Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.
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PMID:Regulation of catalysis by the smallpox virus topoisomerase. 1703 43

Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in Escherichia coli. HipA is a "toxin" that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the hipBA operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg2+-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation.
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PMID:Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. 1704 Oct 39

Vaccinia topoisomerase provides a model system for structure-function analysis of the type IB topoisomerase family. Here we performed an alanine scan of eight positions in the beta4 and beta5 strands of the N-terminal domain (Leu57, Ile58, Phe59, Val60, Gly61, Ser62, Gln69 and Gly73) and eight positions in the alpha8-alpha9 loop of the C-terminal catalytic domain (Ser241, Ile242, Ser243, Pro244, Leu245, Pro246, Ser247, and Pro248). Mutants F59A, G73A, and Q69A displayed rate defects in relaxing supercoiled DNA that were attributed to effects on DNA binding rather than transesterification chemistry. Replacing Gln69 conservatively with Asn, Glu or Lys failed to restore relaxation activity. Gln69 is located along a concave DNA-binding surface of the N-terminal domain and it makes direct contact with the +2A base of the 5'-CCCTT/3-GGGAA target site for DNA cleavage. Gly73 is located at the junction between the N-terminal domain and catalytic domain and it is likely to act as a swivel for the large domain movements that coordinate DNA ingress and closure of the topoisomerase clamp around the duplex. Previous alanine scanning had identified Phe215 in helix alpha7 of the catalytic domain as contributing to DNA relaxation activity. Here we find that F215L resembles F215A in its diminished relaxation activity and its sensitivity to inhibition by salt. The Phe215 side chain makes van der Waals contacts to Ile98, Met121 and Phe101, which we propose stabilize a three helix bundle and promote clamp closure.
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PMID:Vaccinia topoisomerase mutants illuminate roles for Phe59, Gly73, Gln69 and Phe215. 1705 40

Topoisomerase I (Top1) is a ubiquitous enzyme that removes DNA supercoiling generated during transcription and replication. Top1 can be trapped on DNA as cleavage complexes by the anticancer drugs referred to as Top1 inhibitors as well as by alterations of the DNA structure. We reported recently that Top1 cleavage complexes (Top1cc) are trapped during apoptosis induced by arsenic trioxide and staurosporine. In the present study, we generalize the occurrence of apoptotic Top1cc in response to anticancer drugs, which by themselves do not directly interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form in the early phase of apoptosis and persist throughout the apoptotic process. Their formation is prevented by the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone and the antioxidant N-acetyl-L-cysteine. We propose that the trapping of Top1cc is a general process of programmed cell death, which is caused by alterations of the DNA structure (oxidized bases and strand breaks) induced by caspases and reactive oxygen species.
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PMID:Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes. 1717 17

In this study, topoisomerase mutations in ciprofloxacin-resistant and -susceptible Campylobacter jejuni were analysed by DNA sequencing. In certain ciprofloxacin-resistant C. jejuni, the mechanism of resistance was complex. The Thr86-Ala substitution in the GyrA protein appears to play a role in increasing the minimum inhibitory concentration of nalidixic acid only. In addition, isolates with this amino acid change and those resistant to quinolones but lacking a mutation in the GyrA quinolone resistance-determining region could be derived from two different clones. Based on gyrA and gyrB polymorphisms, C. jejuni isolates from the Dakar region of Senegal appeared to be less diverse than those from other countries. Moreover, C. jejuni isolates in Senegal appeared to differ from European isolates by lack of a silent mutation at codon 120 of the gyrA gene.
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PMID:Analysis of topoisomerase mutations in fluoroquinolone-resistant and -susceptible Campylobacter jejuni strains isolated in Senegal. 1722 60

