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...
DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p⅐DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1⌬, rad52⌬ yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug⅐enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p. DNA topoisomerase I (Top1p)1 constitutes the cellular target of the antineoplastic agent camptothecin (CPT) (reviewed in Refs. 1, 2). Top1p catalyzes the relaxation of supercoiled DNA by transiently cleaving a single strand of duplex DNA. This is accomplished by the nucleophilic attack of the enzyme's active site tyrosine on a phosphodiester bond in the DNA backbone. Recent structural studies suggest that the covalent linkage of enzyme to the 3Ј-phosphoryl end of the nicked DNA allows for the rotation of the noncovalently held DNA end to rewind/ unwind the DNA (3-5). A second transesterification resolves the phosphotyrosyl linkage via religation of the nicked DNA.The reversible stabilization of the covalent enzyme⅐DNA intermediate by CPT produces irreversible DNA lesions during S-phase, as a result of collisions between advancing replication forks and drug⅐Top1p⅐DNA complexes. In yeast, deletion of the gene encoding DNA topoisomerase I (TOP1) abolishes the cytotoxic action of the drug (reviewed in Refs. 2, 6). Although CPT sensitivity is restored by the expression of plasmid encoded yeast or human TOP1, cells expressing CPT-resistant mutant enzymes are viable in the presence of the drug. These studies firmly establish DNA topoisomerase I as the cellular target of CPT. Additional factors have been shown to modulate yeast cell sensitivity to CPT, such as double-stranded DNA break repair (7,8), DNA damage cell cycle checkpoints (2), sister chromatin condensation (9), ATP binding cassette transporters (10), nuclear import of Top1p (11), and DNA replication (12). Nevertheless, DNA topoisomerase I poisoning is essential for the cytotoxic...
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