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

A P-32-postlabelling method was developed to measure cisplatin-DNA adducts. Platinated oligonucleotides with different chain lengths were enzymatically digested with deoxyribonuclease I; snake venom phosphodiesterase (SVPD) and prostatic acid phosphatase. We found that SVPD was not able to cut the phosphodiester bond immediately 5' to the platinated nucleotide. As a result the adducts had an attached 5' unmodified nucleotide, while the unmodified nucleotides were digested to nucleosides. 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 unmodified nucleotide at their 5' end can be phosphorylated by T4 polynucleotide kinase and [-P-32]ATP. This method was also shown to be suitable for the detection of cisplatin-adducts in platinated calf thymus DNA.