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 journalArticle

62 Citations (Scopus)

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

Fingerprint

Leishmania donovani
Type I DNA Topoisomerase
Assays
Enzymes
Salts
Camptothecin
Proteins
DNA
Recombinant Proteins
Liver
Yeast
Rats
Plasmids
Genes
Yeasts
Therapeutics

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

Cite this

Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani. / Brata Das, Benu; Sen, Nilkantha; Ganguly, Agneyo; Majumder, Hemanta K.

In: FEBS Letters, Vol. 565, No. 1-3, 07.05.2004, p. 81-88.

Research output: Contribution to journalArticle

Brata Das, Benu ; Sen, Nilkantha ; Ganguly, Agneyo ; Majumder, Hemanta K. / Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani. In: FEBS Letters. 2004 ; Vol. 565, No. 1-3. pp. 81-88.
@article{1e2f2095ed434a87993911dc7278a07c,
title = "Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani",
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.",
keywords = "Cleavable complex, Leishmania, PAGE, polyacrylamide gel electrophoresis, RT-PCR, reverse transcription polymerase chain reaction, Reconstitution, SDS, sodium dodecyl sulfate, Topoisomerase I",
author = "{Brata Das}, Benu and Nilkantha Sen and Agneyo Ganguly and Majumder, {Hemanta K.}",
year = "2004",
month = "5",
day = "7",
doi = "10.1016/j.febslet.2004.03.078",
language = "English (US)",
volume = "565",
pages = "81--88",
journal = "FEBS Letters",
issn = "0014-5793",
publisher = "Elsevier",
number = "1-3",

}

TY - JOUR

T1 - Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani

AU - Brata Das, Benu

AU - Sen, Nilkantha

AU - Ganguly, Agneyo

AU - Majumder, Hemanta K.

PY - 2004/5/7

Y1 - 2004/5/7

N2 - 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.

AB - 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.

KW - Cleavable complex

KW - Leishmania

KW - PAGE, polyacrylamide gel electrophoresis

KW - RT-PCR, reverse transcription polymerase chain reaction

KW - Reconstitution

KW - SDS, sodium dodecyl sulfate

KW - Topoisomerase I

UR - http://www.scopus.com/inward/record.url?scp=2342501361&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2342501361&partnerID=8YFLogxK

U2 - 10.1016/j.febslet.2004.03.078

DO - 10.1016/j.febslet.2004.03.078

M3 - Article

C2 - 15135057

AN - SCOPUS:2342501361

VL - 565

SP - 81

EP - 88

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 1-3

ER -