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)

We have studied the genetic alterations acquired during selection of a cloned human leukaemic cell line (CEM/VP-1) that is 15-fold more resistant to the anticancer topoisomerase II-inhibitor etoposide than parental CCRF-CEM cells. CEM/VP-1 cells exhibit an 'atypical MDR' phenotype: cross resistance to other topo II inhibitors (but not Vinca alkaloids) and expression of a drug-resistant topo II activity. Cytogenetic and molecular studies revealed that the cell line carried multiple genetic changes affecting TOP2 genes encoding both topo II alpha and beta isoforms. CEM/VP-1 was diploid, 47,XX,+20, and appears to have been preferentially selected from a 1% diploid subpopulation present in the tetraploid parental cells. The same chromosomal abnormalities were present in resistant and sensitive cells except for an acquired 3p- change most likely deleting one TOP2 beta allele. PCR/DNA sequence analysis and allele-specific hybridisation showed that one of two TOP2 alpha alleles expressed in CEM/VP-1 cells had acquired a Lys-797-->Asn codon change. This mutation lies close to the catalytic Tyr-804 residue of the protein and may interfere with drug-induced trapping of the cleavable complex. Alternatively, it could exert a loss of function phenotype. CEM/VP-1 cells did not exhibit codon 449 or 486 TOP2 alpha mutations in the ATP binding domain reported in two other resistant cell lines. Diploid selection and multiple changes observed in CEM/VP-1 cells appear to be consequences of the recessive phenotype of at-MDR. These results may be useful in approaching the mechanisms of clinical resistance.
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PMID:Novel selection and genetic characterisation of an etoposide-resistant human leukaemic CCRF-CEM cell line. 838 8

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.
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PMID:Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells. 838 9

The 32-kDa topoisomerase I encoded by vaccinia virus relaxes supercoiled DNA in a manner which is mechanistically equivalent to that utilized by eucaryotic enzymes. Its amino acid sequence contains significant homology to the enzymes encoded by Saccharomyces cerevisiae, Saccharomyces pombe, human cells, and other poxviruses. The small size of the viral enzyme, and its essentiality in the viral life cycle, make it ideally suited for structural and functional analysis. In this report we present the construction and analysis of 15 mutant alleles of the topoisomerase containing amino acid substitutions in a highly conserved region. The enzymes encoded by these alleles were expressed in Escherichia coli and various parameters of their activity were examined. All of the alleles which show diminished (seven alleles) or abrogated (three alleles) DNA relaxation activity are deficient in DNA cleavage and the concomitant formation of the covalent enzyme/DNA intermediate. None are deficient in the prior step of noncovalent interaction with substrate DNA. Five of the mutant enzymes show significant temperature sensitivity in vitro. The extent of in vitro activity of the enzymes shows a good but incomplete correlation with the enzymes' abilities to lethally induce the resident lambda prophage within E. coli BL21(DE3) (via illegitimate recombination). Mutations in 1 amino acid, in particular, impair prophage induction in vivo more significantly than DNA relaxation in vitro. In sum, these studies suggest that this region of the topoisomerase (amino acids 216-225) plays a proximal role in mediating DNA cleavage and the covalent interaction between the 3'-phosphoryl of the nicked DNA and tyrosine 274 of the vaccinia topoisomerase I. The studies also provide useful reagents for the molecular genetic analysis of the role of the topoisomerase within the context of vaccinia virus infection.
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PMID:Biochemical analysis of mutant alleles of the vaccinia virus topoisomerase I carrying targeted substitutions in a highly conserved domain. 839 54

The toxicity of genistein, an inhibitor of tyrosine kinases and topoisomerase-II, on human thymocytes was investigated. Genistein induced marked chromatin fragmentation indicative of apoptosis in human thymocyte cultures. Genistein-induced thymocyte apoptosis is unlikely due to an inhibition of basal tyrosine kinase activity, since another tyrosine kinase inhibitor, herbimycin A, does not induce thymocyte apoptosis, whereas other topoisomerase-II inhibitors do. The thymocyte subpopulation most sensitive to genistein-induced apoptosis exhibited a CD3-CD4+CD8+ phenotype. This subpopulation of thymocytes is also sensitive to glucocorticoid-induced apoptosis; however, differences between genistein- and glucocorticoid-induced apoptosis were noted. In particular, unlike glucocorticoid-induced apoptosis, genistein-induced apoptosis does not involve changes in [Ca2+]i and cannot be blocked by activation of protein kinase C.
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PMID:Genistein induces apoptosis in immature human thymocytes by inhibiting topoisomerase-II. 839 75

