DNA-DAMAGE BY ANTICANCER AGENTS RESOLVED AT THE NUCLEOTIDE LEVEL OF ASINGLE-COPY GENE - EVIDENCE FOR A NOVEL BINDING-SITE FOR CISPLATIN INCELLS

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