TY - JOUR
T1 - N-terminal region of the large subunit of Leishmania donovani bisubunit topoisomerase I is involved in DNA relaxation and interaction with the smaller subunit
AU - Das, Benu Brata
AU - Sen, Nilkantha
AU - Dasgupta, Somdeb Bose
AU - Ganguly, Agneyo
AU - Majumder, Hemanta K.
PY - 2005/4/22
Y1 - 2005/4/22
N2 - 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, LdTOP1Δ39L (lacking amino acids 1-39) and LdTOP1Δ99L (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 LdTOP1Δ99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Δ99L 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 LdTOP1Δ99L and LdTOP1S reveals that LdTOP1Δ99L 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.
AB - 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, LdTOP1Δ39L (lacking amino acids 1-39) and LdTOP1Δ99L (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 LdTOP1Δ99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Δ99L 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 LdTOP1Δ99L and LdTOP1S reveals that LdTOP1Δ99L 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.
UR - http://www.scopus.com/inward/record.url?scp=18144415449&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18144415449&partnerID=8YFLogxK
U2 - 10.1074/jbc.M412417200
DO - 10.1074/jbc.M412417200
M3 - Article
C2 - 15711017
AN - SCOPUS:18144415449
SN - 0021-9258
VL - 280
SP - 16335
EP - 16344
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -