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Trends in Photochemistry & Photobiology   Volumes    Volume 6 
Adaptation of the fluorometric analysis of DNA unwinding (FADU) for the detection of repair-induced DNA strand breaks after UV-irradiation of mammalian cells
Christa Baumstark-Khan, Gerda Horneck, Jens Krug, Uwe Hentschel, Yelizabeta Nikandrova
Pages: 141 - 152
Number of pages: 12
Trends in Photochemistry & Photobiology
Volume 6 

Copyright © 1999 Research Trends. All rights reserved


Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray- induced DNA damage in mammalian cells. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionising radiation as well as to ultraviolet light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in presence and in absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding was processed at pH 12.1 for 30 min at 20 °C. DNA remaining double-stranded after alkaline reaction was detected by binding the Hoechst 33258 dye (bisbenzimide) and measuring its fluorescence. The immediate production of DNA strand breaks observed in all cell lines after exposure to X-rays was followed by restitution of high molecular weight DNA upon post-exposure incubation. In contrast, UV exposure resulted in the time-delayed production of DNA strand breaks related to nucleotide excision repair of DNA photoproducts in repair-proficient cells but not in the repair deficient UV5 cell line, especially when the repair inhibitor aphidicolin is added during repair incubation. These results demonstrate, that the FADU approach is suitable to distinguish between the 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 polymerisation and ligation).

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