Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.
DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.
Pommier
Y
Y
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Kohlhagen
G
G
Kohn
K W
KW
Leteurtre
F
F
Wani
M C
MC
Wall
M E
ME
D000478:Alkylation; D001483:Base Sequence; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D015195:Drug Design; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D059004:Topoisomerase I Inhibitors
C095684:7-chloromethyl-10,11-methylenedioxycamptothecin; D009841:Oligonucleotides; D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin