Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-[9-acridinylamino]-methanesulfon-m-anisidide, mAMSA). Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid. Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.
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PMID:Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II. 131 90

The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features. Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (URF). The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage lambda, which participated in lambda DNA replication, and also with the exonuclease, gp46, of bacteriophage T4. A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the phi 29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.
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PMID:Sequence of the bacteriophage SP01 gene 30. 158 73

Fluoroquinolone-resistant mutants were obtained in vitro from Staphylococcus aureus RN4220 by stepwise selection on increasing concentrations of ciprofloxacin. Results from sequence analysis of the quinolone resistance-determining region of GyrA and of the corresponding region of GrlA, the DNA topoisomerase IV subunit, showed an alteration of Ser-80 to Tyr (corresponding to Ser-83 of Escherichia coli GyrA) or Glu-84 to Lys in GrlA of both low- and high-level quinolone-resistant mutants. Second-step mutants were found to have, in addition to a mutation in grlA, reduced accumulation of norfloxacin or an alteration in GyrA at Ser-84 to Leu or Glu-88 to Lys. Third-step mutants derived from second-step mutants with reduced accumulation were found to have a mutation in gyrA. The results from this study demonstrated that mutations in gyrA or mutations leading to reduced drug accumulation occur after alteration of GrlA, supporting the previous findings (L. Ferrero, B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche, Mol. Microbiol. 13:641-653, 1994) that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.
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PMID:Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. 749 3

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein.DNA complex at sites containing a conserved sequence element 5'-CCCTT decreases. The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive "hinge." The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino-terminal region is punctuated by a trypsin-sensitive "bridge" at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.
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PMID:Proteolytic footprinting of vaccinia topoisomerase bound to DNA. 774 4

Nae I endonuclease must bind to two DNA sequences for cleavage. Examination of the amino acid sequence of Nae I uncovered similarity to the active site of human DNA ligase I, except for leucine 43 in Nae I instead of the lysine essential for ligase activity. Changing leucine 43 to lysine 43 (L43K) changed Nae I activity: Nae I-L43K relaxed supercoiled DNA to yield DNA topoisomers and recombined DNA to give dimeric molecules. Interruption of the reactions of Nae I and Nae I-L43K with DNA demonstrated transient protein-DNA covalent complexes. These findings imply coupled endonuclease and ligase domains and link Nae I endonuclease to the topoisomerase and recombinase protein families.
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PMID:DNA topoisomerase and recombinase activities in Nae I restriction endonuclease. 789 5

The eukaryotic family of type I DNA topoisomerases includes the nuclear type I enzymes and the enzymes encoded by vaccinia and other poxviruses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it likely that this protein constitutes the minimal functional unit of a eukaryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregions were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family members was examined by replacing each conserved residue with alanine. Alanine substitution at the active site Tyr abrogated topoisomerase activity. In contrast, mutations at Ser-270 and Lys-271 had no effect on enzyme activity. The region of the vaccinia topoisomerase from amino acids 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a residue, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitution at each position. Point mutations were identified at three positions, Arg-130, Tyr-136, and Leu-137, which either abrogated or severely reduced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 other amino acids, including conserved residues, had little or no effect on topoisomerase activity.
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PMID:Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis. 796 97

A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent. The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.
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PMID:Protein footprinting by the combined use of reversible and irreversible lysine modifications. 799 55

Titration of Escherichia coli DNA topoisomerase I with PMPS and 65Zn(II) binding showed independent release and binding of the three Zn(II) in each enzyme molecule. Removal of Zn(II) from topoisomerase I or top85 (truncated topoisomerase I with the Zn(II) binding domain at the carboxyl terminal) affected their sensitivity to Glu-C and Asp-N endoproteases but there was no significant effect on their rate of proteolysis by trypsin or Lys-C endoprotease. This suggested that Zn(II) removal did not result in complete unfolding of topoisomerase enzyme structure but only affected folding of small local regions. Digestion with carboxypeptidase Y further demonstrated that the folding of the zinc binding region itself was altered upon Zn(II) removal.
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PMID:Binding of Zn(II) to Escherichia coli DNA topoisomerase I. 808 Dec 8

Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose) polymerase (pADPRp) during the course of chemotherapy-induced apoptosis was examined. Treatment of HL-60 human leukemia cells with the topoisomerase II-directed anticancer agent etoposide resulted in morphological changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the M(r) 116,000 pADPRp polypeptide to an M(r) approximately 25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M(r) approximately 85,000 fragment retained basal pADPRp activity but was not activated by exogenous nicked DNA. Similar cleavage of pADPRp was observed after exposure of HL-60 cells to a variety of chemotherapeutic agents including cis-diaminedichloroplatinum(II), colcemid, 1-beta-D-arabinofuranosylcytosine, and methotrexate; to gamma-irradiation; or to the protein synthesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2'-deoxyuridine and in gamma-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, N-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA. The results suggest that proteolytic cleavage of pADPRp, in addition to being an early marker of chemotherapy-induced apoptosis, might reflect more widespread proteolysis that is a critical biochemical event early during the process of physiological cell death.
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PMID:Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. 835 26

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


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