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)

We have recently shown that DNA containing homopolymeric A-T base pairs can form a parallel-stranded intramolecular duplex [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present paper, we demonstrate that parallel-stranded DNA can also be formed in unconstrained bimolecular DNA of appropriate sequence homology. Three deoxyoligonucleotides, a 21-mer [dCCCATATATATTTTTTTTCCC], a ps-15-mer [dTATATATAAAAAAAA], and an aps-15-mer [dAAAAAAAATATATAT], have been synthesized. Annealing of 21-mer and aps-15-mer results in the formation of a conventional antiparallel duplex (aps); however, the combination of 21-mer and ps-15-mer forms a duplex in which the two strands are in a parallel orientation (ps). The parallel-stranded structure was established from the following criteria: (i) The parallel-stranded structure shows a 1:1 stoichiometry of the constituent strands. (ii) Gel electrophoretic mobility of the ps and aps duplexes are similar under native conditions. (iii) Spectroscopic properties of the ps duplex are characteristics for a base-paired structure but are different from the aps structure. (iv) Both duplexes undergo a thermally induced helix to coil transition; however, the melting temperature for the ps duplex is 22 degrees C lower. (v) The minor groove binding drug Hoechst 33258 shows a reduced affinity for the ps compared to the aps duplex. (vi) The parallel-stranded duplex is not a substrate for DNA Escherichia coli polymerase I (Klenow fragment) or AMV reverse transcriptase. Parallel-stranded DNA can exist under normal solution conditions, but competition experiments show it to be thermodynamically less favorable than the conventional antiparallel form.
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PMID:Relative stability of parallel- and antiparallel-stranded duplex DNA. 246 57

A number of reports have suggested that platelets from polycythemia vera patients contain reverse transcriptase activity that might be correlated with C-type retrovirus-like particles. As described herein, we devised a new assay method for reverse transcriptase activity using MS-2 phage RNA hybridized with synthetic oligodeoxynucleotide (18-mer) as a template/primer. Using this new, sensitive assay method, we examined the platelet enzyme. By the conventional assay method using poly(rA)-oligo(dT), extracts of platelets showed a considerable amount of incorporation. However, by the new assay method using MS-2 RNA, no incorporation was observed. The poly(rA)-oligo(dT)-dependent activity was purified on Mono Q column, and it was shown that this activity coincided with that of DNA polymerase gamma.
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PMID:Re-examination by improved reverse transcriptase assay of DNA polymerase in platelets from myelodysplastic disease patients. 248 74

Effects of phosphorothioate oligodeoxynucleotides of different chain length and base composition on herpes simplex virus (HSV) type 2 (strain 333)-induced DNA polymerase have been examined in vitro. The anti-HSV-2 DNA polymerase activity was related to the base composition of the analogs, with the order of potency: deoxycytidine greater than thymidine greater than deoxyadenosine, for compounds with equal chain length. The potency was also related to oligomer chain length, since it was observed that the longer the chain length, the more potent the inhibition exerted. Among all the compounds tested, the phosphorothioate oligodeoxycytidine 28-mer (S-(dC)28) was the most potent inhibitor of HSV-2-induced DNA polymerase. This inhibition was competitive with an activated DNA template with a Ki value of 7 nM. It was also a competitive inhibitor of the DNA polymerase-associated exonuclease activity with a Ki value of 5 nM. In contrast, this compound showed less inhibition of human DNA polymerase alpha, beta, and gamma, as well as HSV-1 (strain KOS) and Epstein-Barr virus-induced DNA polymerase. The possibility that S-oligomers can serve as primers for DNA elongation was also investigated. Poly(dG).S-(dC)28 and poly(dA).S-(T)28 are poor substrates for DNA elongation catalyzed by HSV-2 DNA polymerase. In summary, phosphorothioate oligonucleotides could be anti-template inhibitors of HSV DNA polymerase. This information may lead to the development of a new class of selective anti-HSV agents.
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PMID:Effect of phosphorothioate homo-oligodeoxynucleotides on herpes simplex virus type 2-induced DNA polymerase. 254 98

