Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding.
The measurement of oxidative DNA base modifications by different methods has received special attention in recent years. Here we describe a procedure to quantify DNA lesions recognized by the bacterial formamido-pyrimidine-DNA glycosylases (Fpg protein). These include 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) as well as some other forms of imidazole ring-opened purines, which are converted into abasic sites and subsequently into DNA single-strand breaks by the associated endonuclease activity. The frequency of DNA strand breaks is determined by the alkaline unwinding technique. The procedure provides a fast and sensitive tool to assess the extent of spontaneous as well as induced oxidative DNA damage in mammalian cells.
Hartwig
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A
University of Bremen, Department of Biology and Chemistry, Germany.
Dally
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H
Schlepegrell
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R
D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000187:drug effects / Q000528:radiation effects; D043382:DNA-Formamidopyrimidine Glycosylase; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*
D004277:DNA, Single-Stranded; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase