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

Broccoli, Sonia; Marquis, Jean-François; Papadopoulou, Barbara; Olivier, Martin; Drolet, Marc

doi:10.1093/nar/27.13.2745

Cited by 33 publications

(23 citation statements)

References 25 publications

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“…To address this possibility, we sequenced the Topo-I genes (LdTOP1A and LdTOP1B) of the wild-type and resistant parasites. LdRCPT.160 revealed the presence of two amino acid substitutions (Gly185Arg and Asp325Glu) in the core domain of LdTOP1A protein, which is highly conserved among known members of the IB (eukaryotic) topoisomerase family (4). Based on recent evidence that the Leishmania Topo-I is an unusual bisubunit enzyme (LdTOP1L and LdTOP1S) (10) and that the mutations that we identified are localized to the gene coding for the long subunit, we hypothesized that this enzyme portion is crucial for the cytotoxic action of CPT in Leishmania.…”

Section: Discussionmentioning

confidence: 99%

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Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in Leishmania donovani

Marquis

Hardy

Olivier

2005

Antimicrob Agents Chemother

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The antitumor compound camptothecin (CPT) is also recognized for its specific activity against Leishmania donovani topoisomerase I (Topo-I). In consequence, defining CPT resistance mechanisms represents an important strategic tool in the acquisition of a better understanding of its mode of action. In the present study, we selected a single highly resistant L. donovani strain termed LdRCPT.160 by stepwise exposure to CPT. Gene sequencing revealed two single nucleotide mutations in the LdRCPT.160 LdTOP1A gene, resulting in two amino acid substitutions (Gly185Arg and Asp325Glu) in the protein. Moreover, these two substitutions observed in the LdTOP1A protein were correlated with a decreased Topo-I DNA relaxation activity in these resistant parasites. Nevertheless, there was no change in the LdTOP1A gene expression level. Interestingly, transfection studies of the LdRCPT.160 LdTOP1A gene in its wild-type counterpart showed that it induced CPT resistance. Sitedirected mutagenesis studies demonstrated that, despite a substantial level of resistance conferred by the Gly185Arg and Asp325Glu substitutions separately, both were essential to reach a high-resistance phenotype. Of interest, the amino acid substitutions observed in LdRCPT.160 LdTOP1A protein occurred near the amino acids previously predicted to interact with CPT, providing new insight into the mechanism of CPT molecular action.

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Section: Discussionmentioning

confidence: 99%

“…Topo-I introduces transient single-stranded DNA breaks in one of the phosphodiester backbones of the duplex DNA and results in a reversible Topo-I/DNA covalent complex (5)(6)(7). In 1999, we cloned and sequenced the first Topo-I gene from L. donovani (LdTOP1A) (4). More recently, a novel Topo-I gene has also been described for this parasite (LdTOP1B) (38).…”

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confidence: 99%

Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in Leishmania donovani

Marquis

Hardy

Olivier

2005

Antimicrob Agents Chemother

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“…Unlike the TopIB proteins described so far, which conserve the catalytic 'tetrad' in a single protein, trypanosomatid's TopIB divide the active amino acid residues into two peptides that are post-translationally assembled. Previous phylogenetic analysis carried out with the gene encoding the large LdTopIB subunit and other eukaryotic TopIB sequences has shown that this branch occurred very early in evolution (Broccoli et al 1999). This early divergence was caused by the absence of the C-terminal domain containing the tyrosine-cleaving residue, which was found later on in a separate chromosome of the leishmanial genome.…”

