Sensitive nonradioactive detection of UV-induced cyclobutane pyrimidine dimers in intact mammalian cells.
In this paper we present a sensitive procedure to determine specifically the induction as well as the removal of cyclobutane pyrimidine dimers in intact mammalian cells without radioactive labeling of the DNA. This technique allows the detection of DNA damage by UV doses as low as 0.1 J/m2. The method consists of gentle lysis of cell monolayers, high-salt treatment and incubation with the cyclobutane pyrimidine dimer-specific repair enzyme T4 endonuclease V, followed by alkaline unwinding, hydroxyapatite chromatography and fluorimetric DNA analysis. The number of T4 endonuclease V-sensitive sites correlates well with the amount of UV-induced cyclobutane pyrimidine dimers reported in the literature, indicating that these cyclobutane pyrimidine dimers are recognized quantitatively by the system. The assay is easily transferable to the detection of other types of DNA adducts by applying different damage-specific repair enzymes, providing a sensitive method to investigate the induction and the repair of DNA lesions without the use of radioactive labeling.
Kasten
U
U
University of Bremen, Department of Biology and Chemistry, Germany.
Beyersmann
D
D
Dahm-Daphi
J
J
Hartwig
A
A
D000468:Alkalies; D006690:Bisbenzimidazole; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004307:Dose-Response Relationship, Radiation; D004706:Endodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D009691:Nucleic Acid Denaturation; D011740:Pyrimidine Dimers / Q000032:analysis*; D014466:Ultraviolet Rays / Q000009:adverse effects*; D014764:Viral Proteins
D000468:Alkalies; D004277:DNA, Single-Stranded; D011740:Pyrimidine Dimers; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D006690:Bisbenzimidazole