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)

Drosophila Rrp1 (Recombination repair protein 1) belongs to a family of DNA repair nucleases that includes Escherichia coli exonuclease III, Streptococcus pneumoniae exonuclease A, bovine BAP, mouse APEX endonuclease, and human APE. Within a 252 amino acid region, colinear homology is shared between all members. Rrp1 is unique in that it includes a 427 amino acid N-terminal region not related to any known sequence. The protein copurifies with an apurinic endonuclease and a double-stranded DNA 3'-exonuclease. In this study, a 5'-end-labeled 37 base pair oligonucleotide substrate containing a single apurinic site was used to characterize the endonuclease activity of Rrp1. This substrate is utilized efficiently by Rrp1: the specific activity observed is 1 x 10(5) units/mg. The abasic double-stranded DNA oligonucleotide is cleaved only at the abasic site to create a single-strand break. Strand breaks are not detected in the complementary strand, in the single-stranded DNA oligonucleotide, or in the base-paired control substrate. After endonucleolytic cleavage at the abasic site, exonucleolytic processing at the nick is slow and requires a molar excess of Rrp1, while exonuclease III degrades the nicked substrate more efficiently. The Rrp1 cleavage product comigrates with a DNaseI cleavage product, and the newly formed terminus supports DNA synthesis by DNA polymerase. Therefore, Rrp1 cleaves the phosphodiester backbone at one position 5' to the apurinic site and leaves a 3'-hydroxyl terminus. Rrp1 is a class II apurinic endonuclease and is likely to be important in DNA repair in Drosophila.
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PMID:Characterization of the apurinic endonuclease activity of Drosophila Rrp1. 769 63

A simple and precise assay is presented for quantification of the relative number of 3'OH ends (breaks) present in DNA molecules. The assay is based on the ability of the Klenow fragment polymerase to initiate random oligonucleotide-primed synthesis from the reannealed 3'OH ends of single-stranded (ss) DNA. After a denaturation-reassociation step, the ssDNA serves as its own primer by randomly reassociating itself or to other ssDNA molecules. Under strictly defined reaction conditions (time, temperature, concentration of precursors) the incorporation of [32P]dNTP into newly synthesized DNA will be proportional to the initial number of 3'OH ends (breaks). The assay is specific for the detection of 3'OH ends and requires only 0.25 micrograms of DNA for analysis. It has application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during normal processes of DNA repair and also for the detection of DNA strand breaks from genotoxic DNA damaging agents. Although specific for 3'OH DNA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or breaks induced by oxidative DNA damaging agents by pretreatment of the DNA with alkaline phosphatase or Escherichia coli exonuclease III. The assay is capable of quantifying first several breaks per 10(5) bp.
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PMID:Quantification of 3'OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay. 863 54

Chloroacetaldehyde (CAA) reacts with DNA bases, forming hydroxyethano derivatives of different stability, which are subsequently converted into etheno (epsilon) adducts: epsilon A, epsilon C, epsilon G. DNA polymerase fingerprint analysis was used to study the distribution of CAA-induced modifications in the p53 sequence. A plasmid bearing cDNA containing the human p53 gene was reacted in vitro with CAA, then dehydrated for conversion of hydroxyethano into etheno adducts, and primer extension by T7 DNA polymerase in the presence of four dNTPs was performed. The DNA repair enzymes methylpurine-DNA glycosylase and Escherichia coli exonuclease III were used to convert epsilon A residues in the template into DNA strand breaks, which enabled precise localization of the epsilon A residues within the p53 gene. Hydroxyethano derivatives of adenine and cytosine in a template blocked T7 DNA polymerase and caused premature chain termination opposite adenine or one base before cytosine. After dehydration, both epsilon A and epsilon C were much more easily by-passed by T7 DNA polymerase. Formation of epsilon G was identified as 'stop bands' one base before guanine residues. Modification of cytosine and guanine was additionally recognized by weakening or disappearance of non-specific stops on an undamaged template, probably due to steric hindrance by the tertiary DNA structure for polymerase. Etheno adduction of cytosine and guanine relaxed the compact DNA structure and enabled DNA polymerase to by-pass. In exons 5-8 of p53, 143 out of 500 sites appeared to be damaged by CAA, with four particularly densely modified regions between codons 135-147, 218-222, 234-255 and 284-292. The pattern of modification followed the pattern of p53 mutations found in vinyl chloride-associated liver angiosarcomas in humans and rats, but only in regions that showed 100% homology with the human sequence. The factors that influence DNA damage and induction of mutations in the p53 gene by CAA and vinyl chloride are discussed.
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PMID:Localization of chloroacetaldehyde-induced DNA damage in human p53 gene by DNA polymerase fingerprint analysis. 1062 28

Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage. Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion. Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini. These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin. Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min. EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons. Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons. Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS. Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P. In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain.
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PMID:In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain. 1065 97

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.
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PMID:Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates. 1087 49


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