Section: Introductionmentioning

confidence: 99%

DNA topoisomerases in apicomplexan parasites: promising targets for drug discovery

et al. 2010

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The phylum Apicomplexa includes a large group of protozoan parasites responsible for a wide range of animal and human diseases. Destructive pathogens, such as Plasmodium falciparum and Plasmodium vivax, causative agents of human malaria, Cryptosporidium parvum, responsible of childhood diarrhoea, and Toxoplasma gondii, responsible for miscarriages and abortions in humans, are frequently associated with HIV immunosuppression in AIDS patients. The lack of effective vaccines, along with years of increasing pressure to eradicate outbreaks with the use of drugs, has favoured the formation of multidrug resistant strains in endemic areas. Almost all apicomplexan of medical interest contain two endosymbiotic organelles that contain their own mitochondrial and apicoplast DNA. Apicoplast is an attractive target for drug testing because in addition to harbouring singular metabolic pathways absent in the host, it also has its own transcription and translation machinery of bacterial origin. Accordingly, apicomplexan protozoa contain an interesting mixture of enzymes to unwind DNA from eukaryotic and prokaryotic origins. On the one hand, the main mechanism of DNA unwinding includes the scission of one-type I-or both DNA strands-type II eukaryotic topoisomerases, establishing transient covalent bonds with the scissile end. These enzymes are targeted by camptothecin and etoposide, respectively, two natural drugs whose semisynthetic derivatives are currently used in cancer chemotherapy. On the other hand, DNA gyrase is a bacterial-borne type II DNA topoisomerase that operates within the apicoplast and is effectively targeted by bacterial antibiotics like fluoroquinolones and aminocoumarins. The present review is an update on the new findings concerning topoisomerases in apicomplexan parasites and the role of these enzymes as targets for therapeutic agents.

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“…A DNA topoisomerase IB-like gene (LdTOP1A), which encodes for a protein lacking the conventional active site "SKXXY," motif has been characterized in L. donovani. However, heterologous expression of LdTOP1A gene in Escherichia coli produced an inactive protein (12).…”

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A Novel Active DNA Topoisomerase I in Leishmania donovani

Villa¹,

Marcos²,

Reguera³

et al. 2003

Journal of Biological Chemistry

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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.DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology by altering the linkage of DNA strands, solving topological problems caused by cellular processes such as DNA replication, transcription, or recombination (1, 2). These enzymes are classified on the basis of the number of DNA strands that they cleave and the covalent bond formed in the enzyme-DNA intermediate. Unlike type II DNA topoisomerases, type I enzymes are ATP-independent, which transiently break a single strand of DNA. Type I DNA topoisomerases are classified into two subfamilies: type IA and type IB. The enzymes of type IA subfamily, including bacterial DNA topoisomerase I and III, eukaryotic DNA topoisomerase III, and reverse gyrase (3, 4), form a tyrosyl linkage with a 5Ј-phosphate group of one of the DNA strands generated due to the enzyme action (2), whereas the enzymes of type IB subfamily, including eukaryotic and vaccinia virus DNA topoisomerases I (5) and DNA topoisomerase V, establish the tyrosyl bond with the 3Ј-phosphate group (2). Type 1A topoisomerases relax only negatively supercoiled DNA with Mg 2ϩ requirement, whereas type IB topoisomerases relax both negatively and positively supercoiled DNA even in the absence of a metallic cofactor, although Mg 2ϩ and Ca 2ϩ stimulate the relaxation activity (6, 7).Type IB DNA topoisomerases are monomeric enzymes, constituted by four domains (8, 9). The nonconserved amino-terminal domain contains putative signals for nuclear localization of the enzyme. The largest domain, the core, is essential for enzyme activity and shows high phylogenetic conservation, particularly in the residues closely interacting with DNA. The third domain is known as the linker, which is poorly conserved and highly variable in length and is not essential for the enzyme activity. Finally, the carboxyl-terminal domain is highly conserved and crucial for the catalytic activity. This domain contains a tyrosine residue (Tyr 723 in the human topoisomerase I), which interacts with one of the DNA strands, creating a transient covalent phosphodiester bond between the enzyme and the DNA.A type I DNA topoisomerase has been purified and characterized from Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis (10). Topoisomerases have...

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

Cited by 33 publications

References 25 publications

Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in Leishmania donovani

Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in Leishmania donovani

DNA topoisomerases in apicomplexan parasites: promising targets for drug discovery

A Novel Active DNA Topoisomerase I in Leishmania donovani

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