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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)

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

In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease. The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene. The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding. Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3%. From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified. The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene.
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PMID:Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis. 224 80

A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared from Bacillus stearothermophilus. The dideoxy sequencing method, combined with the use of M13 vector has proved to be the most powerful one for obtaining the sequences of large genomes. However, the hairpin structure formed along the single-stranded DNA template often prevents the DNA polymerase from moving on, with the result that no sequence information can be obtained. The heat-stable large fragment that we have obtained has proved to be the most active at 65 degrees C. When the sequencing reaction was carried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained were satisfactory.
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PMID:Heat-stable DNA polymerase I large fragment resolves hairpin structure in DNA sequencing. 367 99

Nucleoside diphosphokinase activity is present in highly purified preparations of DNA polymerase from Micrococcus luteus and Escherichia coli, and in a partially purified DNA polymerase from avian myeloblastosis virus. The activity is also observed in the protein fragment of molecular weight 76,000 that is produced by subtilisin cleavage of DNA polymerase I from E. coli. The NDP kinase activity in DNA polymerase preparations from M. luteus uses various ribo- and deoxyribonucleoside di- and triphosphates as substrates. The presence of this activity in preparations of DNA polymerase results in the apparent use of deoxyribonucleoside diphosphates as substrates for DNA synthesis, provided that some triphosphate is present to serve as a phosphate donor.
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PMID:Nucleoside diphosphokinase activity associated with DNA polymerases. 433 52

Purification of DNA polymerase from E. coli B has in two cases each time led to the isolation of two separate polymerase activities, enzyme A and enzyme B. Enzyme A was in contrast to enzyme B almost completely devoid of exonuclease activity. Each of the two enzymes yielded a single symmetrical activity peak in gel filtration chromatograms. From the elution volumes the molecular weights were estimated to be about 70,000 for enzyme A and about 150,000 for enzyme B. Treatment of enzyme B with subtilisin led to an increase of about 30 per cent of the polymerase activity while the exonuclease activity almost completely disappeared. The product of the subtilisin treatment (enzyme C) gave rise to a single symmetrical polymerase activity peak in a gel filtration chromatogram. The elution volume was identical to that obtained with enzyme A. It is concluded that enzyme A and enzyme C are formed by limited proteolysis of enzyme B.
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PMID:Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. 490 67

The primary sequence of DNA polymerase I from Escherichia coli K12 as derived from the DNA sequence (Joyce, C. M., Kelley, W. S., and Grindley, N. D. F. (1982) J. Biol. Chem. 257, 1958-1964) has been verified. Protein sequencing through eight cycles of the Klenow large fragment yields a unique sequence corresponding to residues 324 to 331 from the translated DNA sequence and defines the subtilisin cleavage site for formation of the large and small fragments as Thr323-Val324. Site-specific cleavage of whole enzyme and large fragment at cysteines and sizing of the resulting fragments verify the location of the two cysteines at residues 262 and 907 as assigned by the DNA sequencing. Isolation of tryptic peptides derived from DNA polymerase I yielded unique peptides whose composition exactly corresponded to theoretical tryptic peptides derived from the translated DNA sequence. Identification of the expected carboxyl-terminal tryptic peptide and carboxypeptidase digestion of whole enzyme and large fragment confirm histidine-928 as the carboxylterminus. A secondary structure prediction is made using the available primary sequence data. The model contains 43% alpha helix, 17% beta-structure, 58 beta-turns, and several interesting super-secondary structure elements.
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PMID:Escherichia coli DNA polymerase I. Sequence characterization and secondary structure prediction. 703 56

Previous efforts for biochemical study of the human hepatitis B virus (HBV) DNA polymerase have been limited by its tight association with viral nucleocapsids. We report here that the soluble DNA polymerase from HBV particles was obtained by low pH treatment of the viral particles followed by incubation with small amounts of subtilisin. By these treatments, the approximately 100-kDa band in the activity gel assay was gradually converted to approximately 70 kDa, which subsequently showed reverse transcriptase activity on several exogenous templates. The single approximately 70-kDa active band, which did not show any DNA polymerase activity in endogenous reaction, was eluted through DEAE-Sepharose chromatography. These results suggest that the approximately 100-kDa protein, most likely the product of HBV Pol open reading frame, is tightly associated with viral nucleocapsids, and the approximately 70-kDa protein, the proteolytic cleavage product of approximately 100-kDa enzyme, is solubilized from viral particles as an active enzyme on exogenous templates.
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PMID:Release of the hepatitis B virus-associated DNA polymerase from the viral particle by the proteolytic cleavage. 774 34

Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.
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PMID:Construction of single amino acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'-->3' exonuclease activity. 867 3

We introduce a method for finding weak structural similarities in a set of protein structures. Proteins are considered at their secondary structure level. The method uses a rigorous graph-theoretical algorithm which finds all structural similarities. Protein structures are modelled as undirected labelled graphs, the so-called protein graphs. We suggest that for detecting the similarities between two protein structures it is sufficient to find similarities in the protein core which consists of tightly packed secondary structure elements. Therefore, we can restrict ourselves to solving the maximal common connected subgraph problem instead of the maximal common subgraph problem. We have modified the algorithm by Bron and Kerbosch for solving that problem. The speed of the algorithm increases drastically. After calculating all maximal common connected substructures for all pairwise comparisons in a set of protein graphs the common substructure in all proteins can be calculated by intersecting them. In this paper we characterize the method briefly and explain the modelling of the protein structure in detail. For the pairwise alignment the similarity of porin (1OMF) with bacteriochlorophyll a (3BCL) and BirA protein (1BIB) with DNA polymerase III (2POL) will be discussed. In the case of the multiple structure alignment the similarity in variants of four phosphatases and in subtilisin Carlsberg, carboxypeptidase, elongation factor Tu, and flavodoxin will be represented. Our first experiments show that the method works correctly and fast. The method can be used for arbitrary graphs. Thus, different graph-theoretical models of protein structures can be examined.
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PMID:Detection of distant structural similarities in a set of proteins using a fast graph-based method. 932 32

In the past decade, polymerase chain reaction (PCR) has become an important tool for the identification of previously unknown microorganisms and the analysis of environmental microbial diversity. Several studies published during recent years, however, have demonstrated that products obtained after PCR using Taq or Vent DNA polymerases will contain hybrid molecules when several homologous target sequences such as multigene families, alleles, or RNA viruses are co-amplified. In this report, we examined the recombination frequency and the extent of template switching during PCR using Taq, Pfu and RTth/Vent DNA polymerases. As a test system we constructed a series of plasmids carrying between one and three frame shift mutations in the gene coding for the protease subtilisin or deletions of approximately 100 bp in the lacZ alpha. Highest recombination frequencies were observed when these mutants were co-amplified with Taq followed by RTth/Vent DNA polymerases. Pfu DNA polymerase displayed no discernable recombination activity under normal PCR conditions. Data also suggest that in vivo repair of heteroduplex DNA molecules in Escherichia coli by a RecA-independent mechanism, perhaps the mismatch repair, results in the formation of chimeric molecules. Using Bacillus subtilis as the host, however, can significantly diminish non-PCR RecA-independent in vivo recombination, owing to the fact that transforming DNA molecules enter B. subtilis as single strands. Combined, these results suggest that using Pfu DNA polymerase for amplification and B. subtilis as the host for transformation may significantly reduce chimera formation.
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PMID:Factors affecting PCR-mediated recombination. 1215 89


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