Specific interaction of camptothecin, a topoisomerase I inhibitor, with guanine residues of DNA detected by photoactivation at 365 nm.
Camptothecin-induced DNA photolesions were examined after UVA irradiation at 365 nm. DNA single-strand breaks were induced both in supercoiled and in relaxed SV40 DNA. In uniquely end-labeled human c-myc DNA, camptothecin-induced cleavage occurred exclusively at guanines and was markedly enhanced by hot piperidine treatment. Runs of polyguanines were the most cleaved, especially in their 5' flank. Primer extension experiments in the absence of piperidine treatment confirmed these results and did not show additional lesions. We found that synthetic single-stranded oligonucleotides were more reactive than duplex oligonucleotides. In addition, an excess of dideoxyguanosine triphosphates competed for camptothecin-induced DNA photolesions. Therefore, camptothecin stacking in DNA grooves is more likely than genuine drug intercalation. Groove shielding with sodium or magnesium reduced camptothecin-induced photodamage while minor groove occupancy with spermine extended damages. Photolesion mechanisms were investigated using scavengers. In aerobic conditions, the most effective scavengers were thiourea, sodium azide, and catalase. Protection by superoxide dismutase was weak, and mannitol was ineffective. In anaerobic conditions, lesions were more extensive. Taken together, these results show that photoactivated camptothecin interacts specifically and intimately with guanines. This finding is consistent with preferential stimulation of topoisomerase I cleavage at sites that bear a guanine at their 5'-DNA terminus [Jaxel, C., et al. (1991) J. Biol. Chem. 266, 1465-1469] and with the camptothecin stacking model at topoisomerase I DNA cleavage sites.
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
F
F
Leteurtre
M
M
Fesen
G
G
Kohlhagen
K W
KW
Kohn
Y
Y
Pommier
D000818:Animals; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002412:Cations; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000528:radiation effects; D004249:DNA Damage; D016166:Free Radical Scavengers; D006147:Guanine / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010777:Photochemistry; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D059004:Topoisomerase I Inhibitors; D014466:Ultraviolet Rays
D002412:Cations; D016166:Free Radical Scavengers; D016271:Proto-Oncogene Proteins c-myc; D059004:Topoisomerase I Inhibitors; D006147:Guanine; D004247:DNA; D002166:Camptothecin