracts from pea chloroplasts [ 19]. Here we report the characterization of topoisomerase activity in maize embryonic nuclear extracts.…”

Assessment of the genotoxicity of quinolone and fluoroquinolones contaminated soil with the Vicia faba micronucleus test

“…In addition, these compounds induced a variety of genotoxic effect due to their ability to inhibit topoisomerase II activities, including induction of transient DNA strand breaks during replication, chromosome condensation and disjunction during meiosis (Ferguson and Baguley, 1994;Heisig, 2009). In plants, topoisomerase II exists, and exerts the same role in DNA replication and cell proliferation (Fukata et al, 1986;Reddy et al, 1999).…”

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase

“…Purification and assay of DNA topoisomerases DNA topoisomerase II was isolated from cauliflower influorescence and purified as described previously [8] using AF-blue Toyopearl 650ML column chromatography instead ofphosphocellulose column chromatography in order to obtain the enzyme preparation with higher specific activity. Cauliflower topoisomerase Ia and Ib were purified after the same procedure as topoisomerase II.…”

“…Consequently, topoisomerases are involved intrinsically in DNA replication and transcription processes [10,27]. We have isolated and characterized two types of DNA topoisomerase from cauliflower inflorescence [6][7][8][9]. In this paper, we report on the ability of cauliflower topoisomerase to mediate nucleosome assembly, and provide evidence that nucleosome can be assembled in vitro by incubation of DNA, histones and topoisomerase, and no other assembly factors.…”