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, 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 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 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.Topoisomerases are enzymes that can modify the tertiary structure of DNA without altering the primary structure (1).DNA topoisomerases accomplish their function by passing one strand of DNA duplex through a transient break in the other strand (type I topoisomerase), resulting in changes in linking number in steps of one (2) or by passing of a duplex DNA from the same or another molecule through a double-stranded break generated in the DNA in an ATP-dependent manner (type II topoisomerase), resulting in changes in the linking number in steps of two (3) and are involved in vital cellular processes (4).Trypanosoma and Leishmania are ancient eukaryotes. The distinctive features include structurally and metabolically unusual kinetoplast DNA within the single mitochondrion of the organism. DNA topoisomerases from the kinetoplastid parasites play a key role in many aspects of nucleic acid metabolism (5).Recently, the emergence of the bisubunit topoisomerase I of Trypanosoma (6) and Leishmania (7) in the kinetoplastid family have brought ...

Novel Betulin Derivatives as Antileishmanial Agents with Mode of Action Targeting Type IB DNA Topoisomerase

“…The type I DNA topoisomerases were assayed by decreased mobility of the relaxed isomers of supercoiled pBlueScript (SK ϩ ) [pBS (SK ϩ )] DNA in agarose gel. The relaxation assay was carried out as described previously with LdTOP1LS Das et al, 2005), serially diluted in the relaxation buffer (25 mM Tris-HCl, pH 7.5, 5% glycerol, 0.5 mM DTT, 10 mM MgCl 2 , 50 mM KCl, 25 mM EDTA, and 150 g/ml BSA) and supercoiled plasmid pBS (SK ϩ ) DNA (85-95% was negatively supercoiled, with remainder being nicked circles). The reconstituted enzyme LdTOP1LS was assayed at 50 mM KCl concentration, whereas human topoisomerase I was assayed at 150 mM KCl concentration as described previously .…”

“…The mixture was dialyzed overnight at 4°C, and dialyzed fractions were used for plasmid relaxation activity (Das et al, , 2005.…”

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase

“…The hinge region of topoisomerase IB has important role in opening and closing of the enzyme around DNA and in turn helping the enzyme in attaining precleavage formation (56,58). Near binding site two (hinge region), there is a hydrophobic cluster formed by three Trp residues (Trp-30 and -31 of the large subunit and Trp-253 of the small subunit) that is critical for the DNA binding and strand rotation step of the catalytic cycle of LdTopIB (58,59,60,61).…”

“…Near binding site two (hinge region), there is a hydrophobic cluster formed by three Trp residues (Trp-30 and -31 of the large subunit and Trp-253 of the small subunit) that is critical for the DNA binding and strand rotation step of the catalytic cycle of LdTopIB (58,59,60,61). The benzyl group of compound 4c interacts with this Trp cluster through Trp-30.…”

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts

“…DNA topoisomerase activity was assessed by visualizing the decreased mobility of relaxed isomers of supercoiled plasmid pBR322 (Inspiralis) in an agarose gel. The relaxation assay was carried out as previously described (35,36). Briefly, pBR322 supercoiled DNA was incubated with compounds in relaxation buffer (25 mM Tris- Cl, 5% glycerol, 0.5 mM DTT, 10 mM MgCl 2 , 2.5 mM EDTA, 50 mM KCl, and 150 g/ml bovine serum albumin [BSA] at pH 7.5) with LdTOP⌱LS.…”