Novel chemical mechanisms of DNA damage by natural products
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Leinamycin is a structurally novel natural product isolated from the streptomyces with potent ant tumor and antibiotic activity. In a thiol rich environment, leinamycin generates an episulfonium ion that alkylates N7-deoxyguanosine residues in double strand DNA. In order to understand more about the molecular basis of leinamycin's potent cytotoxicity we set out to characterize the chemical stability of the guanine adduct formed by this compound in double stranded DNA. We report here that depurination of this N7-alkylguanine lesion is unusually facile. The elucidation of molecular mechanism behind the rapid depurination event might help to understand more about the molecule's potent cytotoxicity. So we studied the structure of the nucleoside analogue leinamycin - guanosine by NMR (1D, 2D) and proposed a probable mechanism for its rapid depurination event. It has been shown from earlier studies, that leinamycin does-not alkylate single strand DNA or nucleoside and needs specific non-covalent interaction with the DNA duplex for alkylation of the N7-deoxyguanosisne residues in DNA. Earlier work done in our laboratory shows that leinamycin is a novel non-classical DNA intercalator. Here we provided more evidence for this novel intercalative binding process. Leinamycin is the first natural product that reversibly alkylates N7- deoxyguanosine residues in DNA under physiological conditions. Our work shows that leinamycin-N7-deoxyguanosine adduct, either in duplex DNA or in isolated nucleoside decomposes to regenerate the episulfonium ion derived alkylating agent. We have seen in our studies with the natural product leinamycin, that it is very efficient in generating abasic sites and is one of the most toxic compounds found in nature. Abasic sites resulting from the hydrolysis of the glycoside bonds are one of the most common lesions in cellular DNA. Here we have shown by ³²P labeling studies and negative ion Nan spray mass spectrometric analysis that a basic site can react with the opposing guanine residues in the complementary strand and generate understand crosslink which can be trapped by reduction. We have also shown that guanine N2-amino group is required for cross-link formation. This is very striking because understand DNA cross-links have profound biological consequences. In addition, we came upon one another very interesting natural product, which is fasciculation. Early biological studies suggested that fasciculation's catatonic properties may stem from its ability to damage cellular DNA. Here in our laboratory, we have shown by sequencing gel studies that treatment of a 5'-³²P-labeled DNA duplex with fasciculation in pH 7.0 buffer causes strand cleavage selectively at guanine residues. We further showed by LC/MS & LC/MS/MS that alkylation of guanine residues occurs by a fasicular-inderived aziridinium ion.
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