CLOSING, EXPRESSION, PURIFICATION AND CHARACTERIZATION OF DNA TOPOISOMERASE-I OF MYCOBACTERIUM-TUBERCULOSIS

The complete gene encoding Topoisomerase 1 (Topo I) from Mycobacterium tuberculosis (MTb), Erdman strain, has been isolated and sequenced. T he coding region of this gene is 2700 nt encoding a polypeptide of 900 amino acids with a calculated molecular mass of 99 353 Da. The amino- acid sequence identity compared to E. coli and Synechococcus Topo I is 22 and 30%, respectively. The gene was expressed in E. coli BL21(DE3) and purified to near homogeneity. Recombinant MTb Topo I is enzymatic ally active, relaxing negatively supercoiled DNA in a magnesium-depend ent, ATP-independent reaction. Spermidine, a typical inhibitor of prok aryotic type I DNA topoisomerase, inhibits the activity. Unlike the mo re well-characterized E. coli Topo I, MTb Topo I does not contain a zi nc-finger DNA-binding motif in the C-terminal domain of the protein.