Characterization of the DNA-binding domain and identification of the active site residue in the ‘Gyr A’ half of Leishmania donovani topoisomerase II
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
Characterization of the DNA-binding domain and
identification of the active site residue in the ‘Gyr A’
half of Leishmania donovani topoisomerase II
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| Creator |
Sengupta, Tanushri
Mukherjee, Mandira Das, Rakhee Das, Aditi Majumder, Hemanta K |
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| Subject |
Infectious Diseases and Immunology
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| Description |
DNA topoisomerase II is a multidomain homodimeric
enzyme that changes DNA topology by coupling ATP
hydrolysis to the transport of oneDNAhelix through a
transient double-strandedbreak in another.Toinvestigate
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-
DC1058) 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 (LdDNDC),
the enzyme was no longer active. Moreover, the
removal of 429 aminoacids from the N-terminus
(LdDNDC, core and LdDN429) render the mutant proteinsincapableofperformingATPhydrolysis.
Themutant
proteins show cleavage activities at wide range
of KCl concentrations (25–350 mM). In addition, the
mutant proteins, excepting LdDNDC, 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.
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| Publisher |
Oxford University Press
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| Date |
2005
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/580/1/NUCLEIC_ACIDS_RESEARCH__33(_8)_2364%2D2373;2005[112].pdf
Sengupta, Tanushri and Mukherjee, Mandira and Das, Rakhee and Das, Aditi and Majumder, Hemanta K (2005) Characterization of the DNA-binding domain and identification of the active site residue in the ‘Gyr A’ half of Leishmania donovani topoisomerase II. Nucleic Acids Research, 33 (8). pp. 2364-2373. |
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| Relation |
http://dx.doi.org/10.1093/nar/gki527
http://www.eprints.iicb.res.in/580/ |
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