Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active enzyme with conserved type IB enzyme function Benu B Das1,*, Somdeb Bose Dasgupta1,*, Agneyo Ganguly1, Saumyabrata Mazumder2, Amit Roy1 and Hemanta K Majumder1 Department of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata, India Infectious Diseases Group, Indian Institute of Chemical Biology, Kolkata, India Keywords camptothecin; gene fusion; Leishmania; topoisomerase I; SKXXY motif Correspondence H K Majumder, Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, Raja S.C Mullick Road, Kolkata-700032, India Fax: +91 33 2473 5197 Tel: +91 33 2412 3207 E-mail: hkmajumder@iicb.res.in *These authors contributed equally to this work (Received 12 July 2006, revised November 2006, accepted November 2006) doi:10.1111/j.1742-4658.2006.05572.x All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S) It was found that LdTOPIL-fus-S is active Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS) Interestingly, an N-terminal truncation mutant (1–210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-D(1–210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S Investigation of the reduction in enzyme activity indicated that the nonconserved 1–210 residues of LdTOPIS probably act as a ‘pseudolinker’ domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme–DNA transition state through hydrogen bonding to one of the nonbridging oxygens Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani The type IB DNA topoisomerase family includes eukaryotic nuclear topoisomerase I and the topoisomerases encoded by vaccinia, bacteria and other cytoplasmic poxviruses [1–3] The type IB enzymes relax supercoiled DNA via a multistep reaction pathway entailing noncovalent binding of the topoisomerase to duplex DNA, cleavage of one DNA strand with formation of a covalent DNA-(3-phosphotyrosyl)–protein intermediate, strand passage, and strand religation [1,2,4] Recently, the discovery of the bisubunit topoisomerase I enzymes of Trypanosoma [5] and Leishmania [6] in the kinetoplastid family have brought a new twist in topoisomerase research related to evolution and functional conservation of the type IB family The core DNA-binding domain and the catalytic domain harboring the consensus SKXXY motif are located in separate subunits The two subunits are synthesized by two different genes, and associate with each other through protein–protein interactions to form an active heterodimeric topoisomerase I within the parasite This unusual structure of DNA topoisomerase I may provide a missing link in the evolution of type IB enzymes Abbreviation CPT, camptothecin 150 FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS B B Das et al We have previously demonstrated the in vitro reconstitution of the two recombinant proteins LdTOPIL and LdTOPIS, corresponding to the large and small subunits, and localization of the active enzyme in both the nucleus and kinetoplast [7] The enzyme is conventional in its Mg2+ independence, site specificity for eukaryotic type IB-specific recognition sites and camptothecin (CPT) sensitivity LdTOPIL and LdTOPIS form a direct : heterodimer complex through protein–protein interactions ˚ Davies et al [8] have made a 2.27°A crystal structure of an active truncated Leishmania donovani TOPIL–TOPIS heterodimer bound to nicked doublestranded DNA in the presence of vanadate The vanadate forms covalent linkages between the catalytic Tyr222 residue of the small subunit (LdTOPIS) and the nicked ends of the scissile DNA strand, mimicking the transition state of the topoisomerase I catalytic cycle The structure predicts that the highly conserved constellation of the catalytic residues (Arg314, Lys352, Arg410 and His453 of LdTOPIL and the consensus catalytic residue Tyr222 in LdTOPIS) share a common module between Leishmania and human topoisomerase I Although the details of catalysis for the unusual heterodimeric Leishmania topoisomerase I reaction remain to be elucidated, based on the crystal structure of truncated LdTOPILS, it appears that His453 forms a ˚ 2.6 A hydrogen-bonding contact with a nonbridging oxygen atom of the vanadate [8] This interaction is virtually the same as in the noncovalent complex of ˚ human topoisomerase I, where His632 is 2.6 A from a nonbridging phosphate oxygen atom of the DNA base, and may be responsible for stabilizing the enzyme– DNA interaction [9] Human topoisomerase I is a monomeric structure composed of 765 residues with a molecular mass of 91 kDa Topo70 is a truncated form of human topoisomerase I that lacks residues 1–174 of the N-terminal domain and retains full enzyme activity in vitro The enzyme contains a central DNA-binding core domain and a C-terminal catalytic domain harboring an SKINYL motif The crystal structure of human topoisomerase I demonstrates that the core and C-terminal domains form a clamp-like structure embedding the DNA helix in a central pore, with two lobes of the protein binding to either site of the helix [10] The conserved subdomains I and II contribute the upper part ‘CAP’, which is connected by a flexible hinge to the bottom part of the clamp of subdomain III The linker domain forms a coiled-coil structure that protrudes from the body of the enzyme and connects the core to the highly conserved C-terminal domain close Gene fusion and Leishmania topoisomerase I to the scissile phosphate in the bound DNA This architecture facilitates the opening and closing of the protein clamp during binding and release of DNA [11,12] Champoux and his group have previously reported their findings on human topoisomerase I that has been artificially fragmented into two proteins (topo58 ⁄ 12 or topo58 ⁄ 6.3) The core and the catalytic domain can reconstitute topoisomerase I activity It was shown that detachment of the linker from the core domain makes the enzyme highly distributive, with 20-fold reduced affinity for DNA and less sensitivity to CPT [13] Some of our previous findings on Leishmania topoisomerase I are in keeping with those of reconstituted human topoisomerase I [7], but a closer look reveals that differences exist in the sequences, some biochemical properties and preferential sensitivities to CPT [14,15] Thus, the key questions arise of what will happen to the enzyme architecture and catalytic property if the two subunits are fused to a monomeric structure such as human topoisomerase I, and what the role of the conserved His453 in enzyme catalysis is To address these issues, we describe experiments in which Leishmania bisubunit topoisomerase I large subunit (LdTOPIL) and small subunit (LdTOPIS) genes were fused into a single ORF encoding a new topoisomerase I (LdTOPIL-fus-S) This monomeric enzyme is active