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

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Title Reconstitution and functional characterization of the unusual
bi-subunit type I DNA topoisomerase from Leishmania donovani
 
Creator Das, Benu Brata
Sen, Nilkantha
Ganguly, Agneyo
Majumder, Hemanta K
 
Subject Infectious Diseases and Immunology
 
Description 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 Mg2þ), 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.
 
Publisher Elsevier
 
Date 2004
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1055/1/30_FL_2004.pdf
Das, Benu Brata and Sen, Nilkantha and Ganguly, Agneyo and Majumder, Hemanta K (2004) Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani. FEBS Letters, 565 (1-3). pp. 81-88.
 
Relation http://dx.doi.org/10.1016/j.febslet.2004.03.078
http://www.eprints.iicb.res.in/1055/