Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani

Benu Brata Das, Nilkantha Sen, Agneyo Ganguly, Hemanta K. Majumder

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

Abstract

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 Mg2+), 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.

Original languageEnglish (US)
Pages (from-to)81-88
Number of pages8
JournalFEBS Letters
Volume565
Issue number1-3
DOIs
StatePublished - May 7 2004

Keywords

  • Cleavable complex
  • Leishmania
  • PAGE, polyacrylamide gel electrophoresis
  • RT-PCR, reverse transcription polymerase chain reaction
  • Reconstitution
  • SDS, sodium dodecyl sulfate
  • Topoisomerase I

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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