DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.

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.

Properties of two DNA topoisomerases isolated from cauliflower inflorescence were further investigated using negatively supercoiled pBR322 DNA and its relaxed form as the substrates.It was revealed that the ATPindependent enzyme (topoisomerase-I) has the capacity to relax negatively or positively supercoiled DNA at the presence or absence of ATP. ATPdependent enzyme (topoisomerase-II) also removes supercoils from negatively or positively supercoiled DNA at the presence of ATP, but its reaction products are found to have some different electrophoretic behaviour from the products by topoisomerase-I.Prepared substrates with a unique linking number was catalyzed with topoisomerase-I or -II, and electrophoresed, indicating that simultaneous breaking and rejoining of DNA strands by topoisomerase-I and topoisomerase-II take place on a single-strand of the duplex DNA and on doublestrands, respectively.

The activity and the processivity in DNA relaxation of DNA topoisomerase II, isolated from cauliflower inflorescence, were examined with various ratios of KCl and MgC12 concentrations in the reaction medium. The optimum concentrations for the relaxation were found to be 60 mM for KCl and 5 mM for MgC12 in the same reaction mixture regarding the other elements. The reaction proceeds in a processive manner at low salt concentration, and in a distributive manner at high concentration, in which MgC12 exhibits higher efficiency (five-fold) than KCI.DNA topoisomerases catalyze concerted breaking and rejoining of DNA backbone bonds, and remove superhelical turns in closed circular DNA (Wang and Liu 1979). In a previous article on the characterization of DNA topoisomerase isolated from cauliflower inflorescence, we reported that the optimum salt concentration for the type II enzyme activity was 50-100 mM for KCl

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