super(32)P-postlabeling detection of radiation-induced DNA damage: Identification and estimation of thymine glycols and phosphoglycolate termini

A super(32)P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates. Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and ( gamma - super(32)P)ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC.