A P-32 POSTLABELING ASSAY FOR DNA-ADDUCTS INDUCED BY CIS-DIAMMINEDICHLOROPLATINUM(II)

A P-32-postlabelling method was developed to measure cisplatin-DNA add ucts. Platinated oligonucleotides with different chain lengths were en zymatically digested with deoxyribonuclease I; snake venom phosphodies terase (SVPD) and prostatic acid phosphatase. We found that SVPD was n ot able to cut the phosphodiester bond immediately 5' to the platinate d nucleotide. As a result the adducts had an attached 5' unmodified nu cleotide, while the unmodified nucleotides were digested to nucleoside s. This is a facile enrichment procedure for the adducts, because the normal nucleosides lacking the 3'-phosphate are not substrates for T4 polynucleotide kinase.:Instead, the adduct fragments containing an unm odified nucleotide at their 5' end can be phosphorylated by T4 polynuc leotide kinase and [-P-32]ATP. This method was also shown to be suitab le for the detection of cisplatin-adducts in platinated calf thymus DN A.