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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase alpha (pol-alpha), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase delta, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-alpha extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-alpha, conditions under which pol-alpha was markedly inhibited. These findings suggest that interactions between T antigen, pol-alpha-primase, and HSSB position the pol-alpha-primase complex on the lagging-strand template for RNA primer synthesis.
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PMID:Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation. 131 May 41

We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.
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PMID:A novel DNA helicase from calf thymus. 132 24

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.
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PMID:A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA. 133 Jun 89

A thermostable DNA polymerase was prepared from Bacillus caldotenax by using a four-step chromatography procedure. The protein exists as a monomer of M(r) 94,000, has a pI of 4.9 and has no associated 3'-5' or 5'-3'-exonuclease activities or endonuclease activity. The temperature optimum of the enzyme was about 70 degrees C and the pH for maximum activity was about 7.5. The enzyme has an absolute requirement for a bivalent cation, and maximum activity was obtained at the unusually high concentration of 70 mM-MgCl2. Mg2+ could be replaced by MnCl2 or CoCl2, with decreased activity, at the lower optimal concentrations of 1 mM and 2.5 mM respectively. Enzyme activity was inhibited in the presence of 2',3'-dideoxy-TTP, arabinosyl-CTP and aphidicolin. Enzyme activity was stimulated with KCl concentrations of about 100 mM, and concentrations of univalent salts above about 150 mM inhibited activity. The enzyme could use activated calf thymus DNA, poly(dA).p(dT)10 or primed single-stranded phage M13 DNA as a template and maximum activity was obtained with poly(dA).p(dT)10. The enzyme was inactive on unprimed single-stranded DNA, double-stranded DNA and polyribonucleotide template/primer. The apparent Km values for individual dNTPs, determined with the other dNTPs at saturating concentrations, were 5.7 microM (dCTP), 6.3 microM (dATP, dGTP) and 6.4 microM (dTTP). The Km value for the overall incorporation of each dNTP from an equimolar mixture of all four dNTPs was 24.7 microM. The kcat. value was about 1.05 s-1. The kcat./Km value was 0.16-0.18 M-1.s-1 for individual dNTPs and 0.04 for the incorporation of an equimolar mixture of all four dNTPs. Some of the properties of the enzyme show it may be classified as an alpha-Type DNA polymerase.
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PMID:Purification and properties of DNA polymerase from Bacillus caldotenax. 144 54

The mechanism of tumor cell killing by HO-221, a novel benzoylphenylurea derivative that shows broad-spectrum antitumor activities, was studied. HO-221 strongly inhibited the activity of mammalian DNA polymerase alpha but not that of DNA polymerases beta or gamma. The inhibition was equivalent to that induced by aphidicolin and ara-CTP, which were selective inhibitors of the enzyme. Furthermore, the inhibition by HO-221 of DNA polymerase alpha was found to be non-competitive with respect to dCTP as a substrate, unlike that induced by aphidicolin and ara-CTP. The inhibition was reduced the addition of an excess of DNA polymerase alpha but not by excess amounts of activated DNA as a template primer. These results suggest that HO-221 inhibits the activity of DNA polymerase alpha by direct interaction with the enzyme in contrast to the impairment of template activity through intercalation into DNA induced by anthracycline compounds. On the other hand, HO-221 showed almost no effect on RNA polymerase activity, the reverse transcriptase activity of avian myeloblastosis virus or protein synthesis in a cell-free system. The flow-cytometry analysis revealed that HO-221 accumulated HL-60 cells in G1-S phases at a low concentration but increased the number of cells in the G1 phase at a higher concentration, stopping cell-cycle progression. The results suggest a correlation between cell-cycle progression and inhibition by HO-221 of DNA polymerase alpha, which plays a role in DNA replication during the S phase in living cells.
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PMID:Mechanism of tumor cell killing by HO-221, a novel antitumor compound. 170 66

