Assay for reactive oxygen species-induced DNA damage: measurement of the formamido and thymine glycol lesions

• Published in: Biochimica et biophysica acta 1999-05, Vol.1454 (1), p.80-88
• Main Authors: Maccubbin, Alexander E., Patrzyc, Helen B., Ersing, Noreen, Budzinski, Edwin E., Dawidzik, Jean B., Wallace, John C., Iijima, Herbert, Box, Harold C.
• Format: Article
• Language: English
• Subjects: Acid Phosphatase; Autoradiography - methods; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; DNA - drug effects; DNA - radiation effects; DNA Damage; Phosphorus Radioisotopes; Polynucleotide 5'-Hydroxyl-Kinase; Pyrimidine Nucleosides - chemistry; Reactive Oxygen Species; Reference Standards; Single-Strand Specific DNA and RNA Endonucleases; Thymine - chemistry
• ISSN: 0925-4439; 0006-3002; 1879-260X
• DOI: 10.1016/S0925-4439(99)00022-8

A 32P-postlabeling assay has been developed for the simultaneous detection of the thymine glycol lesion and the formamido remnant of pyrimidine bases in DNA exposed to reactive oxygen species (ROS). The formamido lesion is a principal lesion produced in X-irradiated DNA oligomers when oxygen is available to mediate the damage process. Production of the well-known thymine glycol lesion is less dependent on the concentration of oxygen. These two lesions have the common property that they make the phosphoester bond 3′ to the modified nucleoside resistant to hydrolysis by nuclease P1. Our assay uses 32P-postlabeling to measure these lesions in the form of modified dimers obtained from DNA by nuclease P1 digestion. Appropriate carriers and internal standards have been chemically synthesized to improve the reliability and accuracy of the assay. The measurements were accomplished on 1-μg samples of DNA.