A 32P-postlabelling assay for DNA adducts induced by cis-diamminedichloroplatinum(II).

ABSTRACT

A 32P-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 [-32P]ATP. This method was also shown to be suitable for the detection of cisplatin-adducts in platinated calf thymus DNA.