Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase

Abstract

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.

Plant DNA Topoisomerases: Structure, Function, and Cellular Roles in Plant Development

“…The plant topoisomerases have been purified either partially or to apparent homogeneity from wheat germ (Dynan et al, 1981), chloroplasts (Pyke et al, 1989), and mitochondria (Echeverria et al, 1986(Echeverria et al, , 1991; cauliflower (Fukata and Fukasawa, 1982;Fukata et al, 1986Fukata et al, , 1989Fukata et al, , 1991; cultured tobacco cells (Heath-Pagliuso et al, 1990;Cole et al, 1992); Pisum sativum root (Chiatante et al, 1991(Chiatante et al, , 1993Chiatante and Bryant, 1994), shoot (Rudenko, 1992a(Rudenko, , 1992bLevi et al, 1994), and chloroplasts (Lam and Chua 1987;Neilsen and Tewari 1988;Mukherjee et al, 1994); spinach (Siedlecki et al, 1983), carrot (Carbonera et al, 1988(Carbonera et al, , 1990(Carbonera et al, , 1995; Brassica oleracea (Kieber et al, 1992a); rice (Agris et al, 1990;Yoshida et al, 1991); Downloaded by [Ryerson University Library] at 09:46 05 June 2016 FIG. 5.…”