Measurement of pyrimidine (6-4) photoproducts in DNA by a mild acidic hydrolysis-HPLC fluorescence detection assay.
Pyrimidine (6-4) pyrimidone photoproducts constitute one of the major classes of DNA lesions induced by far-UV irradiation. However, their biological role remains difficult to assess partly because of the lack of a specific and sensitive assay for monitoring their formation in DNA. Here is presented a measurement method based on the release of the (6-4) base adducts from DNA followed by an HPLC separation associated with a sensitive and specific fluorescence detection. The quantitative and mechanistic aspects of the chemical hydrolysis, based on the use of hydrogen fluoride stabilized in pyridine, were investigated, using dinucleoside monophosphate (6-4) photoproducts as model compounds. The final hydrolysis products were isolated and characterized by UV, fluorescence, mass, and 1H NMR spectroscopies. Application of the assay to far-UV irradiated calf thymus DNA provided information on the sequence effect on the rate of formation of three of the four possible bipyrimidine (6-4) photoproducts.
Douki
T
T
CEA/Département de Recherche Fondamentale sur la Matière Condensée, SESAM/LAN, Grenoble, France.
Voituriez
L
L
Cadet
J
J
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D018736:DNA Adducts; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D011740:Pyrimidine Dimers / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
D018736:DNA Adducts; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; D004247:DNA