PURIFICATION AND CHARACTERIZATION OF DNA TOPOISOMERASE-V - AN ENZYME FROM THE HYPERTHERMOPHILIC PROKARYOTE METHANOPYRUS-KANDLERI THAT RESEMBLES EUKARYOTIC TOPOISOMERASE-I

DNA topoisomerase V is a novel prokaryotic enzyme related to eukaryoti c topoisomerase I. The enzyme is a type I DNA topoisomerase and is rec ognized by polyclonal antibody against human topoisomerase I. We descr ibe its purification from the hyperthermophilic methanogen Methanopyru s kandleri. The enzyme has high activity in crude extracts and is pres ent in at least 1,500 copies/cell. Topoisomerase V migrates as a 110-k Da polypeptide in SDS-polyacrylamide gel electrophoresis and as a 142- kDa globular protein in gel filtration. It is active up to at least 10 0-degrees-C on both positively and negatively supercoiled DNA and is n ot inhibited by single-stranded DNA. The enzyme works from 1 to 650 mm NaCl and up to 3.1 m potassium glutamate. It acts processively at low ionic strength and distributively at high NaCl or KCl concentration. Magnesium is not required and does not stimulate the enzymatic activit y. Under DNA denaturing conditions, topoisomerase V catalyzes an unlin king reaction which results in substantial reduction in the linking nu mber of closed circular DNA. The driving force for this process is DNA melting. Camptothecin is not nearly as good an inhibitor for topoisom erase V as it is for eukaryotic topoisomerase 1. The unique occurrence of two major type I topoisomerases (reverse gyrase and topoisomerase V) in M. kandleri may shed new light on the evolution of this family o f enzymes and supports the concept of a distant but significant relati onship between some hyperthermophilic organisms and eukaryotes.