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1.
Breakage of single-stranded DNA by rat liver nicking-closing enzyme with the formation of a DNA-enzyme complex. 总被引:8,自引:5,他引:3
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The DNA nicking-closing enzyme (type I topoisomerase) from rat liver nuclei breaks single-stranded DNA. The broken strand contains a 5'-hydroxyl and tightly bound protein. The stability of this protein-DNA complex to high salt, alkali and detergent suggests a covalent linkage between the DNA and the enzyme. The observed breakage of single-stranded DNA occurs at neutral pH prior to treatment with alkali or detergent, indicating that the breakage may be the result of an interrupted nicking and closing cycle. The resulting covalent complex could represent a reaction intermediate in the overall nicking-closing reaction. 相似文献
2.
Mapping of the active site tyrosine of eukaryotic DNA topoisomerase I 总被引:12,自引:0,他引:12
DNA topoisomerase I from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe was overproduced using the cloned genes. Extracts from cells overproducing DNA topoisomerase I were prepared and incubated with 32P-labeled DNA. Alkali was used to trap the topoisomerase I-DNA covalent intermediate. Most of the DNA was digested with nuclease, and the resultant 32P-labeled topoisomerase I was subjected to cleavage with cyanogen bromide or formic acid. From the molecular weights of the resultant labeled peptides and by comparison of the amino acid sequences derived from the cloned genes, we were able to deduce that the active site tyrosine of eukaryotic DNA topoisomerase I is very near the carboxyl terminus, at amino acid 771 for S. pombe and 727 for S. cerevisiae. Site-directed mutagenesis was used to change tyrosine 727 of S. cerevisiae topoisomerase I to a phenylalanine. The resulting mutant topoisomerase I protein lost all DNA relaxation activity and rendered cells resistant to the topoisomerase I inhibitor, camptothecin. The amino acid sequence of human topoisomerase I has significant similarity to the two yeast topoisomerase I sequences. Based on this similarity, we infer that tyrosine 723 is the active site tyrosine of human enzyme. 相似文献
3.
DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes. 相似文献
4.
Villa H Otero Marcos AR Reguera RM Balaña-Fouce R García-Estrada C Pérez-Pertejo Y Tekwani BL Myler PJ Stuart KD Bjornsti MA Ordóñez D 《The Journal of biological chemistry》2003,278(6):3521-3526
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. 相似文献
5.
Stimulation of intracellular topoisomerase I activity by vasopressin and thrombin. Differential regulation by pertussis toxin.
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Incubation of cultured rat aortic smooth muscle cells (A-10, ATCC CRL 1476) with [8-arginine]vasopressin (AVP) or thrombin increased the amount of DNA strand breakage induced by camptothecin, an inhibitor of topoisomerase I (DNA topoisomerase; EC 5.99.1.2) and transiently stimulated the extractable activity of this enzyme. Both topoisomerase-related responses were prevented by treatment of the cells with AVP or thrombin plus the appropriate receptor antagonist. The increase in strand breakage mediated by AVP and thrombin depended on the concentration of hormone. Neither AVP nor thrombin had any effect on strand breaks obtained with the epipodophyllotoxin VM-26, an inhibitor of topoisomerase II [DNA topoisomerase (ATP-hydrolysing); EC 5.99.1.3]. Pretreatment of the cells with pertussis toxin partially inhibited thrombin-mediated increases in camptothecin-induced strand breakage whereas AVP-mediated increases were unaffected. These results are consistent with the notion that AVP and thrombin induce a transient increase in intracellular topoisomerase I activity via interactions with their respective cell surface receptors and that the effects of the activation of these receptors are mediated by different G-proteins. 相似文献
6.
An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani
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Aditi Das Chhabinath Mandal Arindam Dasgupta Tanushri Sengupta Hemanta K. Majumder 《Nucleic acids research》2002,30(3):794-802
DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes. 相似文献
7.
Allyson D. Cole Sharon Heath-Pagliuso Annette Baich Eric B. Kmiec 《Plant molecular biology》1992,19(2):265-276
The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly. 相似文献
8.
Chrencik JE Burgin AB Pommier Y Stewart L Redinbo MR 《The Journal of biological chemistry》2003,278(14):12461-12466
1-beta-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 A crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2'-hydroxyl of Ara-C and the O4' of the adjacent -1 base 5' to the damage site stabilizes a C3'-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex. 相似文献
9.
The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme. 相似文献
10.
Y H Hsiang R Hertzberg S Hecht L F Liu 《The Journal of biological chemistry》1985,260(27):14873-14878
Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex. 相似文献
11.
Mechanism of strand passage by Escherichia coli topoisomerase I. The role of the required nick in catenation and knotting of duplex DNA 总被引:6,自引:0,他引:6
We studied the interaction between topoisomerase I and a nicked DNA substrate to determine how the nick permits Escherichia coli topoisomerase I to catenate and knot duplex DNA rings. The presence of just a single nick in a 6600-base pair DNA increased the amount of DNA bound to topoisomerase I by 6-fold. The enzyme acts at the nick, as shown by linearization of nicked circles and covalent attachment of an enzyme molecule opposite the nick. DNA breaks are also introduced by the enzyme at sites not opposite to a nick, but three orders of magnitude less efficiently. The break induced by the enzyme is within several base pairs of the nick and on the complementary strand, but the exact site cut is dictated by DNA sequence requirements. Because these sequence requirements are identical to those for cutting of single-stranded DNA, we conclude that the enzyme stabilizes a denatured region at the nick. Breaks in single-stranded DNA occur 98% of the time when a C residue is four bases to the 5' side unless G is adjacent and 5' to the break. For a DNA circle nicked at a unique location, the efficiency of DNA breakage opposite the nick correlates with the rate of catenation. We present a unified model for the relaxation, catenation, and knotting reactions of topoisomerase I in which the enzyme induces a break in a single-stranded region, but bridges that break with covalent and noncovalent interactions and allows passage of one duplex or single-stranded DNA segment. 相似文献
12.
