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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells.
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PMID:Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells. 308 40

Nuclear protein factor type 1 (NPF-1) that simulates IMR-32 primase-associated DNA polymerase alpha 1 and alpha 2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase alpha, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37 degrees C), DNase II (60 min at 37 degrees C), and heat treatment (2 min at 68 degrees C). The 125I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase alpha-primase complex (alpha 1 and alpha 2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of alpha-amanitin (400 micrograms/ml). The labeled duplex containing the poly(dC) template, [32P]-GTP, and [3H]dGTP loses 80% of the 32P label and 70% of the 3H label after treatment with 0.3 M KOH and DNase I, respectively. The products were isolated from reaction mixtures incubated with and without NPF-1 and subjected to alkaline sucrose-density-gradient sedimentation analysis. The results suggest that the rate of synthesis of DNA short chains is increased in the presence of NPF-1 without a concomitant increase in the chain length of the newly synthesized products.
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PMID:Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin. 354 Sep 37

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.
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PMID:Processivity of the DNA polymerase alpha-primase complex from calf thymus. 360 95

In permeable human fibroblasts, novobiocin, coumermycin and nalidixic acid completely inhibit u.v.-induced DNA repair synthesis, with 50% inhibition occurring at 500, 24 and 8800 microM respectively. Novobiocin also inhibits damage-specific incision of DNA in u.v.-irradiated permeable human fibroblasts by at least 75%. It has been suggested that effects of novobiocin on DNA excision repair result from changes in ATP pools; this explanation is not applicable to our data because excision repair in the permeable cell system is entirely dependent on exogenous ATP. It has also been suggested that novobiocin-induced inhibition of repair is mediated by alterations of chromatin structure recognizable by electron microscopy as gross chromatin clumping. There were no ultrastructural alterations, however, in the nuclei of permeable cells that had been incubated with 1 mM novobiocin. We conclude that, in human cells, novobiocin, coumermycin, and nalidixic acid directly inhibit the excision repair of u.v. damage to DNA, and that one locus of inhibition lies at or before the incision step. Because 1 mM novobiocin completely abolishes u.v.-induced repair synthesis in permeable cells, but inhibits damage-specific incision by only 75%, there seems to be a second site of inhibition following the incision step. The similarity between the concentrations of novobiocin, nalidixic acid and coumermycin required to inhibit u.v.-induced excision repair and the concentrations required to inhibit human DNA polymerase alpha suggest that the distal locus of inhibition may be DNA polymerase alpha-mediated repair patch synthesis. The proximal inhibitory site may be a type II DNA topoisomerase.
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PMID:Direct inhibition of u.v.-induced DNA excision repair in human cells by novobiocin, coumermycin and nalidixic acid. 360 79

A primase activity associated to DNA polymerase alpha from rat liver is described. Both activities were absent in normal adult rat liver but were concomitantly induced after partial hepatectomy. As previously shown for polymerase alpha and DNA topoisomerase II, primase activity reached a maximum value 40-43 h after the partial removal of the liver. Primase activity was shown to catalyze dNMP incorporation on unprimed single-stranded DNA template (M13 DNA) in the presence of rNTP. The activity was not detectable on poly(dA) or poly(dG) but was efficient on poly(dT) or poly(dC). However, the reliability of the primase assay in the presence of poly(dC) was dependent upon the degree of purification of the enzyme. The ribo primers were about 10 nucleotides long, and the reaction was completely independent of alpha-amanitin, a strong inhibitor of RNA polymerases II and III. Primase and polymerase were found tightly associated. A cosedimentation on a 5-20% sucrose gradient was always obtained, independent of the ionic strength. There was also a close coincidence between alpha-polymerase and primase activities during phosphocellulose, hydroxylapatite, and single-stranded DNA Ultrogel chromatography. It has been previously demonstrated by us and others that primase and alpha-polymerase are on separated polypeptides. The association of two activities in the replication complex and the conditions allowing their separation are discussed.
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PMID:DNA polymerase alpha associated primase from rat liver: physiological variations. 370 97

