In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells

Abstract

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.

Cloning and characterization of a cell cycle-regulated gene encoding Topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid

“…In animal systems Top I is phosphorylated at serine residue(s) by both PKC (Pommier et al 1990) and CKII (Kordiyak et al 1994) and phosphorylation affects the enzyme activity. In the present study, in vitro phosphorylation did not influence the Top I activity; this also holds for a Top I purified from cultured tobacco cells (Cole et al 1992). It is possible that phosphorylation of Top I may have some other function in plants.…”

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

“…Alkaline phosphatase treatment of nuclear extracts abolished topoisomerase II activity in Zea mays (Carballo et al, 1991). The purified topoisomerase I from cultured tobacco cells were marginally affected by phosphorylation or dephosphorylation with regard to DNA relaxation activity (Cole et al, 1992). A 45-kDa putative topoisomerase I from pea root meristem was shown to be phosphorylated in vitro (Chiatante and Bryant, 1994).…”

“…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.…”