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)

Megaloblastic anaemia is due to a derangement of DNA synthesis caused by insufficient supply of one or other of the four deoxyribonucleoside triphosphate (dNTP) precursors of DNA synthesis or by direct inhibition of one or other DNA polymerase. Reduced supply of the pyrimidine deoxythymidine triphosphate (dTTP) may be caused by folate or vitamin B12 deficiencies or by the action of dihydrofolate reductase inhibitors (e.g. methotrexate, pyrimethamine or trimethoprim), all of which cause reduced supply of the coenzyme 5, 10 methylene tetrahydrofolate (pentaglutamate) needed for thymidylate synthetase. Reduced dTTP supply may also be caused by direct inhibition of thymidylate synthetase by 5-fluorouracil. Reduced supply of both purines, deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP), may be caused by hydroxyurea, 6-mercaptopurine (and probably by another purine antagonist azaserine), whilst reduced supply of both pyrimidine DNA precursors, dTTP and dCTP (deoxycytidine triphosphate) may be due to inherited orotic aciduria or to treatment with azauridine. Cytosine arabinoside directly inhibits DNA polymerase. DNA replication is a discontinuous process and a number of enzymes are concerned with different aspects of the process. The parental strands partly unwind and a large number of initiation points or origins are activated on both strands. A primer RNA is first synthesised using the parental strand of DNA as template. Fragments of new DNA are then synthesised on the parental DNA template, starting at the RNA primer, under the action of one or other DNA polymerase (probably gamma). The RNA primer is then removed and the gap left is filled by further DNA synthesis under the action of a different DNA polymerase (probably alpha). The fragments of new DNA are joined to give newly synthesised stretches of DNA (replicons) which are then liigated together to form bulk DNA of enormous molecular weight. It is suggested here that reduced supply of one or other of the four deoxyribonucleoside triphosphate (dNTP) during the 'S' phase of the cell cycle (due to vitamin B12 or folate deficiency, drug treatment or other congenital or acquired abnormality in synthesis of the dNTP) impairs the cell's ability to elongate newly initiated DNA fragments by preventing gap-filling, the polymerase needed for gap-filling requiring substantially greater concentrations of the deoxyribonucleoside triphosphates than the polymerase involved in chain initiation. Cytosine arabinoside, which also may cause megaloblastosis, may affect principally the synthesis of new DNA fragments. Since active protein synthesis is needed for the cell to enter the S phase and RNA synthesis is needed to prime new DNA synthesis, megaloblastic anaemia may be expected to occur only when DNA synthesis is inhibited but protein and RNA synthesis are relatively unimpaired...
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PMID:Vitamin B12--folate interrelations. 1 Jan 22

The sequence of 129 nucleotides next to the poly(A) tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a phasing primer, [5'-32P]p(dT)8dC. The sequence is in accord with (a) the pyrimidine tracts which were mapped in blocks along the cDNA, (B) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed from the cDNA with RNA polymerase, and (c) a limited amount of sequence deduced by partial spleen phosphodiesterase digestion and depurination of endonuclease IV oligonucleotides. The 3' end shows little secondary structure on its own. Ten nonsense codons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A-A-U-A-A-A with other polyadenylated RNAs is discussed.
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PMID:Sequence of 129 nucleotides at the 3'-terminus of encephalomyocarditis virus RNA. 7 85

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.
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PMID:Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant. 11 Jul 81

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells respond to alkylating and arylalkylating carcinogens with DNA excision repair, as assessed by their stimulation of DNA repair synthesis. In the present work, we have investigated whether DNA repair synthesis in ether-treated E. coli cells can serve as a general indicator to monitor the DNA-binding of carcinogens, mutagens and antitumor agents. Therefore, a standard assay was developed and comparative analyses were performed on 11 ultimate carcinogens, 10 proximate carcinogens, 2 tumor promoters, 6 mutagens, and 12 antitumor agents. All ultimate carcinogens (alkylating, acylating, arylalkylating agents) and mutagens (e.g., hydrogeen peroxide, acridine derivatives) caused DNA excision repair in wild type cells as measured by [3H] dTMP incorporation and simultaneously inhibited replicative DNA synthesis to various extents. Control experiments with the mutant cells uvrA and uvrB were performed to determine whether the pyrimidine-dimer-specific UV-endonuclease was involved in the removal of DNA damage. This was found to be true for the ultimate carcinogens (Ac)2 ONFln, mitomycin C, and for very reactive alkylating carcinogens. None of the ultimate carcinogens induced repair polymerization in mutant cells lacking the 5'-3' exonucleolytic activity of DNA polymerase I. Proximate carcinogens, such as Me2NNO, 4-nitroquinoline-1-oxide and aflatoxins, did not induce excision repair in the standard assay, probably because of the inability of E. coli to perform the activation steps necessary for covalent DNA-binding. However, Me2NNO, when pretreated with Udenfriend's hydroxylating mixture, gave rise to a low level of repair polymerization in ether-treated cells. Intercalating mutagens, such as quinacrine and ethidum bromide, inhibited replicative DNA synthesis. However, they were not found to be repair-inducers. THE TUMOR PROMOters TPA and phorbol-12,13-didecanoate did not cause excision repair, even when applied at high concentrations, nor did they inhibit repair synthesis stimulated by MeNOUr or (Ac)2 ONFln. The antitumor agents may be classified into two groups on the basis of the influence they exert on DNA synthesis: members of the first group (involving BCNU and bleomycin) stimulate repair polymerization and, in addition, inhibit DNA replication. These compounds are known to bind covalently to DNA. The second group of drugs (including adriamycin and cis-Pt(II)diammine complexes) inhibits DNA replication without stimulating repair synthesis. The predominant DNA-interaction of these compounds is known to be a non-covalent (i.e., intercalative, electrostatic) binding. Our experiments show that the ether-permeabilized E. coli cell can be successfully used to test ultimate carcinogens, mutagens and antitumor agents for repair-inducing and replication-inhibiting activity. The standard test might be extended to pre- and proximate carcinogens, provided these can be suitably activated.
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PMID:The nucleotide-permeable Escherichia coli cell, a sensitive DNA repair indicator for carcinogens, mutagens, and antitumor agents binding covalently to DNA. 15 98

