Identification of the gene encoding DNA topoisomerase I from Candida albicans (original) (raw)
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The topoisomerase I gene from Candida albicans
Microbiology, 1997
We report here the cloning of the Candida albicans genomic topoisomerase I gene (TOPI) by use of PCR and subsequent hybridization. The predicted protein sequence shared 58.8 O/ O identity with the Saccharomyces cerevisiae topoisomerase I and 30-50 YO identity with other eukaryotic topoisomerase I proteins. A conditional gene disruption strain (CWJ477) was constructed so that one copy of TOP1 was deleted and the other copy of TOPI was placed under a regulatable promoter. Under repressed conditions, cells grew slowly and cell morphology was abnormal. The virulence of CWJ477 was markedly reduced in a mouse model system, and that of the single gene knockout strain was slightly attenuated, indicating that TOP1 might play a role in the infection of C. albicans in mice in a dose-dependent manner. Despite the reduced virulence of both the single and double knockout strains, viable cells of the pathogen were recovered from the kidneys as late as 22 d post-infection.
FEMS Yeast Research, 2021
The increasing resistance of Candida species to azoles emphasizes the urgent need for new antifungal agents with novel mechanisms of action. The aim of this study was to examine the effect of three DNA topoisomerase inhibitors of plant origin (camptothecin, etoposide and curcumin) on the growth of Candida dubliniensis. The phylogenetic analysis showed a close relationship between the topoisomerase enzymes of C. dubliniensis and Candida albicans. The alignment of the amino acid sequences of topoisomerase I and II of yeasts and humans evidenced conserved domains. The docking study revealed affinity of the test compounds for the active site of topoisomerase I and II in C. dubliniensis. Curcumin and camptothecin demonstrated a stronger in vitro antifungal effect than the reference drugs (fluconazole and itraconazole). Significant synergistic activity between the topoisomerase inhibitors and fluconazole at the highest concentration (750 µM) was observed. Fluconazole induced the petite ph...
FEMS Microbiology Letters, 1996
DNA topoisomerase I is a potential target for therapeutic antifungal agents predicted to have a fungicidal mode of action. This report describes four agents with varying degrees of selectivity for the fungal topoisomerase I compared to the human enzyme: Shydroxy-lH-indole-3-acetic acid (SHIAA), quinizarin, dihenzo-p-dioxin-2-carboxylic acid and 7-amino-4-hydroxy-2-naphthalenesulfonic acid. Taken together with the response of topoisomerase to camptothecin and aminocatechol, these data suggest that there are sufficient structural differences between the topoisomerase I from Can&da albicans and human cells to allow selective targeting of the fungal topoisomerase I over its human counterpart.
Expression of stable and active human DNA topoisomerase I in Pichia pastoris
Protein Expression and Purification, 2018
This study described the isolation of the coding region of human topoisomerase I (TopoI) from MDA-MB-231 and the expression of multiple copy recombinant genes in four Pichia pastoris strains. First, polymerase chain reaction (PCR)-amplification of the enzyme coding region was performed. The PCR fragment was cloned into pPICZ-α-A vector and sequenced. It was then transformed into X33, GS115, SMD1168H and KM71H strains of Pichia. PCRscreening for positive clones was performed, and estimation of multiple copy integrants in each yeast strain was carried out using agar plates containing increasing concentrations of Zeocin ®. The selected clones of multiple copy recombinant genes were then induced for TopoI expression in shaker flasks. GS115 and SMD1168 were found to be better Pichia strains to accommodate the recombinant gene for expression of TopoI extracellularly. However, the DNA relaxation activity revealed that only the target enzyme in the culture supernatants of GS115-pPICZ-α-A-TopoI exhibited consistent enzyme activity over the cultivation time-points. Active enzyme activity was inhibited by Camptothecin. The enzyme produced can be used for affordable gel-based DNA relaxation assay development in performing high throughput screening for target-specific growth inhibitors that display similar effect as the TopoI inhibitors. These inhibitors may contribute to the improvement of the treatment of cancer patients.
The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II
Molecular and cellular biology, 1994
A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a cat...
Cancer research, 1989
Yeast Saccharomyces cerevisiae strains that are permeable to the antitumor alkaloid camptothecin are killed by the drug if they express DNA topoisomerase I, the cellular target of the drug (J. Nitiss and J.C. Wang, Proc. Natl. Acad. Sci. USA, 85: 7501-7505, 1988). We show that in a yeast strain permeable to camptothecin but lacking DNA topoisomerase I, sensitivity to the drug was restored upon expression of human DNA topoisomerase I from a plasmid-borne human complementary DNA clone. When the human enzyme was expressed from a galactose-inducible, glucose-repressible yeast promoter, PGAL1, sensitivity to camptothecin was observed in the presence of galactose but not in the presence of glucose. Expression of human DNA topoisomerase I in Escherichia coli was also demonstrated by the complementation of a conditional lethal E. coli DNA topoisomerase I mutant. These systems can be used in the study of human DNA topoisomerase I-camptothecin interactions and in the screening of additional t...
Genetics, 1999
Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and ...
Genetic analysis of the gyrase A-like domain of DNA topoisomerase II of Saccharomyces cerevisiae
Genetics, 1991
We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the gene encoding yeast DNA topoisomerase II. Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3' two-thirds of the gene. The mutations all appear to be located in the region of DNA topoisomerase II that shows homology to the "A" subunit of bacterial DNA gyrase. The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine. The three other cold-sensitive mutations map farther toward the 3' end of the gene. The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map. We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the ...
Cloning and characterization of an Arabidopsis thaliana topoisomerase I gene
Plant physiology, 1992
cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana Xgtl1 and XFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOPi probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody. DNA topoisomerases are a class of enzymes that share the ability to alter the topological state of DNA. Type I topoisomerases change the linking number of DNA in steps of one by the transient breakage of a single strand of DNA, whereas type II enzymes alter the linking number in integrals of two by introducing double-strand breaks (6, 11, 29, 30). Genes for the type I enzyme have been cloned from bacteria (26), Saccharomyces cerevisiae (25), Schizosaccharomyces pombe (27), and human cells (8, 13). The bacterial and eukaryotic genes are not homologous. However, the yeast clone is able to complement a bacterial topoisomerase I-deficient mutant (4), and recently a novel topoisomerase I gene from yeast with homology to the bacterial gene also was described (28). Topoisomerases have been implicated in a number of processes affecting DNA, including transcription, replication, and recombination. The enzymes are thought to resolve the topological constraints imposed by the double-stranded, helical nature of DNA. For example, topoisomerase II is required for resolution of recombined chromosomes in yeast (23). Topoisomerase I is required for plasmid recombination in Escherichia coli (10) and for the pairing of covalently closed circular plasmids in vitro (7).