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Nucleic Acids Research, 1992, Vol. 20, No. 24 6473-6479
© 1992

CHEMISTRY

Characterization and localization of cis-diamminedichloro-platinum(II) adducts on a purified oligonucleotide containing the codons 12 and 13 of H-ras proto-oncogene

M.-J. Pillaire, G. Villani, J.-S. Hoffmann+, A.-M. Mazard and M. Defais*

Laboratoire de Pharmacologie et de Toxicologie Fondamentales du CNRS 205 route de Narbonne, 31077 Toulouse cedex, France

*To whom correspondence should be addressed

Received October 20, 1992. Revised November 27, 1992. Accepted November 27, 1992.

The use of substrates containing well defined adducts at precise sites, is required to perform a careful analysis of the toxic and mutagenic potential of a lesion. As a first step in this direction the octamer 5'-d(CCGGCGGT), containing the sequence of the codons 12 d(GGC) and 13 d(GGT} of the human H-ras gene, was reacted with the antitumoral drug cis-diamminedichloroplatinum(II). The platinated products have been purified by HPLC. A first set of experiments, including enzymatic digestions with nuclease P1 followed by alkaline phosphatase and acid-catalysed hydrolysis, allowed us to determine which bases were engaged in the cis-DDP lesions. Our results indicate that only guanine residues were chelated with cisplatin to yield bifunctional adducts. Furthermore, by performing enzymatic digestions with phosphodiester ases, we have located the adducts with respect to the 5' end of the octamer. Among the purified and characterized platinated oligonucleotides, three present a particular interest, since we have shown here that the cis-d(GpG) adduct is precisely situated either at the d(GGC) or at the d(GGT) or at both sites of their sequence.

+ Present address: The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, WA 98195, USA


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J.-S. Hoffmann, M.-J. Pillaire, C. Lesca, D. Burnouf, R. P. P. Fuchs, M. Defais, and G. Villani
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