Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: a key molecular target in antileishmanial therapy

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflag ellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an OR F of 3711 bp encoding a putative protein of 1236 amino acids with no intron s. The deduced amino acid sequence of LdTOP2 showed strong homologies to TO P2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma sp p, with estimated identities of 86 and 68%, respectively, LdTOP2 shares a m uch lower identity of 32% with its human homologue, LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome a nd is expressed as a 5 kb transcript, 5'-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the transspli cing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protei n containing the major catalytic portion of the recombinant L.donovani topo isomerase II protein could detect a band on western blots at similar to 132 kDa, the expected size of the entire protein. Use of the same antiserum fo r immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro bio chemical properties of the full-length recombinant LdTOP2 when overexpresse d in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.