DNA damage by anti-cancer agents resolved at the nucleotide level of a single copy gene: evidence for a novel binding site for cisplatin in cells

Abstract

A new PCR based technique has been developed to investigate the sequence selectivity of adduct formation by DNA damaging agents in a single copy gene in isolated genomic DNA or in drug treated cells. Single-strand ligation PCR (sslig-PCR) demonstrated that cisplatin and nitrogen mustards reacted with guanine in an N-ras fragment with varying sequence specificity similar to that observed previously in plasmid DNA. In cisplatin-treated cells sslig-PCR demonstrated all the adducts found in isolated DNA and with the same sequence selectivity showing a preference for GG and AG sites. However, in cells an additional site of DNA binding of cisplatin was observed at the two occurrences of the sequence 5′-TACT-3′ on the transcribed and non-transcribed strands. This sequence is not a recognised target for cisplatin and represents a novel adduct formed in cells.