Measurement of cellular repair activities for oxidative DNA damage.
Two related assays capable of determining cell extract repair activities for different oxidative lesions in DNA are described. Both assays measure the incorporation of radiolabeled nucleotides during repair of an oxidatively damaged template in a cell-free system. The assays differ in the type of oxidative damage present in the DNA. In one, singlet oxygen is used to generate predominantly 8-oxo-2'-deoxyguanosine lesions. In the other, hydroxyl radicals are used to generate a broad spectrum of damage including oxidized bases and strand breaks. Assay conditions were adjusted to ensure that radiolabel incorporation was directly proportional to cell extract repair activity. These assays represent sensitive tools for investigating the regulation of repair systems for oxidative DNA damage.
Institute of Food Research, Norwich Research Park, Colney, Norwich, UK. ruan.elliott@bbsrc.ac.uk
R M
RM
Elliott
S B
SB
Astley
S
S
Southon
D B
DB
Archer
D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004274:DNA, Recombinant / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D005290:Ferric Compounds / Q000494:pharmacology; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D008751:Methylene Blue / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D026082:Singlet Oxygen; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein
D018736:DNA Adducts; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005290:Ferric Compounds; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D026082:Singlet Oxygen; D017665:Hydroxyl Radical; D009571:Nitrilotriacetic Acid; D010100:Oxygen; D008751:Methylene Blue; C020326:ferric nitrilotriacetate