Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damage in vitro: homology with bacterial repair enzymes

Robson, C. N., Milne, A. M., Pappin, D. J., Hickson, I. D. (March 1991) Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damage in vitro: homology with bacterial repair enzymes. Nucleic Acids Res, 19 (5). pp. 1087-92. ISSN 0305-1048 (Print)0305-1048 (Linking)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/1708495
DOI: 10.1093/nar/19.5.1087

Abstract

Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli.

Item Type: Paper
Uncontrolled Keywords: Amino Acid Sequence Animals Base Sequence Bleomycin/pharmacology Cattle Cloning, Molecular DNA/isolation & purification DNA Damage DNA Repair DNA-(Apurinic or Apyrimidinic Site) Lyase DNA-Directed DNA Polymerase/metabolism Deoxyribonuclease IV (Phage T4-Induced) Electrophoresis, Polyacrylamide Gel Endodeoxyribonucleases/genetics/ isolation & purification/metabolism Exonucleases/metabolism Hydrogen Peroxide/pharmacology Molecular Sequence Data Open Reading Frames Oxidation-Reduction Sequence Alignment Thymus Gland/enzymology
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > cDNA
diseases & disorders > pulmonary disease > oxidative stress
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Matt Covey
Date: 11 March 1991
Date Deposited: 18 Sep 2014 18:21
Last Modified: 08 Nov 2017 16:45
PMCID: PMC333785
Related URLs:
URI: http://repository.cshl.edu/id/eprint/30763

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