Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells.
Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).
Baumstark-Khan
C
C
Project Group Human Radiation Risk, Institute of Aerospace Medicine, Medical Faculty of the RWTH Aachen, Germany. christa.baumstark-khan@dlr.de
Hentschel
U
U
Nikandrova
Y
Y
Krug
J
J
Horneck
G
G
D000818:Animals; D006690:Bisbenzimidazole / Q000378:metabolism; D016466:CHO Cells / Q000378:metabolism / Q000528:radiation effects; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000032:analysis / Q000378:metabolism / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D009690:Nucleic Acid Conformation
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D004247:DNA; D006690:Bisbenzimidazole