and shows increased activity compared to the wild-type heterodimeric enzyme (LdTOPILS) Interestingly, an N-terminal truncation mutant (1–210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-D(1–210)S], shows reduced topoisomerase I activity compared to LdTOPIL-fus-S The present study also describes the role of the conserved His453 in the core DNA domain (LdTOPIL) in the reaction catalyzed by the fusion enzyme Hence, this study provides substantial information on the mechanistic details and unusual structure of this bisubunit enzyme Results Purification of recombinant proteins A schematic alignment of monomeric (human, vaccinia and bacterial) topoisomerase IB with that of the heterodimeric topoisomerase IB of Leishmania is shown in Fig 1A, in order to relate the two subunits to the monomeric enzymes All the recombinant constructs used in the present study and the deduced amino acid sequences of the fusion regions are shown in Fig 1B Leishmania bisubunit topoisomerase I fusion constructs were developed as described in Experimental FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 151 Gene fusion and Leishmania topoisomerase I B B Das et al Fig (A) Schematic alignment Monomeric (human vaccinia and bacterial) topoisomerase IB aligned with bisubunit topoisomerase of Leishmania in order to relate the two subunits with their monomeric counterparts The position of active site pentad residues is also shown (B) Protein constructs Structure of recombinant L donovani topoisomerase I proteins The first line shows the full-length larger subunit (dark) as the core DNA-binding subunit with the conserved catalytic His at position 453 The second line shows the Leishmania bisubunit topoisomerase I fusion construct, LdTOPIL (dark) and LdTOPIS (light shaded) and the deduced amino acid sequences of the fusion regions The third line shows the reverse fusion construct of Leishmania bisubunit topoisomerase I, LdTOPIS (light shaded) and LdTOPIL (dark) and the deduced amino acid sequences of the fusion regions The fourth line shows the N-terminal truncated small subunit (amino acids 211–262) fused to intact large subunit to generate an ORF, LdTOPIL-fus-D(1–210)S The fifth and sixth lines show the point mutations generated at the His453 position of the LdTOPIL-fus-S gene to H453A and H453Q, respectively The seventh line shows the smaller catalytic subunit (light shaded) with the active site residue The constructs were developed as described in Experimental procedures (C) Coomassie-stained 10% SDS ⁄ PAGE analysis of the purified recombinant proteins with lg per lane Lanes 1–7, LdTOPIL, LdTOPIL-fus-S, LdTOPIS-fus-L, LdTOPIL-fus-D(1–210)S, H453A and H453Q mutants of LdTOPILfus-S and LdTOPIS proteins purified through an Ni2+–nitrilotriacetic acid column, respectively, followed by a phosphocellulose column The positions and molecular masses of protein standards are indicated on the left A B C as described previously [7] Analysis of the purified proteins by SDS ⁄ PAGE (Fig 1C) showed that all the recombinant proteins are essentially homogeneous LdTOPIL-fus-S fusion protein is a functional topoisomerase I procedures The overexpressed proteins from Escherichia coli BL21(DE3)pLysS cells harboring plasmids pET28cLdTOPIL-fus-S, pET28cLdTOPIS-fus-L and pET28cLdTOPIL-fus-D(1–210)S (1–210 amino acid deletion mutant from the N-terminal region of the small subunit was fused in frame with LdTOPIL) were purified separately through an Ni2+–nitrilotriacetic acid agarose column The proteins were further purified through a phosphocellulose column as described in Experimental procedures A recent crystal structure has identified a conserved His453 of LdTOPIL close to the nonbridging oxygen atom of the vanadate [8] that potentially mimics the transient state of the enzyme–DNA covalent complex To test this possibility directly, we used site-directed mutagenesis to change His453 of LdTOPIL-fus-S to glutamine As a control, we also changed His453 of LdTOPIL-fus-S to alanine, as well to identify its role The other recombinant proteins, i.e LdTOPIL (large subunit) and LdTOPIS (small subunit), were purified 152 We assessed the topoisomerase activity of the Leishmania bisubunit fused protein encoding a new topoisomerase I (LdTOPIL-fus-S) by a plasmid relaxation assay Reconstitution of wild-type Leishmania bisubunit topoisomerase I (LdTOPILS) activity has been described previously [7,14] Time course relaxation experiments were performed in a standard assay mix where the plasmid DNA and the enzymes (LdTOPILS, LdTOPIL-fus-S and LdTOPIS-fus-L) were mixed at a molar ratio of : The velocity for LdTOPIL-fus-S was linear for the first of the reaction It was observed that LdTOPILfus-S relaxed supercoiled DNA at a slower rate than did reconstituted LdTOP1LS (compare lanes 2–9 of Fig 2B with lanes 2–9 of Fig 2A), whereas the reverse fusion LdTOPIS-fus-L failed to show any plasmid DNA relaxation activity (Fig 2C) The smaller number of topoisomer intermediates reacting with LdTOPIL-fus-S indicates that LdTOPIL-fus-S completely relaxes the supercoiled DNA substrate in a processive FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS B B Das et al Gene fusion and Leishmania topoisomerase I A Time (min) Lane LdTOP1LS - 0.5 10 15 20 30 40 RL/NM fashion before dissociating or reassociating with another DNA molecule However, under these conditions, the situation with reconstituted LdTOPILS is different, as partially relaxed topoisomers are visible during the course of the relaxation reaction (compare lanes 2–9 of Fig 2B with lanes 2–9 of Fig 2A) SM B Time (min) Lane Relaxation activity of the mutant topoisomerase I LdTOP1L-fus-S - 0.5 10 15 20 30 40 - 0.5 10 15 20 30 40 RL/NM SM C Time (min) Lane LdTOP1S-fus-L RL/NM SM Fig The LdTOPIL-fus-S fusion protein is a functional topoisomerase I Relaxation of supercoiled pBS (SK+) DNA with reconstituted enzyme LdTOPILS (A), LdTOPIL-fus-S (B), and reverse fusion LdTOPIS-fus-S (C), at a molar ratio of : Lane 1, 80 fmol of pBS (SK +) DNA Lanes 2–9, same as lane 1, but incubated with 20 fmol of topoisomerase I variants at 37 °C for different time periods as indicated in the figure All reactions were stopped by addition of 0.5% SDS; samples were electrophoresed in 1% agarose gel The positions of supercoiled monomer (SM) and relaxed and nicked monomer (RL ⁄ NM) are indicated The effects of mutations on enzyme activity were analyzed by standard plasmid DNA relaxation assays with a molar ratio of DNA to enzyme of : (Fig 3A) LdTOPIL-fus-S completely relaxes the DNA within 10 under these conditions, whereas all three mutant enzymes exhibited slow relaxation kinetics Complete relaxation by the LdTOPIL-fus-D(1–210)S enzyme was not observed