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Nucleic Acids Research, 1994, Vol. 22, No. 12 2311-2317
© 1994

MOLECULAR BIOLOGY

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

Keith A. Grimaldi, Simon R. McAdam, Robert L. Souhami and John A. Hartley*

Department of Oncology, University College London Medical School 91 Riding House Street, London W1P 8BT, UK

*To whom correspondence should be addressed

Received March 15, 1994. Revised May 10, 1994. Accepted May 10, 1994.

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.


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