Recombination activating genes Rag-1 and Rag-2 were isolated on the basis of their ability to confer V(D)J recombination activity when co-expressed in fibroblasts. The mode of action of the confer V(D)J recombination activity when co-expressed in fibroblasts. The mode of action of the products of these genes is not known. Based on sequence comparison data, it was suggested that Rag-1 protein could act like a topoisomerase and that tyrosine in position 998 could be the active site tyrosine. We tested this hypothesis by introducing a point mutation on the Rag-1 cDNA, transforming the tyrosine codon into a phenylalanine codon. We show that the mutation has no effect on site specific recombination implying that Tyr-998 is not essential for the recombination reaction.
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PMID:Rag-1: a topoisomerase? 845 20

The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5' ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5' terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.
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PMID:Mutational analysis of conserved tyrosines in the NS-1 protein of the parvovirus minute virus of mice. 850 71

We studied the effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and cell death in CTLL-2 cells, whose growth is IL-2-dependent. The Flavonoids genistein, biochanin A, and apigenin inhibited topoisomerase II to the same extent as etoposide, a specific inhibitor of the enzyme. Methyl 2,5-dihydroxycinnamate (2,5-MeC) also inhibited topoisomerase II, but was less potent than genistein. Herbimycin A and staurosporine did not inhibit topoisomerase II. None of the inhibitors of protein tyrosine kinases examined inhibited topoisomerase I activity. All the inhibitors induced cell death with internucleosomal DNA fragmentation in the presence of IL-2. Genistein, biochanin A, and apigenin induced DNA fragmentation and cell death early in the incubation period and did not alter the profiles of phosphotyrosine proteins in either the lysate or pelleted fractions, indicating that the early cell death was induced by the inhibition of topoisomerase II activity rather than by the inhibition of protein tyrosine kinase activity. 2,5-MeC similarly induced early cell death and DNA fragmentation, but to a lesser extent than genistein presumably due to the inhibition of topoisomerase II activity. Herbimycin A induced a slow increase in DNA fragmentation and cell death, accompanied by a decrease in phosphotyrosine proteins in the pelleted fraction, suggesting that the inhibition of protein tyrosine phosphorylation, presumably of the nuclear proteins, is related to cell death and DNA fragmentation. Staurosporine-induced DNA fragmentation appeared to be due to mechanism(s) other than the inhibition of topoisomerases and protein tyrosine kinases, since it neither altered the profiles of phosphotyrosine proteins nor inhibited topoisomerase activity.
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PMID:Effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and death of IL-2-dependent CTLL-2 cells. 854 64

A recently described reverse gyrase from the hyperthermophilic methanogen Methanopyrus kandleri is the only known example of a heterodimeric type I topoisomerase. The enzyme is made up of a 42-kDa subunit which covalently interacts with DNA (RgyA) and a 138-kDa subunit which binds ATP (RgyB). We have now cloned and sequenced the genes for both subunits of this enzyme. Surprisingly, the universally conserved type I topoisomerase domain [Lima, C. D., Wang, J. C. & Mondragon, A. (1994) Nature (London) 367, 138-146] which has been found as a contiguous polypeptide in the prokaryotes and eukaryotes is shared between the protomers. The subdomain with the active-site tyrosine is entirely within RgyA, whereas the subdomain implicated in noncovalent binding of the cleaved DNA strand is contained entirely in RgyB. The appearance of this unique structure in a highly conserved enzyme family supports the hypothesis that the methanogens branched from other prokaryotes and eukaryotes very early in evolution.
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PMID:A two-subunit type I DNA topoisomerase (reverse gyrase) from an extreme hyperthermophile. 855 84

We have used ultraviolet laser crosslinking to characterize the DNA-binding properties of highly purified yeast topoisomerase II in the absence of ATP. A single 5 ns, 20 mJ pulse of 266 nm light produced optimal crosslinking to a short DNA duplex, with an efficiency of 0.25%. An equilibrium binding constant (Keq) of 1.2 +/- 0.5 x 10(8) M(-1) was determined from kinetic analysis. Topoisomerase II showed highest affinity for supercoiled DNA. Limited proteolysis of crosslinked topoisomerase II-DNA complexes showed a site of crosslinking to be within a 29-kDa fragment with Leu-681 at its amino-terminal end. This region contains the active Tyr-783 and is homologous to the amino-terminal region of the DNA-binding bacterial gyrase GyrA subunit, suggesting a conserved DNA-binding mechanism.
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PMID:Characterization of topoisomerase II-DNA interaction and identification of a DNA-binding domain by ultraviolet laser crosslinking. 860 20

Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH2-terminal domain (approximately 24 kDa), which is known to be dispensable for activity, (ii) the core domain (approximately 54 kDa), (iii) a linker region (approximately 3 kDa), and (iv) the COOH-terminal domain (approximately 10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH2-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH2-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and approximately 10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH2-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies.
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PMID:The domain organization of human topoisomerase I. 863 94


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