until  40 min, and thus the LdTOPIL-fus-D(1–210)S protein appeared to be fourfold less active than the LdTOPILfus-S H453Q had less activity than LdTOPIL-fus-S, failing to completely relax the supercoiled DNA even after 40 Very little relaxing activity was detectable for the H453A protein We estimate the activity of the H453A protein to be more than 100-fold reduced compared with that of LdTOPIL-fus-S Supercoiled DNA relaxation under conditions of limiting topoisomerase I is stimulated  10-fold in the presence of 10 mm Mg2+, probably because of an increase in the dissociation rate of the enzyme from the DNA [7,16] It follows that the rate-limiting step for DNA relaxation by LdTOPIL-fus-S under normal assay conditions is enzyme dissociation This effect can A B Fig Plasmid relaxation assays for LdTOPILS, LdTOPIL-fus-S and its mutant variants Reaction mixtures containing 90 fmol of supercoiled plasmid DNA in relaxation buffer without (A) and with (B) 10 mM Mg2+ The reactions were initiated by the addition of 30 fmol of topoisomerase I variants incubated at 37 °C for different time periods as indicated in the figure Reactions were stopped by addition of 0.5% SDS; samples were electrophoresed in 1% agarose gel The zero time point was taken prior to the addition of enzyme FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 153 Gene fusion and Leishmania topoisomerase I B B Das et al be seen in Fig 3B, where the addition of 10 mm Mg2+ to the LdTOPIL-fus-S reaction increased the rate approximately 10-fold (reaction complete in min) Although addition of 10 mm Mg2+ enhances the relaxation rate of LdTOPIL-fus-D(1–210)S, it appears to be 20-fold reduced in comparison to LdTOPIL-fus-S (Fig 3B) However, the presence of Mg2+ in the reactions for the His453 mutant proteins (H453Q and H453A) had no effect on the relaxation rates (Fig 3B), suggesting that enzyme chemistry rather than enzyme dissociation was the rate-limiting step for all of the His453 mutant enzymes Moreover, in the presence of 10 mm Mg2+, the differences between the estimated activity for LdTOPIL-fus-S and the activities of the mutant proteins were magnified H453Q was at least 40-fold less active than LdTOPIL-fus-S, whereas H453A was more than 100-fold less active than LdTOPIL-fus-S As a wild-type control, reconstituted LdTOPIL and LdTOPIS were used both in the absence and the presence of Mg2+-containing buffer Effect of CPT on the relaxation activity and equilibrium cleavage activity of fused topoisomerase I variants We examined the effect of CPT on the relaxation activity of wild-type control reconstituted LdTOPILS and the fused enzyme [LdTOPIL-fus-S and LdTOPIL-fusD(1–210)S] The reverse fusion LdTOPIS-fus-L was not included in this experiment, as it was enzymatically inactive in the relaxation assay Time course relaxation experiments were performed in a standard assay mix where the plasmid DNA and the enzyme [LdTOPILS, LdTOPIL-fus-S and LdTOPIL-fus-D(1–210)S] were mixed at a molar ratio of : 2, to circumvent possible effects due to a slow dissociation rate and enzyme turnover number in the presence of CPT The wild-type was more distributive in nature and less sensitive to CPT compared to LdTOPIL-fus-S (Fig 4A) In the absence of CPT, the rate of relaxation of LdTOPIL-fus-S was greater than that of LdTOPIL-fus-D(1–210)S (compare lanes and of Fig 4B with lanes and of Fig 4C) In the presence of CPT, it can be seen that the time required to complete relaxation for LdTOPIL-fus-S was increased approximately 25-fold (from to 25 min; Fig 4B, compare lane with lane 18), whereas the drug had a reduced effect on the rate of relaxation by LdTOPIL-fus-D(1–210)S (compare lane with lane 15 of Fig 4C) CPT, the most established topoisomerase I inhibitor, has been shown to stabilize the cleavable complex Here, we investigated the characteristics of LdTOPILfus-S and LdTOPIL-fus-D(1–210)S in a cleavage assay 154 and compared it with LdTOPIL.S Transesterification was examined under equilibrium conditions by reacting LdTOPIL.S and LdTOPIL-fus-S with 5¢-32P-end-labeled 25-mer duplex oligonucleotides containing the high-affinity topoisomerase IB-binding site [7,14] The fact that cleavage activity with LdTOPIL-fus-S is enhanced in the presence of the drug suggests that CPT binds to the covalent complex between LdTOPILfus-S and DNA (Fig 4D, lanes and 5) CPT enhanced the formation of the cleavable complex by 40% with respect to the extent of cleavage observed in the absence of the drug This result is similar to that obtained for the wild-type enzyme LdTOPILS (Fig 4D, lanes and 3) Interestingly, LdTOPILfus-D(1–210)S showed reduced efficiency in cleaving 25-mer duplex oligonucleotides both in the absence and the presence of CPT (Fig 4C, lanes and 7) The lower cleavage activity obtained for LdTOPIL-fusD(1–210)S was consistent with the modest reduction in relaxation activity and CPT sensitivity These results indicate that the single ORF resulting from fusion of the large and small subunit genes of Leishmania bisubunit topoisomerase I (LdTOPIL-fusS) encodes for a new functional topoisomerase I The enzyme is conventional in its CPT sensitivity and shows cleavage specificity similar to that of LdTOPILS [7,14], whereas 1–210 amino acids residues from the N-terminal end of the small subunit (LdTOPIS) have a probable role in CPT sensitivity in the fused enzyme (LdTOPIL-fus-S) Gene fusion and its analysis for DNA-binding efficiency To test whether the observed changes in relaxing activity of the fused proteins resulted from increased or decreased affinity of the enzymes for DNA, we carried out native gel mobility shift assays with reconstituted LdTOPILS, monomeric LdTOPIL-fus-S, LdTOPIL-fusD(1–210)S, H453Q and H453A mutant LdTOPIL-fus-S complexed with the 5¢-32P-labeled duplex oligomer containing the high-affinity topoisomerase IB binding site [9], as previously described [7,14] Like LdTOPILS, LdTOPIL-fus-S, LdTOPIL-fusD(1–210)S, H453Q and H453A mutant LdTOPIL-fusS are positively charged, and because the bound oligonucleotide only partially neutralizes the positive charge, the protein–DNA complexes failed to enter the native gel Figure 5A shows the extent of unbound oligonucleotide compared to the oligonucleotide control when binding was carried out with increasing concentrations of the enzymes Under these conditions, Kd is equal to the protein concentration FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS B B Das et al Gene fusion and Leishmania topoisomerase I A B C D Fig Effect of CPT on the relaxation activity equilibrium cleavage with LdTOPILS, LdTOPIL-fus-S and LdTOPIL-fus-D(1–210)S Relaxation of supercoiled pBS (SK+) DNA with LdTOPILS (A) LdTOPIL-fus-S (B) or LdTOPIL-fus-D(1–210)S (C) at a molar ratio of : assayed in the presence or absence of CPT Lanes and 11, 50 fmol of pBS (SK+) DNA Lanes 2–10, same as lane but incubated with 100 fmol of LdTOPIL-fus-S or LdTOPIL-fus-D(1–210)S in the absence of CPT Lanes 11–20, same as lanes 2–10, but in the presence of 60 lM CPT incubated at 37 °C for the time periods indicated in the figure All reactions were stopped by addition of SDS to a final concentration of 0.5% (w ⁄ v); samples were electrophoresed in 1% agarose gel (D) Equilibrium cleavage reactions and electrophoresis in a denaturating polyacrylamide gel were performed as described in Experimental procedures Lane 1, 10 nM 5¢-32P-end-labeled 25-mer duplex oligonucleotides as indicated above Lanes 2–3, same as lane 1, but incubated with equal amounts (0.15 lM) of LdTOPILS Lanes 4–5, incubated with LdTOPIL-fus-S Lanes 6–7, incubated with LdTOPIL-fus-D(1–210)S, in the absence or presence of CPT (60 lM) as indicated Positions of uncleaved oligonucleotide (25-mer) and the cleavage product (12-mer oligonucleotide complexed with residual topoisomerase I) and the scheme of the reaction are indicated at which the amount of unbound oligonucleotides observed in the gel has been reduced by a factor of two [17,18] The binding assays yielded a Kd value of 3.2 · 10)7 m for the interaction of LdTOPILS with the DNA substrate, which is about 5.5-fold higher than the value measured for the interaction of LdTOPIL-fus-S (0.6 · 10)7 m) with DNA (Fig 5B), whereas LdTOPIL-fus-D(1–210)S interacts with DNA substrate with a Kd value of 1.9 · 10)7 m, indicating  3-fold lower affinity than LdTOPIL-fus-S Thus, gene fusion increases the DNA-binding efficiency of LdTOPIL-fus-S approximately 5.5-fold compared to the reconstituted enzyme On the other hand, a  3-fold decrease in the DNA-binding efficiency of LdTOPIL-fus-D(1–210)S compared to LdTOPIL-fus-S correlates well with the decrease in topoisomerase activity of LdTOPIL-fus-D(1–210)S in the plasmid relaxation assay (Fig 5B) FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 155 Gene fusion and Leishmania topoisomerase I A B B Das et al B Fig DNA-binding assays The native gel shift assay was carried out as described in Experimental procedures (A) Autoradiograph of the unbound oligonucleotide for each concentration of protein used corresponding to the DNA control for the enzymes LdTOPILS, LdTOPIL-fus-S and LdTOPIl-fus-D(1–210)S (B) The percentage of unbound duplex oligonucleotide present in the gel was quantified by Phosphorimager and plotted against the protein concentrations The binding profiles for LdTOPILS, LdTOPIL-fus-S and LdTOPILfus-D(1–210)S are indicated The binding profiles revealed that the affinity of the H453Q and H453A mutant LdTOPIL-fus-S for DNA substrate was about the same as that of LdTOPIL-fusS, with Kd values of  0.7 · 10)7 m (data not shown) These results demonstrate that the reduction in relaxing activity with the various changes at position 453 of mutant proteins of LdTOPILfus-S did not result from a defect in DNA binding Suicidal cleavage activity of LdTOPIL-fus-S and its mutant variants We examined the transesterification reaction under suicidal conditions by reacting LdTOPILS, LdTOPILfus-S, LdTOPIL-fus-D(1–210)S, H453Q and H453A mutant LdTOPIL-fus-S with synthetic suicide DNA substrate The substrate consisted of a 5¢-32P-labeled 14 bp duplex with an 11 bp 5¢-tail [11,14] Upon cleavage and formation of a covalent protein–DNA complex, the AG dinucleotide at the 3¢-end of the scissile strand is released Cleavage was performed at 230 °C for the time periods given in Experimental procedures The cleavage activities of the enzymes, as determined by the percentage of substrate converted to products, were plotted as a function of time [19] In the suicidal cleavage assay for LdTOPILS, about 75–80% of the input DNA became covalently bound to protein and reached its cleavage plateau after 30 of incubation, whereas LdTOPIL-fus-S completed the reaction after of incubation; however, interestingly, the cleavage pattern with LdTOPIL-fus-D(1–210)S was approximately 10-fold reduced compared to that with the fused enzyme LdTOPIL-fus-S LdTOPIL-fus-D(1–210)S reached its cleavage plateau after 60 These observations indicate that gene fusion leads to a five-fold enhancement of the apparent suicidal cleavage rate of LdTOPIL-fus-S over LdTOPILS, whereas the deletion mutant LdTOPIL-fus-D(1–210)S was defective in the cleavage reaction compared to LdTOPIL-fus-S This difference probably accounts for the relatively slow 156 plasmid relaxation rate caused by LdTOPUIL-fusD(1–210)S The cleavage reaction with H453Q mutant LdTOPILfus-S reached a plateau after 240 of incubation, whereas the cleavage rates for the H453A protein were just detectable above the background compared to LdTOPIL-fus-S (Fig 6A) Thus, the effects of the various changes at position 453 on the cleavage rates quantitatively parallel the reductions in the rates of relaxation described above, indicating its role either in transesterification chemistry per se or in a step in the reaction pathway that occurs after initial binding prior to strand rotation To gain further insight into the fate of the covalent complexes produced by LdTOPILS, LdTOPIL-fus-S, and LdTOPIS-fus-L with labeled oligonucleotide substrate, the reaction mixtures were analyzed by SDS ⁄ PAGE Coomassie blue-stained SDS ⁄ PAGE shows the mobility of free enzymes (Fig 6B, lanes 1–3) An autoradiograph of the same dried gel shows that the label appears to be associated with LdTOPIS and LdTOPIL-fus-S (Fig 6C, lanes and 5), and this association causes slightly slower migration of enzyme– DNA complex compared to free proteins No LdTOPIS DNA or LdTOPIL-fus-S DNA bands are visible with Coomassie blue staining (Fig 6B), as only a small amount of protein became covalently attached to the DNA, and this became visible after autoradiography (Fig 6C, lanes and 5) Suicide cleavage by LdTOPISfus-L was not achieved under the same conditions (Fig 6C, lane 6) These results demonstrate that the reverse fusion product (LdTOPIL-fus-S) was unable to show topoisomerase I cleavage activity Religation activity of LdTOPIL-fus-S and its mutant variants Religation was studied under single-turnover conditions by assaying the ability of the covalent intermediate to attach a 5¢-hydroxyl-terminated 11-mer to the covalently FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS B B Das et al Gene fusion and Leishmania topoisomerase I A A B C B Fig Suicide cleavage assays (A) DNA cleavage rate for LdTOPILS, LdTOPIL-fus-S, LdTOPIL-fus-D(1–210)S, H453Q and H453A mutant LdTOPIL-fus-S with the 5¢-32P-end-labeled suicide DNA substrate (14-mer ⁄ 25-mer) shown in the figure The reaction