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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

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Title 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
 
Creator Bose Dasgupta, Somdeb
Das, Benu Brata
SenGupta, Souvik
Ganguly, Agneyo
Roy, Amit
Tripathi, Gayatri
Majumder, Hemanta K
 
Subject Cell Biology & Physiology
Infectious Diseases and Immunology
 
Description 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 Cterminal- 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 cooperativity of this heterodimeric enzyme.
 
Date 2008
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/290/1/BIOCHEMICAL_JOURNAL%2C_409%2C481%2D489%2C2008[136].pdf
Bose Dasgupta, Somdeb and Das, Benu Brata and SenGupta, Souvik and Ganguly, Agneyo and Roy, Amit and Tripathi, Gayatri and Majumder, Hemanta K (2008) 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. Biochemical Journal, 409. pp. 481-489.
 
Relation http://dx.doi.org/10.1042/BJ20071358
http://www.eprints.iicb.res.in/290/