tyrosyl DNA phosphodiesterase 1 from kinetoplastid parasite Leishmania donovani (LdTdp1) capable of removing topo I-DNA covalent complexes

• Published in: Molecular microbiology 2010-10, Vol.78 (1), p.119-137
• Main Authors: Banerjee, Bijoylaxmi, Roy, Amit, Sen, Nilkantha, Majumder, Hemanta K
• Format: Article
• Language: English
• Subjects: Apoptosis; Biological and medical sciences; Camptothecin - pharmacology; Cell Nucleus - genetics; Cell Nucleus - metabolism; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA damage; DNA Repair; DNA, Protozoan - metabolism; Enzymes; Fundamental and applied biological sciences. Psychology; Gene Knockdown Techniques; Hydrogen Peroxide - pharmacology; Leishmania donovani; Leishmania donovani - enzymology; Leishmania donovani - genetics; Microbiology; Models, Molecular; Mutagenesis, Site-Directed; Oxidative Stress; Parasites; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - metabolism; Phosphorus; Plastids - genetics; Plastids - metabolism; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Sequence Alignment
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/j.1365-2958.2010.07318.x

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3′-DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C-terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H₂O₂-mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT-resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I-mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.