Reaction of apurinic/apyrimidinic sites with [14C]methoxyamine. A method for the quantitative assay of AP sites in DNA.
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 degrees 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.
D000478:Alkylation; D001080:Apurinic Acid / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000378:metabolism*; D007700:Kinetics; D011089:Polydeoxyribonucleotides; D011119:Polynucleotides / Q000378:metabolism*
D002250:Carbon Radioisotopes; D004269:DNA, Bacterial; D006898:Hydroxylamines; D011089:Polydeoxyribonucleotides; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; C005214:methoxyamine