The unusual, heterodimeric topoisomerase IB of Leishmania shows functional activity upon reconstitution of the DNA-binding large subunit (LdTOPIL; or L) and the catalytic small subunit (LdTOPIS; or S). In the present study, we generated N- and C-terminal-truncated deletion constructs of either subunit and identified proteins LdTOPIL39–456 (lacking amino acids 1–39 and 457–635) and LdTOPIS210–262 (lacking amino acids 1–210) as the minimal interacting fragments. The interacting region of LdTOPIL lies between residues 40–99 and 435–456, while for LdTOPIS it lies between residues 210–215 and 245–262. The heterodimerization between the two fragments is weak and therefore co-purified fragments showed reduced DNA binding, cleavage and relaxation properties compared with the wild-type enzyme. The minimal fragments could complement their respective wild-type subunits inside parasites when the respective subunits were down-regulated by transfection with conditional antisense constructs. Site-directed mutagenesis studies identify Lys455 of LdTOPIL and Asp261 of LdTOPIS as two residues involved in subunit interaction. Taken together, the present study provides crucial insights into the mechanistic details for understanding the unusual structure and inter-subunit co-operativity of this heterodimeric enzyme.
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Research Article|
December 21 2007
Amino acids 39–456 of the large subunit and 210–262 of the small subunit constitute the minimal functionally interacting fragments of the unusual heterodimeric topoisomerase IB of Leishmania
Somdeb Bosedasgupta;
Somdeb Bosedasgupta
*Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
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Benu Brata Das;
Benu Brata Das
†Laboratory of Molecular Pharmacology, Centre for Cancer Research, National Cancer Institute, Bethesda, MD, U.S.A.
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Souvik Sengupta;
Souvik Sengupta
*Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
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Agneyo Ganguly;
Agneyo Ganguly
*Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
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Amit Roy;
Amit Roy
*Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
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Gayatri Tripathi;
Gayatri Tripathi
‡Division of Cellular Physiology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
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Hemanta K. Majumder
Hemanta K. Majumder
1
*Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata-700032, India
1To whom correspondence should be addressed (email hkmajumder@iicb.res.in).
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Publisher: Portland Press Ltd
Received:
October 03 2007
Accepted:
October 09 2007
Accepted Manuscript online:
October 09 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 409 (2): 481–489.
Article history
Received:
October 03 2007
Accepted:
October 09 2007
Accepted Manuscript online:
October 09 2007
Citation
Somdeb Bosedasgupta, Benu Brata Das, Souvik Sengupta, Agneyo Ganguly, Amit Roy, Gayatri Tripathi, Hemanta K. Majumder; Amino acids 39–456 of the large subunit and 210–262 of the small subunit constitute the minimal functionally interacting fragments of the unusual heterodimeric topoisomerase IB of Leishmania. Biochem J 15 January 2008; 409 (2): 481–489. doi: https://doi.org/10.1042/BJ20071358
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