About ScienceDirect 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1984,783(3):304-306
Properties of strand breakage in duplex and single-stranded DNA by the wheat germ type 1 DNA topoisomerase were investigated. Strand breakage in duplex DNA is dependent upon the use of denaturing conditions to inactivate the enzyme and terminate the reaction, whereas breakage of single-stranded DNA occurs under the normal reaction conditions and is not dependent upon denaturation. Breakage generates a free 5′ hydroxyl group and enzyme bound to the 3′ side of the break, presumably via the 3′ phosphate group. The location of sites of breakage with both duplex and single-stranded DNA is not random. In all these respects the wheat germ enzyme closely resembles the rat liver type 1 topoisomerase. A comparison of the locations of the sites of breakage in duplex DNA generated by the topoisomerases from wheat germ and rat liver indicates a number of common sites, although the patterns of breakage are not identical. 相似文献
13.
《Anaerobe》2000,6(4):241-247
We have identified a type I DNA Topoisomerase from a mesophilic archaebacteria, Methanosarcina barkeri. The enzyme activity residing in a high molecular mass complex is found to be magnesium-dependent and relaxes negatively supercoiled DNA. The properties of the topoisomerase activity were detected by using the technique of transfering radioactivity from32P labelled DNA to the protein. In presence of the enzyme, nicks are generated in the supercoiled DNA and the enzyme becomes covalently attached to the DNA. A tyrosine residue of the enzyme was found to be responsible for the covalent linkage. 相似文献
14.
Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site 总被引:2,自引:0,他引:2
R P Hertzberg R W Busby M J Caranfa K G Holden R K Johnson S M Hecht W D Kingsbury 《The Journal of biological chemistry》1990,265(31):19287-19295
Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue. 相似文献
15.
DNA strand breakage by wheat germ type 1 topoisomerase 总被引:3,自引:0,他引:3
Properties of strand breakage in duplex and single-stranded DNA by the wheat germ type 1 DNA topoisomerase were investigated. Strand breakage in duplex DNA is dependent upon the use of denaturing conditions to inactivate the enzyme and terminate the reaction, whereas breakage of single-stranded DNA occurs under the normal reaction conditions and is not dependent upon denaturation. Breakage generates a free 5' hydroxyl group and enzyme bound to the 3' side of the break, presumably via the 3' phosphate group. The location of sites of breakage with both duplex and single-stranded DNA is not random. In all these respects the wheat germ enzyme closely resembles the rat liver type 1 topoisomerase. A comparison of the locations of the sites of breakage in duplex DNA generated by the topoisomerases from wheat germ and rat liver indicates a number of common sites, although the patterns of breakage are not identical. 相似文献
16.
The phosphoform of the regulatory subunit RII of cyclic AMP-dependent protein kinase possesses intrinsic topoisomerase activity 总被引:12,自引:0,他引:12
The phosphoform of the type II regulatory subunit (phospho-RII-cAMP) of cAMP-dependent protein kinase from rat liver was found to possess intrinsic topoisomerase activity towards several DNA substrates such as phi X174, pBR322, SV40, and M13. Like the type I topoisomerases from several eukaryotic cells, phospho-RII X cAMP can relax both positive and negative superhelical turns of phi X174 DNA. Topological isomers with a decreasing number of superhelical turns can be identified as transient products. Conditions under which phospho-RII X cAMP relaxes superhelical phi X174 DNA lead to transient formation of a DNA-phospho-RII X cAMP complex via DNA strand breakage and covalent attachment of the DNA to a tyrosine residue of phospho-RII X cAMP via a phospho-RII X cAMP depends on the presence of cAMP and is altered by changes in the degree of phosphorylation of RII. Both dephosphorylation and removal of cAMP from phospho-RII X cAMP abolish its topoisomerase activity. 相似文献
17.
Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors. 相似文献
18.
Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage 总被引:1,自引:1,他引:0
Richter SN Leo E Giaretta G Gatto B Fisher LM Palumbo M 《Nucleic acids research》2006,34(7):1982-1991
Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the −1 position, i.e. immediately 5′ of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria. 相似文献
19.
The human topoisomerase I-mediated DNA relaxation reaction was studied following modification of the enzyme at the active site tyrosine (position 723). A series of unnatural tyrosine analogues was incorporated into the active site of human topoisomerase I by utilizing misacylated suppressor tRNAs in an in vitro protein synthesizing system. The relaxation activities of the modified human topoisomerase I analogues having varied steric, electronic, and stereochemical features were all greatly diminished relative to that of the wild type. It was found that modifications involving replacement of the nucleophilic tyrosine OH group with NH2, SH, or I groups eliminated DNA relaxation activity, as did changing the orientation of the nucleophilic tyrosine OH group. Only tyrosine analogues having the phenolic OH group in the normal position with respect to the protein backbone were active; the relative activities could be rationalized in chemical terms on the basis of the H-bonding and the electronic effects of the substituents attached to the meta position of the aromatic ring. In addition, the poisoning of one of the modified human topoisomerase I analogues, as part of covalent binary complexes with DNA, by CPT and 20-thio CPT was evaluated. 相似文献
20.
Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreover, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme. 相似文献