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)

A novel form of DNA polymerase I (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, DNA nucleotidyltransferase, EC 2.7.7.7) activity has been isolated from Escherichia coli cells that had been activated for expression of the DNA damage-inducible genes. Induction was by treatment of normal cells or cells carrying the spr-51 and tif-1 mutations with nalidixic acid. This activity, DNA polymerase I, seems to be a form of DNA polymerase I because it is insensitive to N-ethylmaleimide, is inhibited by antibody to DNA polymerase I, and does not appear in a polA1 strain. DNA polymerase I activity sediments through sucrose gradients as a broad peak with s20.w = 6.6--10.5, compared with an s20,w = 4.8--5.5 for DNA polymerase I. The fidelity during polymerization reactions of DNA polymerase I is relatively low with a variety of synthetic templates and deoxynucleoside triphosphates, although the enzyme appears to have a normal level of 3' greater than 5' exonuclease. Polymerase I has properties that might implicate it in some form of mutagenic DNA repair.
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PMID:Isolation of an altered form of DNA polymerase I from Escherichia coli cells induced for recA/lexA functions. 628 65

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase alpha from both calf thymus and human lymphoma cells and DNA polymerase beta from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG lesions when Mg2+ is the divalent cation. Substitution of Mn2+ for Mg2+ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase alpha inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase beta is specific, inserting mainly C even in the presence of Mn2+. The Km for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn2+ is about 0.5 mM. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg2+ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn2+. dNTP alpha S and recA protein increase only the insertion of C. We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.
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PMID:A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts. 649 59

Two DNA polymerase activities, polymerases A and B, were separated from the Triton-treated cell homogenate of exponentially growing Tetrahymena pyriformis by phosphocellulose column chromatography. Their properties were as follows. Polymerase A: The molecular weight was about 140,000, the sedimentation value was about 6.2S, the optimum Mg2+ concentration was 15 mM, the optimum K+ (or Na+) concentration was 20 mM, and the optimum pH was 7.4. The enzyme activity was inhibited by cytosine-beta-D-arabinofuranoside-5'-triphosphate (araCTP) or aphidicolin, but not by 2'-3'-dideoxythymidine-5'-triphosphate (ddTTP). Polymerase B: The molecular weight was about 70,000, the sedimentation value was 4.3S, the optimum Mg2+ concentration was 15 mM, the optimum K+ (or Na+) concentration was 150 mM, and the optimum pH was 8.4. The enzyme activity was inhibited by ddTTP, but not by araCTP or aphidicolin. Polymerases A and B were both found to be N-ethylmaleimide-sensitive. These results indicate that at least two N-ethylmaleimide-sensitive DNA polymerases, A and B, are present in exponentially growing Tetrahymena cells. Polymerase A bears many similarities to DNA polymerase alpha of higher eukaryotes and polymerase B also bears similarities to DNA polymerase beta except as regards N-ethylmaleimide sensitivity. Based on the properties of polymerases A and B, the relation of Tetrahymena DNA polymerases reported by several investigators is discussed.
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PMID:DNA polymerases of Tetrahymena pyriformis. I. Characterization of two N-ethylmaleimide-sensitive DNA polymerases from exponentially growing cells. 680 56

DNA polymerase activity was extracted from testis cells of the dogfish Scyliorhinus caniculus. On a sucrose gradient, two main peaks could be separated, corresponding to DNA polymerases beta (3.8 S) and alpha (7.5 S). DNA polymerase gamma could also be detected when poly(A) . (dT)12 was used as template. The properties of alpha and beta polymerases of this primitive vertebrate were similar to those generally described, especially in mammals. The beta enzyme was highly sensitive to N-ethylmaleimide, however, and could use poly(dT) . poly(A) as template. Polymerase alpha was present in spermatogonia, spermatocytes and spermatids. Activity was maximal in spermatocytes. DNA polymerase beta was present in all testis cells with similar activities in spermatogonia and spermatocytes. Decreased activities were observed during spermiogenesis. Some activity remained associated with the chromatin fraction of mature sperm cells.
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PMID:Study of dogfish (Scyliorhinus caniculus) deoxyribonucleic acid polymerase alpha and beta. Extraction, separation, characterization and changes during spermatogenesis. 719 42

Two distinct DNA polymerases, A and B, isolated from a rapidly growing apical tissue of cauliflower (Brassica oleracea var. botrytis) inflorescence have been characterized, and compared with DNA polymerases, alpha and beta, from mouse myeloma. Polymerase-A bears a strong resemblance to polymerase-alpha from mammalian cells in all properties examined. The character of polymerase-B is also quite similar to polymerase-beta of mammalian cells in chromatographic elution properties, template-primer utilization, sensitivity to inhibitors, response to KCl or KPi concentration, and other requirements for maximal activity, although it has a higher molecular weight (approx. 78,000) even in the presence of 0.25 M KCl that polymerase-beta (mol. wt. less than or equal to 50,000) of mammalian cells. This type of DNA polymerase has not been reported to exist in the plant system.
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PMID:Characterization of two DNA polymerases from cauliflower inflorescence. 739 Sep 85

