포도상구균으로부터 DNA topoisomerase I 유전자의 클로닝 및 특성에 관한 연구

Abstract

Type I topoisomerase plays critical roles in DNA metabolism and cell survival. In this study, type I topoisomerase gene from Staphylococcus aureus sp. strain C-66 cells was cloned in pBAD /His A expression vector and expressed in E. coli Top 10 cells. The coding region of this gene was 2,070 nucleotides capable of encoding a polypeptide of 690 amino acids with a predicted molecular mass of 79.1 kDa. The recombinant plasmid named pTP I expressed active type I topoisomerase upon induction with 0.02% L-arabinose. The topoisomerase expressed from pTP I plasmid in E. coli was purified through an affinity chromatography on Hitrap chelating column. The topoisomerase activity of the purified enzyme was Mg2+-dependent and ATP-independent when supercoiled DNA was used as a substrate. The enzyme could relax only negatively supercoiled DNA, not positively supercoiled DNA. The optimal temperature and pH for the enzyme activity were 37℃ and 7.5, respectively. The activity of enzyme was significantly activated in the presence of 50 mM NaCl. The enzyme activity could be clearly inhibited by treatment with camptothecin, but not by nalidixic acid, etoposide, and spermidine. The enzyme made a single-stranded nick on negatively supercoiled DNA and the 5' end of the nick could covalently linked with the enzyme. All these results suggest that the purified enzyme is a typical type I DNA topoisomerase.