Several quinone-based metabolites of drugs and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein and appear to increase levels of enzyme-DNA cleavage complexes by at least two potentially independent mechanisms. Treatment of topoisomerase IIalpha with quinones inhibits DNA religation and blocks the N-terminal gate of the protein by cross-linking its two protomer subunits. It is not known whether these two effects result from adduction of quinone to the same amino acid residue(s) in topoisomerase IIalpha or whether they are mediated by modification of separate residues. Therefore, this study identified amino acid residues in human topoisomerase IIalpha that are modified by quinones and determined their role in the actions of these compounds as topoisomerase II poisons. Four cysteine residues were identified by mass spectrometry as sites of quinone adduction: Cys170, Cys392, Cys405, and Cys455. Mutations (Cys --> Ala) were individually generated at each position. Only mutations at Cys392 or Cys405 reduced sensitivity ( approximately 50% resistance) to benzoquinone. Top2alphaC392A and top2alphaC405A displayed faster rates ( approximately 2-fold) of DNA religation than wild-type topoisomerase IIalpha in the presence of the quinone. In contrast, as determined by DNA binding, protein clamp closing, and protomer cross-linking experiments, mutations at Cys392 and Cys405 did not affect the ability of benzoquinone to block the N-terminal gate of topoisomerase IIalpha. These findings indicate that adduction of Cys392 and Cys405 is important for the actions of quinones against the enzyme and increases levels of cleavage complexes primarily by inhibiting DNA religation.
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PMID:Quinone-induced enhancement of DNA cleavage by human topoisomerase IIalpha: adduction of cysteine residues 392 and 405. 1729 34

Several quinone-based metabolites of industrial and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein, and previous studies suggest that they increase levels of enzyme-associated DNA strand breaks by at least two potential mechanisms. Quinones act directly on the DNA cleavage-ligation equilibrium of topoisomerase II by inhibiting the rate of ligation. They also block the N-terminal gate of the protein, thereby stabilizing topoisomerase II in its "closed clamp" form and trapping DNA in the central annulus of the enzyme. It has been proposed that this latter activity enhances DNA cleavage by increasing the population of enzyme molecules with DNA in their active sites, but a causal relationship has not been established. In order to more fully characterize the mechanistic basis for quinone action against topoisomerase II, the present study characterized the sensitivity of human topoisomerase IIalpha carrying a Cys455-->Ala mutation (top2alphaC455A) toward quinones. Cys455 was identified as a site of quinone adduction by mass spectrometry. The mutant enzyme was approximately 1.5-2-fold hypersensitive to 1,4-benzoquinone and the polychlorinated biphenyl quinone 4'Cl-2,5pQ, but it displayed wild-type sensitivity to traditional topoisomerase II poisons. The ability of 1,4-benzoquinone to inhibit DNA ligation mediated by top2alphaC455A was similar to that of wild-type topoisomerase IIalpha. However, the quinone induced approximately 3 times the level of clamp closure with the mutant enzyme. These findings strongly support the hypothesis that the ability of quinones to block the N-terminal gate of the type II enzyme contributes to their actions as topoisomerase II poisons.
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PMID:Mutation of cysteine residue 455 to alanine in human topoisomerase IIalpha confers hypersensitivity to quinones: enhancing DNA scission by closing the N-terminal protein gate. 1751 63

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.
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PMID: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. 1809 18

Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique bisubunit DNA-topoisomerase IB (LdTopIB). The catalytically active enzyme is a heterodimer constituted by a large subunit (LdTopIL) containing a non-conserved N-terminal end and the phylogenetically conserved core domain, whereas the small subunit (LdTopIS) harbors the C-terminal domain with the characteristic tyrosine residue in the active site. Site-directed mutagenesis was used to substitute the basic amino acid (Arg-314, Lys-352, Arg-410 and His-453) of the LdTopIL subunit by the neutral amino acid alanine. The expression of these mutants in a topoisomerase-free yeast strain produced inactive proteins. Similarly, when the Tyr-222 from small subunit, involved in DNA cleavage, was substituted by Phe no topoisomerase activity was detected in yeast overexpressing extracts. In addition two substitutions involved in camptothecin inhibition were also analyzed. Asp-353 located in the core domain of the large subunit and Asn-221 which heads Tyr-222 in the small subunit, were replaced by Ala and Ser, respectively. These mutants were insensitive to the inhibitor; despite they displayed significant relaxation activity.
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PMID:Mutational study of the "catalytic tetrad" of DNA topoisomerase IB from the hemoflagellate Leishmania donovani: Role of Asp-353 and Asn-221 in camptothecin resistance. 1865 76

DNA viruses adopt various strategies to modulate the cellular environment for efficient genome replication and virion production. Previously, we demonstrated that the BGLF4 kinase of Epstein-Barr virus (EBV) induces premature chromosome condensation through the activation of condensin and topoisomerase IIalpha (C. P. Lee, J. Y. Chen, J. T. Wang, K. Kimura, A. Takemoto, C. C. Lu, and M. R. Chen, J. Virol. 81:5166-5180, 2007). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha- and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses.
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PMID:Epstein-Barr virus BGLF4 kinase induces disassembly of the nuclear lamina to facilitate virion production. 1881 3


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