The effect of NaF on the enzymatic activities of the large fragment of E. coli DNA polymerase I (Klenow enzyme-KE) with different DNA-substrates was studied. It was shown that fluoride ion at concentrations of 5-10 mM efficiently inhibits the 3'----5' exonuclease activity of KE but does not affect the polymerase activity of the enzyme. Selective inhibition of the 3'----5' exonuclease activity of KE is Mg-dependent and is observed with double- or single-stranded DNAs. In reaction with the 14-mer oligonucleotide annealed with single-stranded phage M13 DNA the enzyme was found not only to perform the exonucleolytic hydrolysis of the primers but to catalyse also a limited elongation of some primers, adding a few nucleotide residues in the absence of exogenous dNTP. The primer elongation is inhibited by inorganic pyrophosphatase and is stimulated by micromolar concentrations of exogenous pyrophosphate thus suggesting a possible role of PPi contamination in dNTP generation via pyrophosphorolysis. Traces of precursors in DNA preparations obtained by generally employed methods may serve as another source of nucleotides for the primer elongation.
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PMID:[Selective inhibition of 3'-5'-exonuclease activity of DNA-polymerase I from Escherichia coli by a fluoride ion]. 254 97

A ribonuclease H which degrades RNA specifically in RNA-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers Mg2+ over Mn2+. Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide. [3H]Poly(rA).poly(dT), [3H]poly(rC).poly(dI), and [3H]RNA.M13-DNA are degraded to 3-9-mer oligoribonucleotides with similar kinetics, whereas double- or single-stranded DNA, and double- and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinity-purified calf-thymus DNA-polymerase-alpha-primase complex threefold. When ribonuclease H is present in a three-fold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Ribonuclease H also stimulates the elongation rate of DNA polymerase alpha by a factor of 2-3, independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our results suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in mammalian cells.
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PMID:A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA-polymerase-alpha-primase complex. 255 72

phi X174 viral strand circular DNA can be synthesized in vitro from phi X174 replicative form I (RFI) DNA in the presence of the phi X A protein, the Escherichia coli DNA polymerase III elongation system, the E. coli rep helicase, and the E. coli single-stranded DNA binding protein. M13mp9 or pBR322 RFI DNAs containing a 30-base pair sequence found at the phi X origin of replication supported phi X A protein synthesis as well as the phi X template, giving rise to a net molar excess of deoxynucleotide incorporation. In this paper, we show that mutations in positions 1-3 of the 30-nucleotide origin replicated at a lower efficiency than plasmids containing the wild-type origin, because of a deficiency in the reinitiation reaction. Mutations in positions 4-7, upstream of the phi X A protein cleavage site, failed to support replication because of their inability to support nicking. An origin containing a mutation at the residue to which the phi X A protein is covalently linked to the DNA was an active template that supported a net molar excess of incorporation. Mutations at the 3' end of the origin region, retaining only the first 21-25 nucleotides of the 30-base pair origin, failed to support replication because of impaired binding of the phi X A protein to the template and consequently poor nicking. A construct bearing the first 28 nucleotides of the origin supported wild-type replication, as did a plasmid containing a 28-mer origin with a point mutation at position 26, but this latter construct also appeared to be partially deficient in phi X A protein binding activity. These results are consistent with the presence of a phi X A protein binding domain at the 3' end of the origin.
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PMID:Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. II. Effects of DNA replication of mutations in the 30-nucleotide icosahedral bacteriophage origin. 297 12

