N-terminal Region of the Large Subunit of Leishmania donovani Bisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
N-terminal Region of the Large Subunit of Leishmania donovani
Bisubunit Topoisomerase I Is Involved in DNA Relaxation and
Interaction with the Smaller Subunit
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| Creator |
Das, Benu Brata
Sen, Nilkantha Bose Dasgupta, Somdeb Ganguly, Agneyo Majumder, Hemanta K |
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| Subject |
Infectious Diseases and Immunology
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| Description |
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,
LdTOP1�39L (lacking amino acids 1–39) and
LdTOP1�99L (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 LdTOP1�99LS was unable to show
topoisomerase I activity. Investigation of the loss of activity
indicates that LdTOP1�99L 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 LdTOP1�99L and LdTOP1S reveals
that LdTOP1�99L 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.
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| Publisher |
American Society for Biochemistry and Molecular Biology
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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/375/1/JOURNAL_OF_BIOLOGICAL_CHEMISTRY_280_(16)16335%2D16344;2005[70].pdf
Das, Benu Brata and Sen, Nilkantha and Bose Dasgupta, Somdeb and Ganguly, Agneyo and Majumder, Hemanta K (2005) N-terminal Region of the Large Subunit of Leishmania donovani Bisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit. The Journal of Biological Chemistry, 280 (16). pp. 16335-16344. |
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| Relation |
htp://dx.doi.org/10.1074/jbc.M412417200
http://www.eprints.iicb.res.in/375/ |
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