N-terminal Region of the Large Subunit of Leishmania donovani Bisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit Export

@article{citeulike:4327144, abstract = {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{Delta}39L (lacking amino acids 1-39) and LdTOP1{Delta}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{Delta}99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1{Delta}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{Delta}99L and LdTOP1S reveals that LdTOP1{Delta}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. 10.1074/jbc.M412417200}, author = {Das, Benu B. and Sen, Nilkantha and Dasgupta, Somdeb B. and Ganguly, Agneyo and Majumder, Hemanta K.}, citeulike-article-id = {4327144}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M412417200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/280/16/16335}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/15711017}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=15711017}, day = {22}, doi = {10.1074/jbc.M412417200}, journal = {J. Biol. Chem.}, keywords = {cepa}, month = {April}, number = {16}, pages = {16335--16344}, posted-at = {2009-04-16 16:36:24}, priority = {2}, 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}, url = {http://dx.doi.org/10.1074/jbc.M412417200}, volume = {280}, year = {2005} }

TY - JOUR ID - citeulike:4327144 L3 - citeulike-article-id:4327144 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, LdTOP1{Delta}39L (lacking amino acids 1-39) and LdTOP1{Delta}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{Delta}99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1{Delta}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{Delta}99L and LdTOP1S reveals that LdTOP1{Delta}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. 10.1074/jbc.M412417200 IS - 16 JF - J. Biol. Chem. EP - 16344 TI - N-terminal Region of the Large Subunit of Leishmania donovani Bisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit VL - 280 SP - 16335 KW - cepa AU - Das, Benu AU - Sen, Nilkantha AU - Dasgupta, Somdeb AU - Ganguly, Agneyo AU - Majumder, Hemanta PY - 2005/04/22/ UR - http://dx.doi.org/10.1074/jbc.M412417200 ER -