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 from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.
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PMID:Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences. 49 22

We describe a method leading to the formation of closed circles of rDNA starting from total DNA of Xenopus laevis. Linear DNA molecules were digested with exonuclease 3 and self-annealed. Open circles were enriched and covalently closed by the simultaneous use of polynucleotide kinase, DNA polymerase and polynucleotide ligase. Closed circles of rDNA1 were shown to be alkali-resistant, to have higher density than linear molecules in cesium chloride density gradients containing ethydium bromide, and to have the sedimentation constant expected for a single repeat unit of rDNA comprehensive of its spacer.
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PMID:Preparation and isolation of covalently closed circular rDNA molecules from DNA of Xenopus laevis. 67 51

Predefined changes in a known DNA sequence were introduced by a general method. Oligodeoxyribonucleotides complementary to positions 582 to 593 of the viral DNA strand of the bacteriophage phiX174 am3 mutant (pGTATCCTACAAA), and to the wild type sequence in this region (pGTATCCTACAAA), were synthesized and used as specific mutagens. Each of these oligonucleotides was incorporated into a complete circular complementary strand when used as primer on a genetically heterologous viral strand template, by the combined action of subtilisin-treated Escherichia coli DNA polymerase I and T4 DNA ligase. Incomplete duplexes were removed or were inactivated by nuclease S1 and the products were used to transfect spheroplasts of E. coli. Both oligonucleotides induced specific mutations at high efficiency when used with heterologous template (15% mutants among progeny phage). The am phages isolated by this procedure are phenotypically gene E mutants, and contain A at position 587 of the viral strand. They thus appear identical with am3 and provide evidence that the change G leads to A at position 587 is sufficient to produce a defective E function. Since the template for the induction of am mutants carried another genetic marker (sB1), the strains carrying the induced mutations have the new genotype am3 sB1. It should be possible to introduce the am3 mutation into any known mutant strain of phi174 using this same oligonucleotide. Both possible transition mutations were induced in these experiments. In principle, the method could also induce transversions, insertions, and deletions. The method should be applicable to other circular DNAs of similar size, for example recombinant DNA plasmids.
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PMID:Mutagenesis at a specific position in a DNA sequence. 68 66

DNA, gamma-irradiated in vitro or in isolated thymocytes was treated with several enzymes to achieve repair of the radiation-induced single strand braks. Whereas an incubation with polynucleotide ligase can join only 25% of the single strand breaks, a combined treatemnt with exonuclease III (EC 3.1.4.1), DNA polymerase I (EC 2.7.7.7), and polynucleotide ligase leads to repair of 80% of the breaks. For this in vitro repair the exonuclease III has to remove several, probably damaged, nucleotides from the 3'-terminal producing a single-stranded gap, which will be filled in by DNA polymerase I and joined by ligase. Tests for successful rejoining of the strand breaks were performed by showing the loss of primer 3'-OH sites for DNA polymerase I, by the resistance of incorporated nucleotides in the gap to removal by a second exonuclease III treatment, and by strand break determination in the analytical ultracentrifuge. 20% of the radiation-induced strand breaks will not be repaired by this combined treatment possibly due to an incomplete binding of the ligase on the 5'-terminals and/or an incomplete removal of the damaged 3'-terminals by exonuclease III.
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PMID:In vitro repair of radiation-induced strand breaks in DNA. 77 32

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.
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PMID:Lysogenization of Escherichia coli by bacteriophage Lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins Q and P. 77 26

Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.
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PMID:Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand. 77 43

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.
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PMID:Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template. 77 32

Excision repair of UV-damaged Bacillus subtilis transforming DNA has been carried out by a sequential enzyme system in vitro. Incision adjacent to the pyrimidine dimer in the DNA strand by correndonuclease II-initiated excision of the damage by the 5' in equilibrium 3'-directed exonuclease of the Micrococcus luteus DNA polymerase. Reinsertion of nucleotides into the gap in the strand by the DNA polymerase at 10 degrees C terminated in a single-strand break which was sealed by a polynucleotide ligase, thereby repairing the DNA strand. This restored biological activity to damaged DNA up to doses resulting in 60% inactivation of transforming activity. At higher doses, less repair was achieved, due to the development of double-strand breaks during the in vitro incision and excision steps.
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PMID:Enzymatic repair of UV-irradiated DNA in vitro. 81 Dec 6

Escherichia coli B/r (suo) was infected, at 30 degrees C, with T4Dam+, T4DamB24-amN82 (I-, 44-, DNA-negative phenotype), and T4DamN134amBL292 (33-, 55-, maturation-defective phenotype). A genetic ('transformation') assay was used to monitor transcription of genes 30 (polynucleotide ligase), 42 (deoxycytidylate hydroxymethylase), 43 (DNA polymerase), rIIA, rIIB, and e (endolysin). The principal results are: (I) All of the genes studied were transcribed exlusively from the so-called l-strand of phage DNA. (2) DNA synthesis and the maturation-defective proteins were required to turn-off transcription of genes 42, rIIA, tIIB, and 43. Experiments performed with chloramphenicol suggested that all phage-specific proteins required to turn-off transcription of these genes were not present until 6 to 8 min post infection (p.i.). (3) During a normal developmental programme, gene 30 was transcribed throughout the eclipse. DNA-negative and maturation-defective conditions had no obvious effect on transcription of this gene. (4) During a normal lytic event, two discrete waves of gene e transcription were observed. The late wave was dependent upon DNA-synthesis and presence of functional maturation-defective proteins. The early wave was unaffected by DNA-negative or maturation-defective conditions. Experiments with chloramphenicol indicated that, if any virus-specific proteins are involved with regulation of early e transcription, such proteins are present by 3 min p.i. The data are interpreted to mean that early gene transcription is regulated by a minimum of two mechanisms. One of these mechanisms is fully operational by the 3rd min and, among the genes studied, controlled early e transcription. A second mechanism becomes operational between 6 and 8 min p.i. and controls transcription of genes 42, 43, rIIA, and rIIB.
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PMID:Transcriptional control of T4 coliphage-specific genes 30, 42, 43, rIIA, rIIB, and e. 94 27

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.
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PMID:Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro. 105 35


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