Fluorescence postlabeling assay of DNA damage induced by N-methyl-N-nitrosourea.
We have used a new technique to assay DNA adduct by combining enzymatic digestion of DNA to nucleotides and fluorescence postlabeling. The assay relies on the selectivity of nucleotide chromatography by high-performance liquid chromatography and the sensitivity of fluorescence detection. This report describes the fluorescence postlabeling assay of N7-(N7-methyldGuo) and O6-methyl-2'-deoxyguanosine (O6-methyldGuo) in calf thymus DNA exposed to N-methyl-N-nitrosourea. Using a conventional fluorescence detector, fluorescence postlabeling assay detected 1 modified nucleotide/10(6) normal nucleotides in 100 micrograms DNA. Laser induced fluorescence detection offers a linear response of the chromatographic signal from 10(-13)-10(-16) mol fluorescent nucleotides (correlation coefficient, 0.998) improving the detection limit of the assay to 1 modified nucleotide/10(8) normal nucleotides in 10 micrograms DNA. Fluorescence postlabeling analysis of the authentic markers shows that both N7- and O6-methyldGuo can be assayed with similar detection sensitivity. However, the inherent nature of the instability of N7-methyldGuo, even under physiological conditions, makes its quantitation difficult by any postlabeling technique. O6-MethyldGuo, on the other hand, can be detected with sufficient selectivity and sensitivity by a fluorescence postlabeling assay. Quantitative efficiency of enzymatic excision and chemical postlabeling of O6-methyldGuo were validated by fluorescence postlabeling analysis of synthetic model DNA. Fluorescence postlabeling assay complements 32P-postlabeling assay for O6-methyldGuo without requiring the handling and disposal of radiolabel. Fluorescence postlabeling assay has potential, therefore, to monitor the level of O6-alkyldGuo in human DNA exposed to both environmental and chemotherapeutic alkylating agents.
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263.
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Sharma
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005453:Fluorescence; D009682:Magnetic Resonance Spectroscopy; D008770:Methylnitrosourea / Q000633:toxicity
C048746:7-methyl-2-deoxyguanosine; D008770:Methylnitrosourea; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; D003849:Deoxyguanosine