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)

Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.
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PMID:Immunoaffinity purification and properties of Drosophila melanogaster DNA polymerase alpha-primase complex. 212 88

A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 and 8 of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-protein complex as template, Mg2+, and four deoxyribonucleoside triphosphates. No in vitro DNA synthesis was observed when deproteinized PRD1 DNA was used as template. The origin and direction of PRD1 DNA replication in vitro was determined by restriction enzyme analysis of 32P-labeled PRD1 DNA synthesized in this system. Replication starts at both ends of the linear PRD1 DNA template. Alkaline sucrose gradient centrifugation and agarose gel electrophoresis showed that full-length PRD1 DNA is synthesized in vitro. DNA synthesis in this system is inhibited by the drug aphidicolin. We also observed that dimethyl sulfoxide (DMSO) stimulates in vitro DNA synthesis, although it inhibits bacterial DNA polymerase.
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PMID:Protein-primed replication of bacteriophage PRD1 genome in vitro. 249 14

The effect of dimethyl sulfoxide (DMSO) on the interaction of human cytomegalovirus (HCMV) with host cell was studied. Confluent state of a human rhabdomyosarcoma cell line (A204) showed a much lower susceptibility to HCMV infection when compared to that in subconfluent actively growing cell cultures. Treatment of confluent cultures with DMSO, however, converted many nonproductive cells in these cultures to a productive state for virus replication. Infectious center assay revealed that approximately 100-fold more cells in the compound-treated cultures are able to produce infectious virus. The amount of infectious virus produced in DMSO-treated confluent cultures was enhanced by approximately 10,000-fold over production in untreated cultures and recovered to the level of that produced in subconfluent cultures productive state for virus replication. This cell physiology-dependent inhibition of HCMV replication and enhancement of virus growth by DMSO did not occur with herpes simplex virus type 2. Immunofluorescence staining, gel electrophoresis, and DNA analyses indicate that block of HCMV replication in confluent cultures probably occurs at the level of early transcription or translation of the viral genome. In contrast, in DMSO-treated confluent cultures appreciable amounts of HCMV DNA polymerase (an early virus function), viral DNA, and late antigens were synthesized. Pretreatment of confluent cultures with DMSO enabled the cells to support HCMV replication. In addition, the most effective enhancement by DMSO was found in cultures that had been treated with the compound up to 5 hr after infection. These results suggest that the enhancing effect by DMSO is primarily expressed through some host cellular function(s) and the early stages in virus growth cycle are most likely under control by DMSO action.
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PMID:Effect of dimethyl sulfoxide on interaction of human cytomegalovirus with host cell: conversion of a nonproductive state of cell to a productive state for virus replication. 299 16

DNA synthesis and DNA polymerase activities were followed in FL cell cultures (clone 5.86) at different stages of differentiation. A temporary block of growth and DNA synthesis was observed after the addition of the inducers (DMSO or HMBA). This delay in the growth and in the DNA synthesis initiation is not observed in cultures of DMSO-resistant variants after treatment with DMSO. In both uninduced and induced cultures, during growth, the DNA gamma-polymerase activity is constant and the alpha-polymerase activity is strictly dependent on the DNA synthesis rate. On the other hand, a different behaviour between induced and uninduced cultures is observed for the DNA polymerase beta: its activity is constant in uninduced cultures, whereas it changes in induced cultures at various stages of differentiation, dropping to lower values at early times and rising to values similar to those observed in uninduced cultures at later times. This behaviour is not observed in cultures of a DMSO-resistant variant clone: in this case the beta-polymerase activity is constant in both the absence and the presence of DMSO or HMBA.
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PMID:DNA polymerase activities in Friend cells during the differentiation process. 696 73

