Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani.
Leishmania donovani topoisomerase I is an unusual bi-subunit enzyme. The activity of the enzyme has been detected when the genes of the individual subunits were co-expressed in yeast [J. Biol. Chem. 278 (2003) 3521]. Here, we report for the first time, the in vitro reconstitution of the two recombinant proteins, LdTOP1L and LdTOP1S, corresponding to the large and small subunits and localization of the active enzyme in both the nucleus and kinetoplast. The proteins were purified from bacterial extract and the activity was measured by plasmid DNA relaxation assay. LdTOP1L and LdTOP1S form a direct 1:1 heterodimer complex through protein-protein interaction. Under standard relaxation assay condition (50 mM KCl and 10 mM Mg(2+)), reconstituted enzyme (LdTOP1LS) showed reduced processivity as well as 2-fold reduced affinity for DNA compared to eukaryotic monomeric rat liver topoisomerase I (RLTOP1). Cleavage assay at various salt concentrations reveals that Camptothecin (CPT) enhanced the formation of "cleavable complex" at low salt. Interaction between the two subunits leading to the formation of an active complex could be explored as an insight for development of new therapeutic agents with specific selectivity.
Das
Benu Brata
BB
Department of Molecular Parasitology, Indian Institute of Chemical Biology 4, Raja S.C. Mullick Road, Kolkata 700032, India.
Sen
Nilkantha
N
Ganguly
Agneyo
A
Majumder
Hemanta K
HK
D000818:Animals; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D004247:DNA / Q000737:chemistry; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D018105:DNA, Kinetoplast / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007893:Leishmania donovani / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008856:Microscopy, Fluorescence; D009532:Nickel / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013997:Time Factors
D018105:DNA, Kinetoplast; D004278:DNA, Superhelical; D011994:Recombinant Proteins; D012492:Salts; D012967:Sodium Dodecyl Sulfate; D011189:Potassium Chloride; D009532:Nickel; D004247:DNA; D012685:Sepharose; D004264:DNA Topoisomerases, Type I; D011188:Potassium; D002166:Camptothecin