We have previously demonstrated that nonamers can prime manual T7 polymerase sequencing reactions. We next wanted to determine whether nonamers could be used to prime sequencing reactions for the Applied Biosystems Model 373A fluorescence-based sequencer. Both the Applied Biosystems T7 and Taq DNA Polymerase DyeDeoxy Terminator methodologies were tested, and successful results were obtained using a modified Taq DNA polymerase cycle sequencing procedure. The best nonamer cycle sequencing reaction conditions found, thus far, include denaturation at 96 degrees C for 30 s, primer-template annealing at 20 degrees C for 5 min, a 5-min ramp to the extension temperature, extension at 60 degrees C for 4 min and repeating this cycle 50 times. Furthermore, we found that the results were greatly improved by using linear (restriction enzyme cut) and alkaline-denatured, double-stranded DNA in combination with a doubling (2x) of the standard cycle sequencing reaction mixture containing a 2% final concentration of dimethyl sulfoxide. A total of 121 nonamer primers were tested, and approximately 50% primed successful cycle sequencing reactions. An average reading length of 257 bp was obtained from the successful reactions.
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PMID:Fluorescence-based cycle sequencing with primers selected from a nonamer library. 766 98

Positive diagnosis of Herpes simplex virus (HSV) encephalitis was rarely obtained in the past, when brain biopsy had been performed. Other tests (HSV antigen and HSV antibodies detection and interferon alpha measurement, in cerebrospinal fluid) failed to prove HSV infection. Polymerase chain reaction has been proposed for accurate and rapid diagnosis of HSV encephalitis. With 35 cycles of a DNA polymerase sequence duplication, sensitivity reaches 95% and specificity 100%. HSV PCR is a useful tool for the diagnosis of acute encephalitis. This should be available in many neurologic clinics. Therapeutic consequences include rapid disruption of aciclovir when clinical features, MRI study and negative PCR suggest non herpetic encephalitis.
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PMID:[Value of gene amplification of herpesviruses in the diagnosis and treatment of acute viral encephalitis]. 767 39

Denaturing gradient gel electrophoresis (DGGE) separates (DNA) molecules based on their sequence. Using the proper conditions, all base-pair substitutions can be resolved from the wild-type sequence using DGGE. Polymerase chain reaction (PCR) permits rapid amplification of a given region of the genome. In this paper, we demonstrate the utility of DGGE combined with PCR for mutation analysis by presenting different examples: (i) analysis of mouse p53 cDNA for mutations, (ii) simultaneous analysis of thousands of 4NQO-induced mutants for mutations in HPRT exon 3, (iii) examination of the fidelity of the thermostable DNA polymerase isolated from Pyrococcus furiosus (Pfu), (iv) purification of mutant DNA from contaminating wild-type DNA from mouse spleenic T-cell clones.
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PMID:Mutational analysis using denaturing gradient gel electrophoresis and PCR. 768 54

Detection of Mycobacterium tuberculosis by microscopy is difficult in specimens containing fewer than 10(4) bacteria/mL and growth in culture can take up to 6 wks. In this study the Polymerase Chain Reaction (PCR) was investigated as a rapid diagnostic technique for the detection of M. tuberculosis. The presence of DNA polymerase inhibitors in sputum specimens poses a potentially serious problem as false negative results can occur. In this study polymerase inhibitors were detected by inclusion of an internal plasmid control in each test. DNA from specimens in which the internal control failed to amplify was purified with a DNA binding matrix before retesting by PCR. A total of 169 sputum specimens were examined and 4 were found to have inhibitors. The correlation between detection of M. tuberculosis by PCR with a combination of culture, Ziehl-Neelsen (ZN) staining and patient history was 97.6%. This study confirms that PCR offers a more sensitive and rapid alternative for the detection of M. tuberculosis to ZN staining and culture, with results being available within 24 hrs of a specimen being received in the laboratory.
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PMID:The diagnostic use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis. 789 54

We have examined the molecular mechanism that enables the T4 bacteriophage DNA polymerase holoenzyme to synthesize DNA processively on the leading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from one Okazaki fragment to the next in less than 4 s. We use a perfect hairpin helix of 15 base pairs to mimic the encounter of the polymerase with the end of a previously synthesized Okazaki fragment. Polymerase dissociation is monitored during the stall at the hairpin helix by the addition of excess T4 gene 32 protein (SSB protein), which rapidly melts the helix and allows a stalled polymerase molecule to continue DNA synthesis. In the accompanying paper, we show that polymerase holoenzyme dissociation is slow (half-life of 2.5 min) when this enzyme is stalled by nucleotide omission (Hacker, K. J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 24209-24220). In contrast, the holoenzyme dissociates with a half-life of 1 s after hitting the hairpin helix, a rate sufficient to allow efficient polymerase recycling on the lagging strand in vivo. We conclude that, upon completing each Okazaki fragment, the holoenzyme senses an encounter with duplex DNA and then switches to a state that rapidly dissociates.
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PMID:The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment. 792 78


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