mixtures were incubated with the topoisomerase I variants for 1, 5, 10, 15, 30, 60, 120, 180, 240, 300 at 23 °C as described in Experimental procedures Cleavage products were analyzed by denaturating PAGE, and the percentage of cleaved DNA substrate was plotted as a function of time The results depicted were from experiments performed three times, and representative data from one set of these experiments are expressed as means ± SD Variations among different set of experiments were < 5% (B) Coomassie blue-stained SDS-polyacrylamide gel (C) Autoradiograph of the same gel Lanes 1–3, 5¢-32P-end-labeled suicide DNA substrate (14-mer ⁄ 25-mer) was incubated with lg of reconstituted LdTOPILS, LdTOPIL-fus-S and LdTOPIS-fus-L, respectively, in the reaction buffer for h at 23 °C, and reactions were stopped with SDS ⁄ PAGE sample buffer; samples were boiled and loaded onto 10% SDS ⁄ PAGE gel cleaved 12-mer to form a 23-mer product [11,14] The ligation reactions of LdTOPILS, and LdTOPIL-fus-S and LdTOPIL-fus-D(1–210)S, were performed as described in Experimental procedures The results indicated that the religation kinetics for LdTOPILS was approximately two-fold faster than that of LdTOPIL-fus-S However, the religation kinetics for LdTOPIL-fus-D(1–210)S were more or less similar to those of LdTOPIL-fus-S (Fig 7A,B) Therefore, the five-fold faster cleavage rate and the two-fold reduced religation rate of Leishmania fused topoisomerase I (LdTOPIL-fus-S) accounts for a small shift in Fig Religation activity (A) Religation activity of LdTOPILS and LdTOPIL-fus-S Active cleavage complexes containing LdTOPILS or LdTOPIL-fus-S covalently attached to the covalently cleaved 12-mer of the suicide substrate were reacted with 5¢-hydroxyl-terminated 11-mer to form a 23-mer product for 15, 30 and 60 s at 37 °C, and the products were analyzed as above Religated product, active covalent complex and uncleaved product are indicated (B) The relative amount of cleavage product converted to ligation product in each sample for LdTOPILS and LdTOPIL-fus-S was plotted as function of time The religation reactions were stopped after 15, 30, 60, 120, 150 s at 37 °C, and the products were analyzed as above The results depicted were from experiments performed three times, and representative data from one set of these experiments are expressed as means ± SD Variations among different set of experiments were < 5% the cleavage–religation equilibrium towards cleavage compared to the reconstituted enzyme, and correlates with the increase of activity in the plasmid DNA relaxation assay Role of His453 of LdTOPIL in the fused enzyme construct The crystal structure of Leishmania heterodimeric topoisomerase I shows that the Ne2 atom of His453 of FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 157 Gene fusion and Leishmania topoisomerase I B B Das et al Fig Effect of pH on suicide cleavage rate for LdTOPIL-fus-S and H632Q mutants of LdTOPIL-fus-S proteins The rate of suicide cleavage was measured as described in Experimental procedures, and the logarithm (base 10) of the rates was plotted as a function of pH LdTOPIL forms a hydrogen bond with the nonbridging oxygen atom of the vanadate Hence, we assumed that the His side chain might possibly serve as a general acid that donates a proton to the leaving 5¢-hydroxyl as cleavage occurs [10] If His453 were to act as a general acid, deprotonation of the imidazole ring with increased pH should reduce the rate of the cleavage reaction for LdTOPIL-fus-S, but a similar increase in pH should have no effect on cleavage by the H453Q mutant enzyme To test this prediction, we measured the cleavage rates of both LdTOPIL-fus-S and the H453Q mutant LdTOPIL-fus-S proteins at the following pH values: 6, 6.5, 7, 7.5, 8, 8.5, and 9.5 As shown in Fig 8, the activity of LdTOPIL-fus-S decreases slightly over the pH range from 7.5 to 9.5, but the response of the H453Q mutant enzyme was very similar Thus, it appears unlikely that His453 of LdTOPIL for the fused enzyme acts as a general acid that donates a proton to the leaving 5¢-oxygen In eukaryotic type IB enzymes, the conserved His residue is involved solely in phosphate binding and transition state stabilization Some bacterial type IB enzymes have an Asn residue [3] in place of this His residue This further suggests a generalized role of His453 rather than a specific role as a proton donor Discussion The crystal structure of monomeric human topoisomerase I seems compatible with a rotational model for the relief of supercoils during DNA relaxation Modeling studies have indicated that the DNA would probably contact both the CAP and linker regions of the protein during strand rotation [10,12] The linker 158 domain, which is poorly conserved and variable in length, links the core and catalytic domains of the monomeric enzyme and is responsible for the activity of the enzyme and CPT sensitivity [10] However, interestingly, L donovani topoisomerase I is an unusual bisubunit enzyme in which the functional linker is absent between the core DNA-binding domain and the catalytic domain which is harbored in a separate subunit [6,7,23] Our recent findings reveal that 1–39 amino acid residues of the large subunit that resemble the CAP region of the monomeric enzymes have a modulating role in noncovalent interactions with DNA and sensitivity towards CPT [14] Thus, it is interesting to observe the change in the catalytic properties of the heterodimeric enzyme when the two subunits are fused We also investigated the role of the conserved His453 residue in the large subunit (LdTOPIL) during enzyme catalysis Our studies provide insights into the mechanistic conservation of topoisomerase IB function in the Leishmania heterodimeric enzyme Change in the catalytic efficiency due to gene fusion We describe here the significant modulation of in vitro DNA relaxation due to gene fusion We have previously shown that the reconstituted LdTOPILS has reduced activity in plasmid relaxation assays LdTOPILS appears to leave intermediate substrates after removing only a few supercoils at a time This accounts for the higher dissociation rate, yielding a higher turnover number [7], whereas under the same conditions, the relaxation mode of the fused enzyme LdTOPIL-fus-S was found to be more processive, and is going through multiple rounds of relaxation before dissociating from its substrate DNA (Fig 2B), which is well manifested by decreases in the enzyme dissociation rate and turnover number (data not shown) These observations were further supported by a  5.5-fold increase in the DNA-binding affinity of LdTOPIL-fus-S (Kd of 0.6 · 10)7 m) compared to the reconstituted enzyme LdTOPILS (Kd of 3.2 · 10)7 m) This observation is consistent with that of reconstituted human topoisomerase I that has been artificially divided into two proteins (topo58 ⁄ 12 or topo58 ⁄ 6.3), which are highly distributive, and bind DNA at a lower affinity than that of the intact enzyme [13] The enhanced activity in the plasmid DNA relaxation assay shown by LdTOPIL-fus-S correlated well with the increase in cleavage rates seen under singleturnover condition; that is, LdTOPIL-fus-S shows a five-fold increase in cleavage rate over reconstituted LdTOPILS Interestingly, the fused enzyme shows an FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS B B Das et al approximately two-fold slower religation rate compared to the reconstituted Leishmania enzyme Therefore, the greater cleavage rates for LdTOPIL-fus-S and slower religation compared to LdTOPILS account for a shift in the cleavage–religation equilibrium towards cleavage, as observed in monomeric human topoisomerase I [10,12] However, both the Leishmania enzymes (LdTOPILS and LdTOPIL-fus-S) show functional conservation, i.e substrate specificity and CPT sensitivity similar to those of eukaryotic topoisomerase IB (Fig 4A,C) Therefore, gene fusion may account for the control of noncovalent DNA binding or coordination of DNA contacts by other parts of the enzyme Comparing the crystal structure of human and vaccinia topoisomerase I enzymes, it is evident that a precleavage conformational change in the core and catalytic domains is necessary to establish the correct position of the active site Tyr for nucleophilic attack on DNA [19–21] This implies that the reverse fusion (LdTOPIS-fus-L) may lead to a conformational change in the topoisomerase architecture, leading to loss of activity Effect of deletion of 1–210 amino acids from the N-terminus of LdTOPIS The small subunit (LdTOPIS) shares 43.5% sequence identity with the C-terminal domain of human topoisomerase I, including alignment of conserved sequences surrounding the catalytic Tyr residue LdTOPIS contains a large nonconserved N-terminal extension (startMet-Asn210), enriched in serine residues that might be potential sites of phosphorylation [8] Reconstitution of LdTOPIL with truncated LdTOPI-D(1–210)S shows topoisomerase I activity (data not shown) Interestingly when LdTOPI-D(1–210)S was fused to intact LdTOPIL to create a single ORF LdTOPIL-fus-D(1– 210)S, it showed decreased topoisomerase I activity and sensitivity towards CPT in plasmid DNA relaxation experiments compared to LdTOPIL-fus-S (Fig 4) The reduced relaxation activities of LdTOPIL-fus-D(1– 210)S (Fig 3) correlated well with decreased cleavage rates under suicidal conditions; that is, LdTOPIL-fusD(1–210)S showed a 10-fold reduction in cleavage rate compared to LdTOPIL-fus-S This finding is consistent with the results of the 25-mer duplex oligonucleotide equilibrium cleavage assay A low level of cleavage was observed for LdTOPIL-fus-D(1–210)S in the presence or absence of CPT compared to cleavage by LdTOPIL-fus-S or LdTOPILS (Fig 4D) Hence, we surmise that 1–210 amino acid residues from the N-terminal end of the small subunit probably act as a ‘pseudolinker’ in the fused LdTOPIL-fus-S Gene fusion and Leishmania topoisomerase I construct Owing to gene fusion, some additional contacts (1–210 amino acid of LdTOPIS) perhaps account for the prominent role in the cleavage step or in the steps preceding cleavage, i.e DNA binding The later possibility is supported by  3-fold decreased binding affinity of the mutant LdTOPIL-fus-D(1–210)S (Kd of 1.9 · 10)7 m) compared to that of LdTOPILfus-S (Kd of 0.6 · 10)7 m) These findings are in keeping with those for human topoisomerase I, where it was demonstrated that the linker domain participates in a network of correlated movements with key regions of the enzyme involved in the human topoisomerase I catalytic cycle, providing a structural–dynamic explanation for the better DNA relaxation activity and CPT sensitivity of topo70 when compared to topo58 ⁄ 6.3 [21] Role of His453 (LdTOPIL) in enzyme catalysis The catalytic activity of type IB topoisomerases is derived chiefly from five strictly conserved amino acid residues In human topoisomerase I, the residues constituting this active site pentad are Arg488, Lys532, Arg590, His632, and Tyr723 The analogous residues Arg314, Lys352, Arg410 and His453 are also conserved in the large subunit of the Leishmania enzyme, and the smaller catalytic subunit harbors the consensus SKXXY motif [8,23] In the present study we also investigated the role of His453 of LdTOPIL in the transesterification reaction Point mutations leading to changes in His265 of the structurally similar vaccinia topoisomerase I and His632 of human topoisomerase I have adverse effects on the transesterification reaction catalyzed by the two enzymes, and changes at this position appear to perturb the corresponding active sites somewhat differently [8,9,21] Unlike His, the Glu and Ala side chain mutants of LdTOPIL-fus-S show appreciable variation in their effects on concerted topoisomerase I action We found that replacing His453 in fused Leishmania topoisomerase I with Glu caused a 40-fold reduction in the rate of relaxation and suicide cleavage With Ala, both relaxation and suicide cleavage were reduced to nearly undetectable levels From the mutational analysis, it seems most likely that the active site His453 of LdTOPIL plays a major role in stabilizing the pentavalent transition state of the enzyme through an interaction with the nonbridging oxygen of the scissile phosphate of the DNA [10] In conclusion, our gene fusion studies improve our knowledge of the unusual structure of L donovani heterodimeric topoisomerase I Our study also shows that unconserved N-terminal extended regions of the small subunit (amino acids 1–210) have a role in controlling noncovalent DNA binding and CPT sensitivity Thus, FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 159 Gene fusion and Leishmania topoisomerase I B B Das et al fusion of two subunits leads to better coordination between the two subunits of the Leishmania enzyme, permitting the fused enzyme to remain associated with DNA for a larger number of cleavage and religation rounds These probably account for the increase in activity of the fused enzyme On the other hand, our point mutational studies demonstrate that His453 of LdTOPIL contributes to the active site of the Leishmania heterodimeric topoisomerase I by stabilizing the enzyme–DNA transition state through hydrogen bonding to one of the nonbridging oxygens, and are in agreement with the common theme of eukaryotic type IB topoisomerase I action Experimental procedures Site-directed mutagenesis Construction of recombinant plasmid The full-length large subunit gene (LdTOPIL) and small subunit gene (LdTOPIS) waere cloned in the NdeI–BamHI site of the bacterial expression vector pET16b [6] For preparation of the fusion construct LdTOPIL-fus-LdTOPIS, the region corresponding to amino acids 1–634 of LdTOPIL was first amplified by PCR using pET16bLdTOPIL as a template The sense primer was 5¢-CGGGATCC TGATGAAGGTGGAGAATAGC-3¢, containing a BamHI site created at the initiation codon, and the antisense primer was 5¢-CGGAATTCCACCCTCAAAGCTGCAAGAGG3¢, with an EcoRI site immediately downstream before the termination codon, so that the stop codon was removed in the PCR-amplified product The amplified products were cloned in the BamHI–EcoR1 site of pET28c, resulting in the construct pET28cLdTOPIL Next, the ORF of the small subunit (LdTOPIS) was PCR amplified using sense primer 5¢-CGGAATTCATGCAGCCTGTTCAAAGTCCT3¢, containing an EcoRI site created at the initiation codon of the ORF, and an antisense primer 5¢-CCCAAGCTTAC TAAAATCGAAGTTCTCGGC-3¢, with a HindIII site immediately downstream from the termination codon The PCR-amplified fragment was cloned in the EcoRI–HindIII site of bacterial expression vector pET28cLdTOPIL, resulting in the fused construct pET28cLdTOPIL-fus-S Thus, a single ORF consisting of 2685 bp encoding a new topoisomerase I was generated Similarly, a reverse fusion construct LdTOPIS-fus-LdTOPIL was generated by PCR amplification, where the stop codon of LdTOPIS was removed and fused to LdTOPIL in bacterial expression vector pET28c under the restriction site of BamH1–EcoR1– HindIII, resulting in the reversed-fused construct pET28c LdTOPIS-fus-L The resultant constructs pET28cLdTOPIL-fus-S and pET28cLdTOPIS-fus-L were transformed in E coli BL21 (DE3) pLysS as previously described [6,13] For construction of the N-terminal truncation mutant of small subunit LdTOPIS, the region corresponding to amino 160 acids 211–262 was amplified by PCR using pET16bLdTOPIS as a template The sense primer was 5¢-CGGA ATTCATGAAGGCTGTGTCGCTCGGCACC-3¢, containing an EcoRI site created at the initiation codon of the ORF, and the antisense primer was 5¢-CCCAAGCT TACTAAAATCGAAGTTCTCGGC-3¢, with a HindIII site immediately downstream from the termination codon The PCR-amplified fragment was cloned in the EcoRI–HindIII site of the bacterial expression vector harboring the intact large subunit (pET28cLdTOPIL), resulting in the fused construct pET28cLdTOPIL-fus-D(1–210)S, was transformed in E coli BL21(DE3)pLysS as previously described [7,14] In all cases, the integrity of the constructs was confirmed by DNA sequencing Single mutations were introduced into the Leishmania heterodimeric fused topoisomerase I at position His453 of LdTOPIL Mutagenesis was performed using the Stratagene (La Jola, CA, USA) QuikChange kit, following the manufacturer’s protocol To carry out the desired mutations using the QuikChange kit, the N-terminal His-tagged expression plasmid (pET28c) harboring the LdTOPIL-fusLdTOPIS gene (pET28cLdTOPIL-fus-S) was used directly as the template The following sense primers (along with their antisense counterparts), with their substitution sites in bold, were used: H453A, 5¢-GCCATTCTGTGCAACGCT CAGAACTCCGTCTCG-3¢, and H453Q, 5¢-GCCATTCT GTGCAACCACCAGAACTCCGTCTCG-3¢ Mutations were confirmed by automated DNA sequencing in a Perkin Elmer (Norwalk, CT, USA) ABI PrismTM DNA sequencer Overexpression and purification of recombinant proteins Escherichia coli BL21(DE3)pLysS cells harboring pET28cLdTOPIL-fus-S, pET28cLdTOPIS-fus-L, pET28cLdTOPIL-fus-D(1–210)S, pET28cLdTOPIL-fus-S(H453A), pET28c LdTOPIL-fus-S(H453Q), pET16bLdTOPIL and pET16b LdTOPIS were separately induced at A600 ¼ 0.6 with 0.5 mm isopropyl thio-b-d-galactoside (IPTG) at 22 °C for 12 h Cells harvested from L of culture were separately lysed by lysozyme ⁄ sonication, and the proteins were purified with an Ni2+–nitrilotroacetic acid agarose column (Qiagen, Valencia, CA, USA) and a phosphocellulose column (P11 cellulose, Whatman, Maidstone, UK), as described previously [7] Finally, the purified proteins LdTOPIL-fus-S, LdTOPIS-fus-L, LdTOPIL-fus-D(1–210)S, H453A (LdTOPIL-fus-S), H453Q (LdTOPIL-fus-S), LdTOPIL and LdTOPIS were stored at ) 70 °C The concentrations of purified proteins were quantified by the Bradford reaction using a Bio-Rad Protein Estimation Kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s protocol FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS B B Das et al Reconstitution of topoisomerase I activity Purified LdTOPIL was mixed with purified LdTOPIS separately at a molar ratio of : and at a total protein concentration of 0.5 mgỈmL)1 in reconstitution buffer (50 mm potassium phosphate, pH 7.5, 0.5 mm dithiothreitol, mm EDTA, 0.1 mm phenylmethanesulfonyl fluoride, 10% glycerol) The mixtures were dialyzed overnight at °C, and the dialyzed fractions were used for determination of plasmid relaxation activity [7,14] Plasmid relaxation assay The type I DNA topoisomerase was assayed by decreased mobility of the relaxed isomers of supercoiled pBluescript (SK+) DNA in an agarose gel The relaxation assay was carried out as previously described [7,14] with LdTOPILS, LdTOPIL-fus-S and LdTOPIS-fus-L, LdTOPIL-fus-D(1– 210)S, H453A (LdTOPIL-fus-S) and H453Q (LdTOPILfus-S) serially diluted in the relaxation buffer (25 mm Tris ⁄ HCl, pH 7.5, 5% glycerol, 0.5 mm dithiothreitol, 10 mm MgCl2, 2.5 mm EDTA, 150 lgỈmL)1 BSA), supercoiled pBluscript (SK+) DNA (85–95% were negatively supercoiled, with the remainder being nicked circles), and 50 mm KCl Analysis of duplex oligonucleotides ) cleavage assay The 25-mer duplex of oligonucleotide (5¢-GAAAAAAG ACTT_AGAAAAATTTTTA-3¢) and oligonucleotide (5¢-TAAAAATTTTTCTAAGTCTTTTTTC-3¢) containing a topoisomerase I-binding motif was [c-32P]ATP labeled and annealed as previously described [11,14] Cleavage was carried out using a 20-fold molar excess of the wild-type (LdTOPILS) and fused enzymes [LdTOPIL-fus-S and LdTOPIL-fus-D(1–210)S] over duplex 25-mer DNA (enzymes, 0.2 lm; DNA, 10 nm) in the presence and absence of 60 lm CPT, as described previously [14] All the reactions were stopped by addition of SDS to a final concentration of 2% (w ⁄ v) Samples were precipitated with ethanol, digested with lL of mgỈmL)1 trypsin, and analyzed with 12% denaturing polyacrylamide gel The amount of strand cleavage in the presence of drugs for LdTOPILS, LdTOPILfus-S and LdTOPIL-fus-D(1–210)S were quantified by using a Phosphorimager (Bio-Rad Molecular Imager system) Analysis of topoisomerase I–DNA interaction by electrophoretic mobility shift assay The c-32P end labeling and annealing of the 25-mer duplex of oligonucleotide were performed as described above The DNA-binding assay