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J. Biol. Chem., Vol. 280, Issue 16, 16335-16344, April 22, 2005

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N-terminal Region of the Large Subunit of Leishmania donovani Bisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit^*

Benu Brata Das, Nilkantha Sen, Somdeb Bose Dasgupta, Agneyo Ganguly, and Hemanta K. Majumder

From the Department of Molecular Parasitology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India

Leishmania donovani topoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstituted in vitro to show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP139L (lacking amino acids 1–39) and LdTOP199L (lacking amino acids 1–99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1–39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitro DNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP199LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP199L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni²⁺-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S in Escherichia coli BL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP199L and LdTOP1S reveals that LdTOP199L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1–39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.

Received for publication, November 3, 2004 , and in revised form, February 11, 2005.

^* This work was supported by Department of Science and Technology, Government of India, Grant SP/SO/D11/2000 (to H. K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a University Grant Commission,Government of India, Senior Research Fellowship.

To whom correspondence should be addressed. Tel.: 91-33-2412-3207; Fax: 91-33-2473-5197; E-mail: hkmajumder{at}iicb.res.in.

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				A. Ganguly, B. B. Das, N. Sen, A. Roy, S. B. Dasgupta, and H. K. Majumder 'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani Nucleic Acids Res., December 4, 2006; 34(21): 6286 - 6297. [Abstract] [Full Text] [PDF]

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