P-32 POSTLABELING ASSAY FOR FREE RADICAL-INDUCED DNA-DAMAGE - THE FORMAMIDO REMNANT OF THYMINE

A prominent lesion in DNA exposed to oxidative free radicals results f rom the degradation of thymine leaving a formamido remnant. A P-32-pos tlabeling assay has been developed for the detection of the formamido lesion. The assay is based on the circumstance that the lesion prevent s hydrolysis by nuclease P1 of the phosphoester bond 3' to the damaged nucleoside. Thus, a nuclease P1 plus acid phosphatase digest of DNA g enerates mostly nucleosides whereas the formamido lesion is rendered a s a modified dinucleoside monophosphate. Dinucleoside monophosphates, but not nucleosides, are apt substrates for P-32-postlabeling by polyn ucleotide kinase. The assay was applied to calf thymus DNA X-irradiate d in oxygenated solution. The formamido lesion could be detected down to a dose of a few Gy.