The chemicals 9, 10-dimethylbenzanthracene (DMBA), ethionine, daunorubicin, actinomycin D, 1-(2-chloroethyl-1)-nitrosourea (CCNU), steroids, croton oil and dimethyl-sulfoxide (DMSO) were used in order to correlate their effect on the in vitro synthesis of normal and cancer DNA, on DNA strand separation and on accelerated in vivo multiplication of cancer cells. All of the compounds tested strongly stimulate the synthesis of cancer DNA in vitro catalyzed by DNA-dependent DNA polymerase I and measured as an acid-precipitable labeled product. Under the same conditions, the synthesis of DNA originating from healthy tissues is only slightly enhanced, except in the case of croton oil and DMSO. These substances are almost equally active on cancer and normal DNA. Although both cancer and normal DNA contain a large amount of double-stranded regions, the extent of DNA strand separation measured by the increase in UV absorbance (hyperchromicity) in the presence of each compound tested is much higher for all cancer DNA than for corresponding normal DNA. In contrast, DMSO and croton oil do not appear to distinguish cancer DNA from normal DNA. Additive and differential effects of various compounds on cancer DNA strand separation can be observed. Small doses of DMBA and CCNU stimulate the multiplication of Ehrlich ascites tumor cells in vivo in mice. There is thus a possible correlation between DNA strand separation, DNA synthesis, multiplication and differentiation of cancer cells in the presence of the above compounds, which is different from the response of normal cells to these compounds.
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PMID:Correlation between in vitro DNA synthesis, DNA strand separation and in vivo multiplication of cancer cells. 725 Apr 82

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
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PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

Automated base calling algorithms are more sensitive to the quality of the DNA sequencing data than are the labor intensive visual methods of base calling. To improve this quality, data from DNA sequencing reactions have been compared in order to determine the effects of the inclusion of dimethyl sulfoxide (DMSO). Inclusion of 10% DMSO into the reaction cocktail resolves at least one type of sequence compression. This compression may be due to the lack of ability in T7 DNA polymerase to read through certain sequences correctly. The poor quality of these data is seen as radioactive bands or fluorescent signal peaks that have an abnormal alignment, either in the wrong order or as single bands/peaks. The inclusion of DMSO also resolves sequences where the peak signal is absent or severely diminished, leading to a "gap" in the chromatogram profile. DMSO is better than deaza-dITP for resolving certain compressions. Addition of DMSO is a cheaper and more efficient method for high-throughput DNA sequencing than repeating reactions with base analogs.
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PMID:DMSO resolves certain compressions and signal dropouts in fluorescent dye labeled primer-based DNA sequencing reactions. 761 23

A comparison was made of partially purified DNA polymerases alpha, delta, epsilon and from normal Chinese hamster ovary cells and mutant cells (JK3-1-2A) resistant to aphidicolin, araA, and araC. In vitro the pol alpha from the mutant cells (1) was resistant to aphidicolin and araCTP but was sensitive to araATP, (2) showed a 1.6 to 2.6-fold reduced specific activity, and (3) was more sensitive to carbonyldiphosphonate, DMSO and SJK 287-38 anti-pol alpha antibody inhibition, but was less sensitive to alkylphenyl nucleotide analogs BuPdGTP and BuAdATP. On the other hand, pol delta and pol epsilon of the mutant cells did not show increased aphidicolin-resistance but differed from the wild type enzymes with regard to their 3' exonuclease activity. The 3' exonuclease/DNA polymerase activity ratio was increased 6-fold for pol delta and 3.3-fold for pol epsilon for enzymes from the mutant cells in comparison to wild type values. It is suggested that these altered properties of the DNA polymerases of the alpha-family are responsible for in vivo aphidicolin resistance of the mutant cells. The higher 3' exonuclease activity may explain the observed antimutator phenotype of this cell line. In view of the proficient 3' exonuclease activities of the DNA pol delta and epsilon, the present aphr mutant is unique among all mammalian DNA polymerase mutants.
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PMID:An aphidicolin-resistant mutant of Chinese hamster ovary cell with altered DNA polymerase and 3' exonuclease activities. 764 Mar 4

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase, Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.
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PMID:Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. 812 24

This paper describes the cytotoxicity of ranunculin (RAN) and its mechanism of action. The IC50 of RAN against the KB and Bel7402 cells in colony test were found to be 0.21 and 0.35 mumol/L respectively. RAN inhibited the incorporation of 3H-labeled precursors into DNA and RNA of L1210 cells. RAN (15 mumol/L) markedly decreased DNA synthesis catalyzed by DNA polymerase I and promoted the generation of superoxide anions in DMSO/KO2 system. In the meantime, SOD and CAT were shown to partly revoke the inhibitory effects of RAN upon the incorporation of 3H-TdR into DNA. No direct reaction between RAN and DNA template was observed and no effect of RAN on DNA TOPO II or RNA polymerase was found. Our results suggest that the cytotoxicity of RAN in vitro may be due to inhibition of DNA polymerase and increase of oxygen free radicals.
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PMID:[The cytotoxicity and action mechanism of ranunculin in vitro]. 823 75


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