We have modified current methods to create a very efficient technique for cloning cDNAs in a defined orientation, into plasmid vectors bearing phage SP6 and T7 polymerase promoters. First strand synthesis is primed at the poly(A) tail with a 26-mer synthetic oligonucleotide linker/primer, the RNA is hydrolyzed and the cDNA is tailed with 10 to 15 dG residues. The cDNA is then annealed to two prepared vector fragments specific for the two ends of the cDNA (one bearing a dC10-15 tail and the other bearing a 14-nucleotide cohesive end complementary to the linker/primer). After ligation the second strand is synthesized with the large fragment of DNA polymerase I. Libraries of up to 8 x 10(6) independent transformants have been obtained from 1 microgram of Drosophila poly(A)+ RNA. The design of the method and careful optimization of first strand synthesis have permitted cloning of several large (4.3 to 6.5 kb), low abundance cDNAs. Transcription of essentially full-length clones with phage SP6 RNA polymerase produces RNAs that are efficiently translated in vitro to give complete, unfused products, thus permitting rapid characterization of the clones via the encoded polypeptides. Antisense RNAs can also be produced by transcription with phage T7 RNA polymerase.
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PMID:Functional cDNA libraries from Drosophila embryos. 319 41

Under appropriate conditions synthetic oligonucleotide hybridization probes display essentially absolute hybridization specificity. That is, every nucleotide must form a Watson-Crick base pair in order that the probe forms a stable duplex. All of the non-Watson-Crick base pairs, including G-T, have a destabilizing effect. Thus, it is possible to choose stringent conditions of hybridization such that, while a perfectly matched duplex between an oligonucleotide and complementary DNA will form, duplexes mismatched at one or more position will not. Mutations in a single base in the DNA sequence of a gene can and do result in genetic diseases. The hybridization of oligonucleotides to the region of DNA containing these base changes would be affected by the mutations and thus, oligonucleotide hybridization provides a means of detecting single base changes. In an attempt to develop a non-radioactive method for the detection of human genetic diseases, we have prepared biotinylated-oligonucleotides by an enzymatic method. An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3'-terminus was prepared by a primer extention reaction using E. coli DNA polymerase I (Klenow fragment). The probe could be specifically and tightly bound with Avidin D in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing 5 non-consecutive non-complementary bases. The hybridized biotinylated probe could be visualized by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmol) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.
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PMID:Application of synthetic oligonucleotides to the diagnosis of human genetic diseases. 386 11

Single-stranded DNA of coliphage M13mp8 was treated with the oxidizing agent, KMnO4, under conditions that selectively form cis-5,6-dihydro-5,6-dihydroxythymine (thymine glycol). Treatment of DNA with 0.7 and 1.4 mM KMnO4 introduced approximately 200 and 400 thymine glycol residues, respectively, per genome. When these DNAs were used to transform Escherichia coli, it was observed that phage survival was reduced in a dose-dependent manner. In studies designed to investigate the effect of DNA oxidation products on replication in vitro, a complementary 15-mer oligodeoxynucleotide was annealed to the oxidized template and extended with the Klenow fragment of DNA polymerase I from E. coli. It was observed that lesions in oxidized DNA strongly inhibited DNA elongation and that DNA synthesis was stopped opposite thymine residues. This is taken as suggestive evidence that the thymine glycol is inhibitory to DNA replication.
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PMID:Possible role for thymine glycol in the selective inhibition of DNA synthesis on oxidized DNA templates. 390 79

An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3' terminus was prepared by a primer extention reaction using Escherichia coli DNA polymerase I (Klenow fragment). For efficient synthesis of the probe, it was necessary to add about 16-fold molar excess of the template oligonucleotide (pentadecanucleotide) to the primer oligonucleotide (nonadecanucleotide) in the reaction mixture and to continue the reaction for 2.5 hr at 4 degrees C. The probe was purified by polyacrylamide gel electrophoresis under denaturing conditions. The probe could be specifically and tightly bound with Avidin D (Vector Laboratories) in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing five consecutive noncomplementary bases. The hybridized biotinylated probe could be detected by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmoles) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.
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PMID:Biotin-labeled oligonucleotides: enzymatic synthesis and use as hybridization probes. 674 43


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