Dose-response curves were compared for deletions [ColBR (resistant to colicin B) mutations being more than 80% deletions] and base changes (reversion of argFam to prototrophy argplus) induced in the same set of E. coli strains (wild-type for DNA repair, uvrA-, polA- and recA-) by N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), hydroxylamine (HA), 4-nitroquinoline I-oxide (4NQO), mitomycin C (MTC, UV and X-rays. All these agents induced deletions as well as base changes in the wild-type strain. Thus chemical mutagenesis differed in E. coli and bacteriophages in vitro, for HA, NTG, EMS and perhaps UV produced only point mutations in phage Tr. The patterns of deletion and base-change mutability in E. coli were surprisingly similar. (I) The recombination less recA- strain was mutable by only three (NTG, EMS, HA) of the seven mutagens for either deletions or base changes. (2) The uvrA- strain, unable to excise pyrimidine dimers, was very highly mutable by 4NQO and UV but immutable by MTC for both deletions and base changes. (3) The polA- strain, defective in DNA polymerase I due to a non-suppressible mutation, was very highly mutable by HA and highly mutable by MTC and 4NQO for both deletions and base changes but was highly mutable only for deletions by UV and X-rays, remaining normally mutable by the other agents for both deletions and base changes despite its high sensitivity to their inactivating action. We conclude that errors in the recA-dependent repair of induced DNA damage (after 4NQO, MTC, UV and X-rays) or errors in replication enhanced by damage to the replication system or to the template strands (after NTG, EMS, and HA) give rise to deletions as well as to base changes. From a comparative analysis of 14 dose-response curves for deletions and base changes, we conclude that the order of mutagenic efficiency relative to killing is (EMS, NTG) greater than (UV, 4NQO) greater than HA greater than (X-rays, MTC), and that X-rays, 4NQO, HA and MTC induce more ColBR deletions than Argplus base changes, whereas UV and EMS induce ColBR deletions and Argplus base changes at nearly equal rates and the specificity of NTG is intermediate between these two types.
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PMID:Comparative analysis of deletion and base-change mutabilities of Escherichia coli B strains differing in DNA repair capacity (wild-type, uvrA-, polA-, recA-) by various mutagens. 16 24

The sequence of 18 nucleotides in the region preceding the initiation of transcription of the gene N of bacteriophage lambda has been determined to be as follows (see article). The basic approach used for the sequence determination involved Escherichia coli DNA polymerase I-catalyzed elongation of the octadecanucleotide primer, dT-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, possessing the appropriate polarity and nucleotide sequence corresponding to the 5' end of the gene N transcript. Following hybridization of the primer to the r-stand of bacteriophage lambda CI85657, sequences of the newly grown ollgonucleotide chains were determined by a) partial exonuclease digestion followed by two-dimensional fingerprinting; b) determination of pyrimidine tracts; and c) nearest neighbor analyses. Primer elongation was carried out in a controlled manner, the size of the newly grown chains being kept short by the following techniques: a) insertion of a ribonucleotide unit as the 3' terminus of the primer; b) use of a limited number of deoxynucleoside 5'-triphosphates in the elongation reaction; and c) enlongation of the primer using all the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.
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PMID:The nucleotide sequence in the promoter region of the gene N in bacteriophage lambda. 16 18

A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).
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PMID:Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. 20 61

The phosphorylation of arabinofuranosylthymine (araThd) has been studied both in non-infected cells and in those infected with herpes simplex virus (HSV-1, Lennette; HSV-1, IES and HSV-2, D-316). In these experiments, HSV strains were used which either contain (Lennette, TK+ and D-316 TK+) or lack (IES, TK-) the capacity to induce pyrimidine deoxyribonucleoside kinase. It was found that extracellularly administered araThd is phosphorylated to ara TTP via araTMP and araTDP in both non-infected and in HSV-infected cells. The phosphorylating capacity is more than tenfold lower in non-infected cells than in infected cells. Interestingly, cells infected with the TK- strain have a tenfold higher phosphorylating capacity than normal, uninfected cells, a fact which might indicate that host cell deoxythymidine kinase is induced during HSV infection. AraTMP is incorporated into cellular DNA but not into HSV DNA. This finding is in contrast to observations with arabinofuranosyladenine, which is incorporated into both cellular and HSV DNA. In vitro experiments with HSV-induced DNA polymerase show that araTTP strongly inhibits the enzyme activity. Therefore we conclude that the inhibition of HSV DNA polymerase by araTTP (formed intracellularly from araThd) is the explanation for the observed antiviral activity of araThd.
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PMID:Phosphorylation of arabinofuranosylthymine in non-infected and herpesvirus (TK+ and TK-)-infected cells. 22 22

Deoxyribonucleic acid (DNA) from bacteriophage T7 has been used to monitor the capacity of gently lysed extracts of Escherichia coli to perform repair resynthesis after ultraviolet (UV) irradiation. Purified DNA damaged by up to 100 J of UV radiation per m2 was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. It was found that incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original molecular weight. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional tothe number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymerase I since it does no occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.
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PMID:Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli. 32 26

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.
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PMID:Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA. 32 59


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