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.
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, LdTOP1Delta39L (lacking amino acids 1-39) and LdTOP1Delta99L (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 LdTOP1Delta99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Delta99L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni(2+)-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 LdTOP1Delta99L and LdTOP1S reveals that LdTOP1Delta99L 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.
authors.affiliation: Department of Molecular Parasitology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.
authors.forename: Benu Brata
authors.initials: BB
authors.lastname: Das
authors.forename: Nilkantha
authors.initials: N
authors.lastname: Sen
authors.forename: Somdeb Bose
authors.initials: SB
authors.lastname: Dasgupta
authors.forename: Agneyo
authors.initials: A
authors.lastname: Ganguly
authors.forename: Hemanta K
authors.initials: HK
authors.lastname: Majumder
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D017384:Sequence Deletion; D059004:Topoisomerase I Inhibitors
chemical_list: D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin