'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.     

A novel active DNA topoisomerase I in Leishmania donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.     

Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovani

The amino acid sequences of the C-terminal domain (CTD) of the type II DNA topoisomerases are divergent and species specific as compared with the highly conserved N-terminal and central domains. A set of C-terminal deletion mutants of Leishmania donovani topoisomerase II was constructed. Removal of more than 178 amino acids out of 1236 amino acid residues from the C-terminus inactivates the enzyme, whereas removal of 118 amino acids or less has no apparent effect on the ability of the parasite enzyme to complement a temperature-sensitive mutation of the Saccharomyces cerevisiae topoisomerase II gene. Deletion analysis revealed a potent nuclear localization signal (NLS) within the amino acid residues 998–1058. Immunomicroscopy results suggest that the removal of an NLS in the CTD is likely to contribute to the physiological dysfunction of these proteins. Modeling of the LdTOP2 based on the crystal structure of the yeast type II DNA topoisomerase showed that the parasite protein assumes a structure similar to its yeast counterpart harboring all the conserved residues in a structurally similar position. However, a marked difference in electrostatic potential was found in a span of 60 amino acid residues (998–1058), which also do not have any homology with topoisomerase II sequences. Such significant differences can be exploited by the structure-based design of selective inhibitors using the structure of the Leishmania enzyme as a template.     

Topoisomerases and site-specific recombinases: similarities in structure and mechanism

The processes of DNA topoisomerization and site-specific recombination are fundamentally similar: DNA cleavage by forming a phospho-protein covalent linkage, DNA topological rearrangement, and DNA ligation coupled with protein regeneration. Type IB DNA topoisomerases are structurally and mechanistically homologous to tyrosine recombinases. Both enzymes nick DNA double helices independent of metal ions, form 3′-phosphotyrosine intermediates, and rearrange the free 5′ ends relative to the uncut strands by swiveling. In contrast, serine recombinases generate 5′-phospho-serine intermediates. A 180° relative rotation of the two halves of a 100?kDa terameric serine recombinase and DNA complex has been proposed as the mechanism of strand exchange. Here I propose an alternative mechanism. Interestingly, the catalytic domain of serine recombinases has structural similarity to the TOPRIM domain, conserved among all Type IA and Type II topoisomerases and responsible for metal binding and DNA cleavage. TOPRIM topoisomerases also cleave DNA to generate 5′-phosphate and 3′-OH groups. Based on the existing biochemical data and crystal structures of topoisomerase II and serine recombinases bound to pre- and post-cleavage DNA, I suggest a strand passage mechanism for DNA recombination by serine recombinases. This mechanism is reminiscent of DNA topoisomerization and does not require subunit rotation.     

Topoisomerases and site-specific recombinases: similarities in structure and mechanism

The processes of DNA topoisomerization and site-specific recombination are fundamentally similar: DNA cleavage by forming a phospho-protein covalent linkage, DNA topological rearrangement, and DNA ligation coupled with protein regeneration. Type IB DNA topoisomerases are structurally and mechanistically homologous to tyrosine recombinases. Both enzymes nick DNA double helices independent of metal ions, form 3'-phosphotyrosine intermediates, and rearrange the free 5' ends relative to the uncut strands by swiveling. In contrast, serine recombinases generate 5'-phospho-serine intermediates. A 180° relative rotation of the two halves of a 100 kDa terameric serine recombinase and DNA complex has been proposed as the mechanism of strand exchange. Here I propose an alternative mechanism. Interestingly, the catalytic domain of serine recombinases has structural similarity to the TOPRIM domain, conserved among all Type IA and Type II topoisomerases and responsible for metal binding and DNA cleavage. TOPRIM topoisomerases also cleave DNA to generate 5'-phosphate and 3'-OH groups. Based on the existing biochemical data and crystal structures of topoisomerase II and serine recombinases bound to pre- and post-cleavage DNA, I suggest a strand passage mechanism for DNA recombination by serine recombinases. This mechanism is reminiscent of DNA topoisomerization and does not require subunit rotation.     

A tyrosyl DNA phosphodiesterase 1 from kinetoplastid parasite Leishmania donovani (LdTdp1) capable of removing topo I–DNA covalent complexes

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3′‐DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C‐terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H₂O₂‐mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT‐resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I‐mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.     

The structure of the transition state of the heterodimeric topoisomerase I of Leishmania donovani as a vanadate complex with nicked DNA

Type IB topoisomerases are essential enzymes that are responsible for relaxing superhelical tension in DNA by forming a transient covalent nick in one strand of the DNA duplex. Topoisomerase I is a target for anti-cancer drugs such as camptothecin, and these drugs also target the topoisomerases I in pathogenic trypanosomes including Leishmania species and Trypanosoma brucei. Most eukaryotic enzymes, including human topoisomerase I, are monomeric. However, for Leishmania donovani, the DNA-binding activity and the majority of residues involved in catalysis are located in a large subunit, designated TOP1L, whereas the catalytic tyrosine residue responsible for covalent attachment to DNA is located in a smaller subunit, called TOP1S. Here, we present the 2.27A crystal structure of an active truncated L.donovani TOP1L/TOP1S heterodimer bound to nicked double-stranded DNA captured as a vanadate complex. The vanadate forms covalent linkages between the catalytic tyrosine residue of the small subunit and the nicked ends of the scissile DNA strand, mimicking the previously unseen transition state of the topoisomerase I catalytic cycle. This structure fills a critical gap in the existing ensemble of topoisomerase I structures and provides crucial insights into the catalytic mechanism.     