Inhibitors of DNA polymerase alpha (aphidicolin, phosphonoacetic acid, phosphonoformic acid) efficiently inhibit initiator-induced amplification of SV40 DNA sequences in the SV40-transformed Chinese hamster cell line CO631. Amplification is also inhibited by various protease inhibitors (antipain, leupeptin, aprotinin, alpha-I-antitrypsin, epsilon-amino-caproic acid, soy-bean protease inhibitor), by the non-initiating but DNA-damaging agent caffeine, and by sodium butyrate, which inhibits DNA synthesis by histone modification. In contrast, an inhibitor of topoisomerase II, nalidixic acid, enhances amplification when applied simultaneously with initiating treatment. This latter compound does not induce amplification when applied without initiator. Cycloheximide induces DNA amplification in the same way as chemical and physical carcinogens. This amplification can still be observed when protein synthesis is completely blocked. The data suggest a complex mechanism of selective DNA amplification. The possible involvement of proteases leading to a functional modification of DNA polymerase alpha is discussed.
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PMID:Selective DNA-amplification induced by carcinogens (initiators): evidence for a role of proteases and DNA polymerase alpha. 389 46

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.
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PMID:Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins. 630 11

We have previously shown that a fraction from the nuclei of S phase (DNA-synthesizing) Chinese hamster embryo fibroblasts (CHEF/18 cells) can be obtained that has a number of the enzyme activities required for DNA biosynthesis, and can catalyse the incorporation of labelled precursors into DNA (refs 1-4, also see ref. 8). This fraction, which we have termed the 'replitase', contains spherical particles of diameter approximately 25 nm, apparently multienzyme complexes for de novo DNA biosynthesis. Here we present evidence for the functional association of one of the enzyme activities, thymidylate synthase, with several of the other enzyme activities. Hydroxyurea, novobiocin and aphidicolin, inhibitors of ribonucleotide reductase, topoisomerase and DNA polymerase alpha, respectively, all inhibit thymidylate synthase in intact S phase CHEF/18 cells, but not in their soluble extracts. We suggest that these results reflect allosteric interactions between the subunits of a multienzyme DNA-synthesizing complex, which can be modulated by the specific inhibitors of individual enzyme activities in intact cells.
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PMID:Inhibitor evidence for allosteric interaction in the replitase multienzyme complex. 640 86

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
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PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

We have extended our permeable cell system for measuring DNA excision repair [Roberts, J. D., & Lieberman, M. W. (1979) Biochemistry 18, 4499-4505] so that steps of the repair process, beginning with incision and extending at least through the "rearrangement" of repaired nucleosomes which follows repair synthesis, all take place in permeable cells. In the revised protocol, human fibroblasts are made permeable, damaged with UV or chemicals in suspension, and incubated with a reaction mix containing ATP and the four deoxyribonucleoside triphosphates, one of which is labeled with 32P. By reducing the exogenous dNTP concentration to 3 microM and including 15 mM KCl in the reaction mixture, we have greatly reduced background incorporation in undamaged cells without significantly reducing repair synthesis. This permits us to measure repair synthesis without separating it from replicative synthesis by isopycnic centrifugation. Repair synthesis in this system is very similar to that occurring in intact cells: in response to DNA damage, nucleotides are incorporated into DNA of parental density (when analyzed by the BrdUrd density shift technique), incorporation increases with increasing DNA damage, synthesis is dependent on the presence of all four dNTPs, and the system accurately reflects the genetic UV repair deficiency of xeroderma pigmentosum (XP) cells. Furthermore, as has been observed in intact cells, repair-incorporated nucleotides in these permeable cells are initially overrepresented in staphylococcal nuclease sensitive regions of chromatin and are subsequently redistributed to give a nearly uniform distribution between nuclease-sensitive and -resistant regions. The UV dose curve of permeable cells differs somewhat from that of intact cells; however, the dose differs somewhat from that of intact cells; however, the dose curve for permeable cells treated with N-methyl-N-nitrosourea is very similar to that of intact cells. Repair synthesis in UV-damaged, permeable normal and XP cells is stimulated by addition of Micrococcus luteus UV endonuclease, indicating that the damaged DNA is accessible to exogenous repair enzymes and suggesting that incision, or an obligatory preincision step, is rate limiting for excision repair in these permeable cells. Repair synthesis in this system is inhibited by aphidicolin, but not by high levels of dideoxy-TTP, suggesting involvement of DNA polymerase alpha in excision repair. Novobiocin is also inhibitory alpha and the HeLa cell type II DNA topoisomerase.
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PMID:Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts. 709 2


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