Hirosuke Fukasawa

Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences.

SummaryType 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.

Cauliflower RNA polymerase: Partial purification and preliminary characterization of DNA-dependent enzymes

Abstract DNA-dependent RNA polymerase was easily solubilized from the apical part of cauliflower inflorescences into homogenizing medium containing ammonium sulfate. A distinct RNA polymeraseshave been isolated by DEAE-cellulose column chromatography. DEAE-polymerase II was more effectively primed when denatureded calf-thymus DNA, rather than native DNA, was used as a template. Cauliflower DNA extracted from the inflorescence was more effectively utilized when cauliflower RNA polymerase, rather than calf-thymus DNA, was used as a template for RNA synthesis.

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase.

Nucleosome-like structures have been efficiently assembled in vitro by interaction of cauliflower histones, pBR322 DNA and cauliflower DNA topoisomerase, as assayed by supercoiling of relaxed circular DNA and by digestion with micrococcal nuclease. The optimum ionic strength for supercoiling was 150 mM KCl and the optimum weight ratio of histone to DNA was approximately 1.0. Four histones, H2A, H2B, H3 and H4, were necessary for the optimum assembling conditions, and the nucleosomes assembled protected DNA fragments of approximately 150 bp in length. It was found that cauliflower DNA topoisomerase acts not only as a DNA-relaxing enzyme but also as a chaperon factor for nucleosome assembly.

Template-specific inhibitor for DNA polymerase isolated from cauliflower inflorescence

A novel inhibitory factor which greatly inhibits DNA polymerase activity was isolated from the apical portion of the cauliflower inflorescence during purification of DNA polymerases. It can be adsorbed on a DEAE-cellulose column, but not on CM-Sephadex or DNA-cellulose. The factor exclusively inhibits the incorporation of [3H]dTTP into DNA when poly(rA, dT10) is used as the template primer, but not when activated DNA, heat-denatured DNA or native DNA is used as a template. The concentration of the factor in the reaction medium required for 50% inhibition is approx. 8 microgram/ml. The factor is heat-stable, is inactivated by trypsin, and has a maximum ultraviolet absorption at 278 nm. The molecular weight was estimated as 2500-3000 by Sephadex gel chromatography.

Characterization of an α-amanitin insensitive RNA polymerase from cauliflower

Abstract α-Amanitin insensitive RNA polymerase (polymerase I isolated from apical parts of the cauliflower inflorescence was highly stable for several months at − 18°. The DEAE-cellulose fraction was more effective in utilizing denatured DNA than native DNA as a template. Optimum pH for RNA synthesis was ca 7 in the reaction mixture with Tris-HCl or with Tris-maleate buffer. From the properties examined, it seems that DNA-dependent RNA polymerase I of cauliflower differs from other eucaryotic RNA polymerases.