was performed by incubating the labeled oligo1 ⁄ oligo2 (1 nm) in 25 lL of reaction mixture as Gene fusion and Leishmania topoisomerase I described previously [7,14] The concentrations of LdTOPILS, LdTOPIL-fus-S, LdTOPIL-fus-D(1–210)S, H453A and H453Q mutants of LdTOPIL-fus-S protein used in the assay ranged from lm to 0.02 lm The reaction mixtures were incubated at 15 °C for 15 min, and electrophoresed in 6% nondenaturating polyacrylamide gel at °C Owing to the high pI values for the Leishmania topoisomerase I proteins (> 9.0), free protein and protein–DNA complexes migrated to the cathode, and therefore only the free oligonucleotides entered the gel The unbound oligonucleotides in the gel were quantified by using a Phosphorimager (BioRad Molecular Imager system) The Kd was estimated from the protein concentration at which one half of the amount of duplex oligonucleotide was bound to the protein [18] Suicidal cleavage assay A 14-mer (5¢-GAAAAAAGACTTAG-3¢) oligonucleotide containing a topoisomerase IB-specific cleavage site was 5¢-32P end labeled and annealed to a 25-mer (3¢-CTT TTTTCTGAATCTTTTTAAAAAT-5¢) oligonucleotide as previously described [11,14] The suicidal cleavage reaction was carried out with nm DNA substrate and 0.18 lm enzymes [LdTOPILS, LdTOPIL-fus-S, LdTOPIL-fus-D(1– 210)S, H453A (LdTOPIL-fus-S) and 453Q (LdTOPIL-fusS)] in 20 lL of reaction mix under standard assay conditions (10 mm Tris ⁄ Cl, pH 7.5, 10 mm MgCl2, 0.5 mm EDTA, 50 mm KCl) at 23 °C for the indicated time periods All reactions were stopped by addition of SDS to a final concentration of 2% (w ⁄ v) Samples were precipitated with 0.4 mm NaCl and three volumes of ethanol, and subsequently digested with lL of mgỈmL)1 trypsin in 10 mm Tris ⁄ Cl (pH 7.5) and mm EDTA for 30 at 37 ¢C For analysis, samples were mixed with lL of formamide dye (80% deionized formamide, 50 mm Tris ⁄ borate, pH 8.3, mm EDTA, 0.05% xylene cyanol, 0.05% bromophenol blue), boiled, electrophoresed in 12% denaturing polyacrylamide gel, and autoradiographed [24] The percentage of substrate converted to cleavage product was determined with a Phosphorimager (Bio-Rad Molecular Imager system) by using Bio-Rad quality one software, and was plotted as a function of time [24] To determine the effect of pH on the suicide cleavage rate by LdTOPIL-fus-S and H453Q (LdTOPIL-fus-S), the cleavage buffer was modified using the following buffers (50 mm) to the indicated pH values: sodium 2-(N-morpholino)ethanesulfonic acid, pH 6.0, 6.5; and Tris ⁄ HCl, pH 7, 7.5, 8, 8.5; and sodium 3-(cyclohexylamino)-1-propanesulfonic acid, pH 9.5 The percentage cleavage (Cl%) was quantified as described above, and the logarithm of the cleavage rate (Kcl) was determined by fitting the data from the first three time (t) points to the equation ln(100 ) Cl%) ¼ 4.605 ) (Kclt) as described previously [9], and plotted as a function of pH FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 161 Gene fusion and Leishmania topoisomerase I B B Das et al For further analysis, the suicidal cleavable complexes formed with the enzymes (LdTOPIL-fus-S and LdTOPISfus-L) after h of incubation at 23 °C were boiled with lL of SDS loading buffer (5% SDS, 20% glycerol, 100 mm Tris ⁄ Cl, pH 8.0, 5% 2-mercaptoethanol, 0.12% bromophenol blue) and analyzed by 10% SDS ⁄ PAGE The gel was stained with Coomassie blue to visualize the protein bands, and dried before exposure to the film to detect the radiolabeled proteins [14] Single-turnover religation For religation experiments, covalent complexes were generated by incubating nm suicide DNA substrate with 0.18 lm enzymes [LdTOPILS, LdTOPIL-fus-S and LdTOPIL-fus-D(1–210)S] for h at 23 °C as above Under these conditions, suicidal cleavage of 70–80% of the input DNA was observed Religation was initiated by the addition of a 300-fold molar excess of the 11-mer religation acceptor oligonucleotides (5¢-OH-AGAAAAATTTT-3¢) in the same reaction mix and incubation for indicated time periods as described previously [14] The reaction was carried out under standard conditions (10 mm Tris ⁄ HCl, pH 7.5, 10 mm MgCl2, 0.5 mm EDTA and 50 mm KCl) in a 30 lL reaction volume at 37 °C for the indicated time periods All the reactions were stopped by adding SDS, and DNAs were subsequently precipitated by ethanol Finally, samples were precipitated, digested with lL of mgỈmL)1 trypsin, electrophoresed in 12% denaturing polyacrylamide gel, and analyzed by using a Phosphorimager (Bio-Rad Molecular Imager system) Acknowledgements We thank Professor S Roy, the director of our institute, for his interest in this work This work was supported by grants from the Department of Biotechnology, Government of India (BT ⁄ PR6399 ⁄ BRB ⁄ 10 ⁄ 434 ⁄ 05) and the Network Project SMM-003 of the Council of Scientific and Industrial Research (CSIR), Government of India, to HKM BBD and SBDG are supported by Senior Research Fellowship from the Council for Scientific and Industrial Research, Government of India References Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective Nat Rev Mol Cell Biol 3, 430–440 Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism Annu Rev Biochem 70, 369– 413 Krogh BO & Shuman S (2002) A poxvirus-like type IB topoisomerase family in bacteria Proc Natl Acad Sci USA 99, 1853–1858 162 Corbett KD & Berger JM (2004) Structure, molecular 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for covalent catalysis by vaccinia topoisomerase Gene fusion and Leishmania topoisomerase I and is conserved in all eukaryotic type I enzymes J Biol Chem 272, 3891–3896 22 Ferst A (1999) Structure and Mechanism in Protein Science: a Guide to Enzyme Catalysis and Protein Folding WH Freeman, New York, NY 23 Das A, Dasgupta A, Sengupta T & Majumder HK (2004) Topoisomerases of kinetoplastid parasites as potential chemotherapeutic targets Trends Parasitol 20, 381–387 24 Frohlich RF, Andersen FF, Westergaard O, Andersen HA & Knudsen BR (2004) Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding J Mol Biol 336, 93–103 FEBS Journal 274 (2007) 150–163 ª 2006 The Authors Journal compilation ª 2006 FEBS 163 ... compilation ª 2006 FEBS 151 Gene fusion and Leishmania topoisomerase I B B Das et al Fig (A) Schematic alignment Monomeric (human vaccinia and bacterial) topoisomerase IB aligned with bisubunit topoisomerase. .. describe experiments in which Leishmania bisubunit topoisomerase I large subunit (LdTOPIL) and small subunit (LdTOPIS) genes were fused into a single ORF encoding a new topoisomerase I (LdTOPIL-fus-S)... insights into the mechanistic conservation of topoisomerase IB function in the Leishmania heterodimeric enzyme Change in the catalytic efficiency due to gene fusion We describe here the significant