Resolution of Holliday junction substrates by human topoisomerase I

Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases.     

p-Thiophenylalanine-induced DNA cleavage and religation activity of a modified vaccinia topoisomerase IB

Vaccinia DNA topoisomerase IB is the smallest of the type IB topoisomerases. Because of its small size (314 amino acids) and target site specificity (5'(C/T)CCTTp(↓) sites), it constitutes an excellent model for studying the interaction of type IB enzymes with duplex DNA. In this study, p-thiophenylalanine was incorporated into the enzyme active site (position 274) by in vitro translation in the presence of a chemically misacylated tRNA. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate (k(cl) = 1 × 10(-4) s(-1)) was 30 times lower than for the wild-type enzyme (k(cl) = 3 × 10(-3) s(-1)). In contrast, thiophenylalanine-induced DNA religation was faster than that of the wild-type enzyme. We propose that the change in kinetics reflects the difference in bond energies between the O-P and S-P bonds being formed and broken in the reactions catalyzed by the wild-type and modified enzymes. We also studied the effect of adding Mg(2+) and Mn(2+) to the wild-type and modified topoisomerases I. Divalent metal ions such as Mg(2+) and Mn(2+) increased DNA relaxation activity of the wild-type and modified enzymes. However, the pattern of increases failed to support the possibility that metal ion-heteroatom interaction is required for catalysis.     

Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I

The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining.     

Crystal structure of a bacterial type IB DNA topoisomerase reveals a preassembled active site in the absence of DNA

Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-A crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) beta-sheet domain (amino acids 1-90) and a predominantly alpha-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an "open" conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.     

Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase.

In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.     

Characterization of mimivirus DNA topoisomerase IB suggests horizontal gene transfer between eukaryal viruses and bacteria

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5'-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.     

Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I

Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase–DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.     

Inhibition of Flp recombinase by the topoisomerase I-targeting drugs, camptothecin and NSC-314622

Recombinases of the lambda-Int family and type IB topoisomerases act by introducing transient single strand breaks in DNA using chemically identical reaction schemes. Recent structural data have supported the relationship between the two enzyme groups by revealing considerable similarities in the architecture of their catalytic pockets. In this study we show that the Int-type recombinase Flp is inhibited by the two structurally unrelated topoisomerase I-directed anti-cancer drugs, camptothecin (CPT) and NSC-314622. The interaction of these drugs with topoisomerase I is very specific with several single amino acid substitutions conferring drug resistance to the enzyme. Thus, the observed interaction of CPT and NSC-314622 with Flp, which is comparable to their interaction with the cleavage complex formed by topoisomerase I, strongly supports a close mechanistic and evolutionary relationship between the two enzymes. The results suggest that Flp and other Int family recombinases may provide model systems for dissecting the molecular mechanisms of topoisomerase I-directed anti-cancer therapeutic agents.     

Thirteen-exon-motif signature for vertebrate nuclear and mitochondrial type IB topoisomerases

DNA topoisomerases contribute to various cellular activities that involve DNA. We previously identified a human nuclear gene that encodes a mitochondrial DNA topoisomerase. Here we show that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. A 13-exon topoisomerase motif was identified as a characteristic of genes for both nuclear and mitochondrial type IB topoisomerases. The presence of this signature motif is thus an indicator of the coexistence of nuclear and mitochondrial type IB DNA topoisomerases. We hypothesize that the prototype topoisomerase IB with the 13-exon structure formed first, and then duplicated. One topoisomerase specialized for nuclear DNA and the other for mitochondrial DNA.     

Topoisomerases of kinetoplastid parasites: why so fascinating?

DNA topoisomerases are the key enzymes involved in carrying out high precision DNA transactions inside the cells. However, they are detrimental to the cell when a wide variety of topoisomerase-targeted drugs generate cytotoxic lesions by trapping the enzymes in covalent complexes on the DNA. The discovery of unusual heterodimeric topoisomerase I in kinetoplastid family added a new twist in topoisomerase research related to evolution, functional conservation and their preferential sensitivity to Camptothecin. On the other hand, structural and mechanistic studies on kinetoplastid topoisomerase II delineate some distinguishing features that differentiate the parasitic enzyme from its prokaryotic and eukaryotic counterparts. This review summarizes the recent advances in research in kinetoplastid topoisomerases, their evolutionary significance and the death of the unicellular parasite Leishmania donovani induced by topoisomerase I inhibitor camptothecin.     

An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity

Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.     

Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.