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

Content Provider	Semantic Scholar
Author	Sengupta, Tanushri Mukherjee, Mandira Das, Rakhee Das, Aditi Majumder, Hemanta Kumar
Copyright Year	2005
Abstract	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 LdDeltaC1058) 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429 aminoacids from the N-terminus and 451 aminoacids from the C-terminus were removed (LdDeltaNDeltaC), the enzyme was no longer active. Moreover, the removal of 429 aminoacids from the N-terminus (LdDeltaNDeltaC, core and LdDeltaN429) 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 LdDeltaNDeltaC, 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 Tyr(775) 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.
Starting Page	2364
Ending Page	2373
Page Count	10
File Format	PDF HTM / HTML
PubMed reference number	15860773
Volume Number	33
Journal	Nucleic acids research
Alternate Webpage(s)	http://ftp.ncbi.nlm.nih.gov/pub/pmc/fc/0a/gki527.PMC1087781.pdf
Language	English
Access Restriction	Open
Subject Keyword	Adenosine Triphosphate Amino Acids Anatomy, Regional CD4 stimulated ATP immune response:MCnc:Pt:Bld:Qn DNA, Kinetoplast Direct Sequencing Etoposide Leishmania donovani Potassium Chloride Protein Truncation Abnormality Topoisomerase II mutant
Content Type	Text
Resource Type	Article