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.
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.
Department of Oncology, University College London Medical School, UK.
K A
KA
Grimaldi
S R
SR
McAdam
R L
RL
Souhami
J A
JA
Hartley
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA; D004249:DNA Damage; D008466:Mechlorethamine / Q000494:pharmacology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D000970:Antineoplastic Agents; D008466:Mechlorethamine; D004247:DNA; D002945:Cisplatin