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Identification of a Functional Type IA Topoisomerase, LdTopIIIβ, from Kinetoplastid Parasite Leishmania donovani.

Banerjee B, Sen N, Majumder HK - Enzyme Res (2011)

Bottom Line: Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid.A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite.Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Parasitology Laboratory, Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
DNA topoisomerases of kinetoplastids represent a family of DNA processing enzymes that essentially solve the topological problems not only in nuclear DNA but also in kinetoplast DNA. We have, for the first time, identified a Leishmania donovani homologue of bacterial and eukaryotic IA type of topoisomerase III protein and termed as LdTopIIIβ. Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid. A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite. Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

No MeSH data available.


DNA relaxation assay by recombinant LdTopIIIβ. (a) Negatively super coiled DNA was incubated with 1, 2, 3, 4, 5, 7 and 10 μL of recombinant LdTopIIIβ containing lysate for 30 min (lanes 2–8). Lane 1 is the DNA control. (b) DNA relaxation assay carried out with recombinant LdTopIIIβ (lane 2), and empty vector containing lysate (lane 3). Lane 1 is the DNA control.
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fig6: DNA relaxation assay by recombinant LdTopIIIβ. (a) Negatively super coiled DNA was incubated with 1, 2, 3, 4, 5, 7 and 10 μL of recombinant LdTopIIIβ containing lysate for 30 min (lanes 2–8). Lane 1 is the DNA control. (b) DNA relaxation assay carried out with recombinant LdTopIIIβ (lane 2), and empty vector containing lysate (lane 3). Lane 1 is the DNA control.

Mentions: LdTopIIIβ was cloned in bacterial expression vector pET-16b and overexpressed in BL21 (DE3)-pLysS strain and induced with IPTG. But the overexpressed protein went to inclusion body and were found in the pellet as insoluble protein which could not be recovered in the soluble fraction in active state. However, to test the activity of the recombinant protein, we have used in vitro transcription-translation kit, which is specially designed for in vitro transcription and translation of target DNA to protein in a single reaction. The crude lysate containing the newly synthesized proteins were used for DNA relaxation assay. Figure 6(a) shows DNA relaxation by increasing amount of recombinant LdTopIIIβ (lanes 2–8). Lane 1 is the DNA control. The results clearly show that the recombinant protein containing lysates were able to relax the negatively supercoiled DNA. To test that the activity was not coming from the lysate itself, we have carried out DNA relaxation activity with the empty vector containing lysate which contained insignificant amount of activity, shown in Figure 6(b) (lane 3). Lane 2 shows DNA relaxation activity by recombinant LdTopIIIβ.


Identification of a Functional Type IA Topoisomerase, LdTopIIIβ, from Kinetoplastid Parasite Leishmania donovani.

Banerjee B, Sen N, Majumder HK - Enzyme Res (2011)

DNA relaxation assay by recombinant LdTopIIIβ. (a) Negatively super coiled DNA was incubated with 1, 2, 3, 4, 5, 7 and 10 μL of recombinant LdTopIIIβ containing lysate for 30 min (lanes 2–8). Lane 1 is the DNA control. (b) DNA relaxation assay carried out with recombinant LdTopIIIβ (lane 2), and empty vector containing lysate (lane 3). Lane 1 is the DNA control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102327&req=5

fig6: DNA relaxation assay by recombinant LdTopIIIβ. (a) Negatively super coiled DNA was incubated with 1, 2, 3, 4, 5, 7 and 10 μL of recombinant LdTopIIIβ containing lysate for 30 min (lanes 2–8). Lane 1 is the DNA control. (b) DNA relaxation assay carried out with recombinant LdTopIIIβ (lane 2), and empty vector containing lysate (lane 3). Lane 1 is the DNA control.
Mentions: LdTopIIIβ was cloned in bacterial expression vector pET-16b and overexpressed in BL21 (DE3)-pLysS strain and induced with IPTG. But the overexpressed protein went to inclusion body and were found in the pellet as insoluble protein which could not be recovered in the soluble fraction in active state. However, to test the activity of the recombinant protein, we have used in vitro transcription-translation kit, which is specially designed for in vitro transcription and translation of target DNA to protein in a single reaction. The crude lysate containing the newly synthesized proteins were used for DNA relaxation assay. Figure 6(a) shows DNA relaxation by increasing amount of recombinant LdTopIIIβ (lanes 2–8). Lane 1 is the DNA control. The results clearly show that the recombinant protein containing lysates were able to relax the negatively supercoiled DNA. To test that the activity was not coming from the lysate itself, we have carried out DNA relaxation activity with the empty vector containing lysate which contained insignificant amount of activity, shown in Figure 6(b) (lane 3). Lane 2 shows DNA relaxation activity by recombinant LdTopIIIβ.

Bottom Line: Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid.A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite.Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Parasitology Laboratory, Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
DNA topoisomerases of kinetoplastids represent a family of DNA processing enzymes that essentially solve the topological problems not only in nuclear DNA but also in kinetoplast DNA. We have, for the first time, identified a Leishmania donovani homologue of bacterial and eukaryotic IA type of topoisomerase III protein and termed as LdTopIIIβ. Complementation study of wild-type and mutant LdTopIIIβ with slow-growing topoisomerase III mutant yeast S. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase IIIβ protein, the 327 tyrosine being the active site amino acid. A C-terminal deletion construct of LdTopIIIβ could not suppress the slow-growth phenotype of mutant yeast, indicating the requirement of C-terminal region for the enzyme function in vivo.LdTopIIIβ localized inside the nucleus and kinetoplast of the parasite. Taken together, our study indicates functional conservation and possible role of LdTopIIIβ in parasite DNA processing.

No MeSH data available.