A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology by altering the linkage of DNA strands, solving topological problems caused by cellular processes such as DNA replication, transcription, or recombination (1, 2). These enzymes are classified on the basis of the number of DNA strands that they cleave and the covalent bond formed in the enzyme-DNA intermediate. Unlike type II DNA topoisomerases, type I enzymes are ATP-independent, which transiently break a single strand of DNA. Type I DNA topoisomerases are classified into two subfamilies: type IA and type IB. The enzymes of type IA subfamily, including bacterial DNA topoisomerase I and III, eukaryotic DNA topoisomerase III, and reverse gyrase (3, 4), form a tyrosyl linkage with a 5Ј-phosphate group of one of the DNA strands generated due to the enzyme action (2), whereas the enzymes of type IB subfamily, including eukaryotic and vaccinia virus DNA topoisomerases I (5) and DNA topoisomerase V, establish the tyrosyl bond with the 3Ј-phosphate group (2). Type 1A topoisomerases relax only negatively supercoiled DNA with Mg 2ϩ requirement, whereas type IB topoisomerases relax both negatively and positively supercoiled DNA even in the absence of a metallic cofactor, although Mg 2ϩ and Ca 2ϩ stimulate the relaxation activity (6, 7).Type IB DNA topoisomerases are monomeric enzymes, constituted by four domains (8, 9). The nonconserved amino-terminal domain contains putative signals for nuclear localization of the enzyme. The largest domain, the core, is essential for enzyme activity and shows high phylogenetic conservation, particularly in the residues closely interacting with DNA. The third domain is known as the linker, which is poorly conserved and highly variable in length and is not essential for the enzyme activity. Finally, the carboxyl-terminal domain is highly conserved and crucial for the catalytic activity. This domain contains a tyrosine residue (Tyr 723 in the human topoisomerase I), which interacts with one of the DNA strands, creating a transient covalent phosphodiester bond between the enzyme and the DNA.A type I DNA topoisomerase has been purified and characterized from Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis (10). Topoisomerases have...