Fluorescence postlabeling assay of cis-thymidine glycol monophosphate in X-irradiated calf-thymus DNA.

DNA damage was induced by irradiating calf-thymus DNA with a GE Maxitron-250 as an X-ray source. The use of nitrous oxide as a scavenger of solvated electrons in the irradiation process, resulted in essentially a monoreactant system of the biologically important hydroxyl radical. A novel approach combining the enzymatic digestion of the irradiated DNA to nucleoside 5' monophosphates and fluorescence postlabeling was applied to detect a specific modified nucleotide induced by ionizing radiation, namely the 5,6-dihydroxy-5,6-dihydrothymine lesion. This modification, often referred to as the glycol lesion, is polar and is generated mainly as the cis stereoisomers. In order to demonstrate the detection of this lesion in DNA by fluorescence labeling, the lesion was first produced chemically in a DNA model compound d(CGTA). The modified oligomers were isolated intact by HPLC and characterized by NMR as cis stereoisomers of glycol derivatives of d(CGTA). The major isomer of the modified d(CGTA) was enzymatically digested to yield 5' monophosphates. The digest was chromatographed by HPLC to enrich the modified nucleotide. The fraction containing the modified nucleotide was labeled with dansyl chloride. The fluorescent labeled nucleotide was chromatographed by HPLC. The same overall procedure was applied to DNA X-irradiated in aqueous solution. With a conventional fluorescence detector, HPLC analysis of the fluorescence labeled nucleotides detected 1 modified nucleotide/10(6) normal nucleotides from 100 micrograms DNA. The two cis glycol lesions were detected in the irradiated DNA by co-chromatography with fluorescent labeled markers. The initial assay of the modified oligomer demonstrated that the same stereoisomer of cis glycol was induced as a major modified nucleotide by both chemical oxidation and ionizing radiation.