Reaction of apurinic/apyrimidinic sites with [C]methoxyamine: A method for the quantitative assay of AP sites in DNA |
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Authors: | Myriam Talpaert-Borl Michel Liuzzi |
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Institution: | Myriam Talpaert-Borl ,Michel Liuzzi |
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Abstract: | A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA. The method involves the reaction of 14C]methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss. Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37°C, pH 7.2) untreated DNA is almost unreactive and the 14C]methoxyamine incorporation in DNA is proportional to the number of AP sites. Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity. |
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Keywords: | DNA damage AP site Methoxyamine |
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