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Nucleic Acids Research 2005 33(8):2364-2373; doi:10.1093/nar/gki527
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Published online 28 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Characterization of the DNA-binding domain and identification of the active site residue in the ‘Gyr A’ half of Leishmania donovani topoisomerase II

Tanushri Sengupta, Mandira Mukherjee, Rakhee Das, Aditi Das1 and Hemanta K. Majumder*

Molecular Parasitology Laboratory, Indian Institute of Chemical Biology 4 Raja S.C. Mullick Road, Kolkata-700032, India 1Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, USA

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

Received January 19, 2005. Revised April 4, 2005. Accepted April 4, 2005.

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. To investigate the biochemical properties of the individual domains of Leishmania donovani topoisomerase II, four truncation mutants were generated. Deletion of 178 aminoacids from the C-terminus (core and Ld{Delta}C1058) had no apparent effect on the DNA-binding or cleavage activities of the enzymes. However, when 429 aminoacids from the N-terminus and 451 aminoacids from the C-terminus were removed (Ld{Delta}N{Delta}C), the enzyme was no longer active. Moreover, the removal of 429 aminoacids from the N-terminus (Ld{Delta}N{Delta}C, core and Ld{Delta}N429) render the mutant proteins incapable of performing ATP hydrolysis. The mutant proteins show cleavage activities at wide range of KCl concentrations (25–350 mM). In addition, the mutant proteins, excepting Ld{Delta}N{Delta}C, can also act on kDNA and linearize the minicircles. Surprisingly, the mutant proteins fail to show the formation of the enhanced cleavable complex in the presence of etoposide. Our findings suggest that the conformation required for interaction with the drug is absent in the mutant proteins. Here, we have also identified Tyr775 through direct sequencing of the DNA linked peptide as the catalytic residue implicated in DNA-breakage and rejoining. Taken together, our results demonstrate that topoisomerase II are functionally and mechanistically conserved enzymes and the variations in activity seem to reflect functional optimization for its physiological role during parasite genome replication.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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