The effects of fludarabine triphosphate (Fara-ATP), 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer RNA and DNA synthesis in human CCRF-CEM leukemia cells were investigated. RNA-primed Okazaki fragment synthesis was monitored by first incubating whole cell lysates for 10 min in the presence or absence of the compound and then following the incorporation of [alpha-32P]ATP and [3H]dTTP into the primer RNA and DNA portions, respectively, of the Okazaki fragments. In whole cell lysates the degree of DNA synthesis inhibition induced by Fara-ATP was directly related to the extent of primer RNA synthesis inhibition over the entire range of Fara-ATP concentrations tested (10-50 microM). In contrast, primer RNA formation was stimulated by concentrations of ara-CTP (25-200 microM) and aphidicolin (0.5-5 micrograms/ml) that inhibited DNA synthesis. The primer RNA recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or aphidicolin was of normal length, predominately 11 nucleotides. Fara-ATP was a more potent inhibitor of the polydeoxythymidylate primase activity than of the DNA polymerase alpha/delta activities present in the 100,000 x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive inhibitor of DNA primase with respect to ATP [50% inhibitory concentration, 2.3 +/- 0.3 (SD) microM, Ki = 6.1 +/- 0.3 (SE) microM] and the Km(ATP)/Ki (Fara-ATP) was 25. The 50% inhibitory concentration values of Fara-ATP for DNA polymerases alpha/delta activities on calf thymus DNA were 43 +/- 1.6 (SD) microM and greater than 100 microM with respect to dATP and dTTP. The effects of ara-CTP and aphidicolin on these enzymes were opposite those seen with Fara-ATP, since 50% inhibitory concentrations of either ara-CTP or aphidicolin for DNA polymerases alpha/delta did not inhibit polydeoxythymidylate primase activity. The results provide evidence that fludarabine phosphate blocks DNA synthesis in CCRF-CEM cells through inhibition of primer RNA formation. In contrast, the accumulation of primer RNA and RNA-primed Okazaki fragments that is induced by ara-CTP and aphidicolin could lead to the rereplication and amplification of chromosomal DNA segments.
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PMID:Inhibition of primer RNA formation in CCRF-CEM leukemia cells by fludarabine triphosphate. 170 19

1. Pharmacodynamics and pharmacokinetics of antimetabolites. Antimetabolites are administered in the form of a base or its riboside, which is incorporated into the cell and converted to an active or inactive metabolite. The active metabolite remain in the cell inhibiting the enzymes to catalyze nucleotide synthesis for nucleotide triphosphate formation, but the inactive metabolites are rapidly excreted out of the cell. The inhibitory effect of antimetabolites on nucleotide formation is correlated with factors, such as maintenance of drug blood level, incorporation of the drug into the cell, activation and inactivation of the drug, affinity of the active form to the corresponding enzyme, and change in pool size of the intermediate metabolites in nucleotide synthesis. The salvage synthesis occurring at the higher level of the enzymes catalyzing nucleotide synthesis to counteract the inhibition by the drug is also correlated with the nucleotide formation. II. Pyrimidine antagonists 1. Cytosine arabinoside (ara-C) and its derivatives Ara-C is rapidly converted to ara-CTP and ara-U. The former remains in the cell and inhibits DNA polymerase, but the latter is excreted rapidly out of the cell. A small portion of ara-C is incorporated into DNA, which results in the degradation of DNA as demonstrated by reduced sedimentation of bulk DNA in alkaline sucrose gradient centrifugation and the ladder DNA fragmentation with a minimum fragment of approximately 180 base pairs and its conjugates in agarose gel electrophoresis. Behenoyl ara-C (BHAC) is highly lipophilic and highly distributed in the erythrocyte stroma and membrane fraction of leukocytes after iv infusion. The incorporated BHAC is released after the plasma BHAC level decreases, which suggests that erythrocytes can be a drug reservoir after iv infusion. Therefore, severe anemia should be treated before BHAC chemotherapy for longer maintenance of the plasma BHAC level. 2. 5-Fluorouracil (5-FU) and its derivatives Activation of 5-FU in the cells is metabolized by uracil metabolizing enzymes to FUMP and FdUMP. FUMP is further metabolized to FdUMP and is also incorporated to RNA. FdUMP produces a ternary complex with thymidylate synthetase and leucovorin; subsequently, conversion of dUMP to dTMP is strongly inhibited. Thus, FUMP and FdUMP inhibit RNA and DNA metabolism, respectively. Enzyme activity during 5-FU metabolism and consequently the degree of inhibition of DNA and RNA syntheses markedly differ with the tumor cell species. This should be taken into consideration when performing chemotherapy of malignancies.
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PMID:[Clinical pharmacology of anticancer agents (Part 4). Antimetabolites (1)]. 173 42

P388 murine leukemic cells 28.6-fold resistant to vincristine were cross-resistant to doxorubicin and etoposide. Although the intracellular ara-CTP in P388 murine leukemic cells resistant to vincristine (P388/VCR) was significantly higher than that in the parent cells, the cytotoxic effect of ara-C was not significantly different between the parent and resistant cells. Therefore, we investigated the DNA synthesis in P388/VCR and its parent cell line using the permeable cell system. The DNA synthesis in P388/VCR cells was less sensitive to ara-CTP and dTTP than that in P388 parent cells. These results suggested that the characteristics of DNA polymerase in P388/VCR cells might change when the cells developed multiple drug resistance.
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PMID:Decreased DNA polymerase sensitivity to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in P388 murine leukemic cells resistant to vincristine. 189 49

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.
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PMID:The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells. 190 85


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