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Fluorescence Postlabeling Assay of DNA Damage Induced by N-Methyl-N-nitrosourea¹

Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263

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/10⁶ normal nucleotides in 100 µg 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/10⁸ normal nucleotides in 10 µg 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.

¹ Supported in part by National Cancer Institute Grant CA46896.

² To whom requests for reprints should be addressed.

Received 12/18/92. Accepted 4/ 9/93.