Single-Strand Ligation PCR for Detection of DNA Adducts
Many anticancer drugs, chemical carcinogens, and UV irradiation form covalent adducts on nucleotides in DNA. These adducts can interfere with transcription and replication of DNA and in so doing can trigger cell death or potentially carcinogenic mutations. Studies with anticancer drugs such as cisplatin and the nitrogen mustards have shown that even relatively simple DNA damaging agents show a degree of sequence preference in adduct formation on isolated plasmid DNA (Cullinane et al., 1993; Hartley et al., 1986; Mattes et al., 1986; Ponti et al., 1991). Agents with a greater degree of sequence selectivity are under development with the aim of improving the specificity of anticancer therapy. Carcinogens such as benzo[a]pyrene also show some selectivity for particular nucleotides when reacted with isolated plasmid DNA (Lobanenkov et al., 1986; Puisieux et al., 1991). These observations illustrate the importance of studying DNA damage at the nucleotide level of a single-copy gene in cells where DNA exists in a highly ordered structure complexed with many proteins, and where other intracellular components could affect the reactivity of drugs and carcinogens. It has also become clear that it is important to be able to study the repair of individual lesions since repair mechanisms play an important part in determining sensitivity of a tumor cell to chemotherapy, and in preventing mutation. Recent work has shown that mutation hot spots in the E. coli lacI gene and the human p53 gene corresponded to sites of slow repair of UV-induced pyrimidine dimers (Kunala and Brash, 1992; Tornaletti and Pfeifer, 1994). It would be interesting to learn how widespread this phenomenon is and whether similar correlations exist for chemical carcinogens.
KeywordsAdduct Formation Ligation Oligonucleotide Terminal Amine Group Thermostable Polymerase Slow Repair
Unable to display preview. Download preview PDF.
- Roberts, J., and Friedlos, F. (1981). Quantitative aspects of the formation and loss of DNA interstrand crosslinks in Chinese-hamster cells following treatment with cis-diamminedichloroplatinum(II) (cisplatin). 1. Proportion of DNA-platinum reactions involved in DNA crosslinking. Biochim. Biophys. Acta 655:146–151.PubMedCrossRefGoogle Scholar
- Smellie, M., Grimaldi, K., Bingham, J., McAdam, S., Thompson, A., Thurston, D., and Hartley, J. (1994). Cross-linking and sequence specific binding in isolated DNA and intact cells by C8-linked pyrrolobenzodiazepine dimers DSB-120 and AT-486. Proc. Am. Assoc. Cancer Res. 35:534.Google Scholar