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Possible Role for Thymine Glycol in the Selective Inhibition of DNA Synthesis on Oxidized DNA Templates¹

Laboratory of Toxicology, Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Single-stranded DNA of coliphage M13mp8 was treated with the oxidizing agent, KMnO₄, under conditions that selectively form cis-5,6-dihydro-5,6-dihydroxythymine (thymine glycol). Treatment of DNA with 0.7 and 1.4 mM KMnO₄ introduced approximately 200 and 400 thymine glycol residues, respectively, per genome. When these DNAs were used to transform Escherichia coli, it was observed that phage survival was reduced in a dose-dependent manner. In studies designed to investigate the effect of DNA oxidation products on replication in vitro, a complementary 15-mer oligodeoxynucleotide was annealed to the oxidized template and extended with the Klenow fragment of DNA polymerase I from E. coli. It was observed that lesions in oxidized DNA strongly inhibited DNA elongation and that DNA synthesis was stopped opposite thymine residues. This is taken as suggestive evidence that the thymine glycol is inhibitory to DNA replication.

¹ This work was supported by Grant CA33821 from the NIH and by a fellowship to P. R. from Ministere de la Recherche et de l'Industrie, Paris, France.

² Present address: Laboratoire de Biophysique, Institute de Biologie Moleculaire et Cellulaire, C. N. R. S., 15 rue Descartes, 67084 Strasbourg Cedex, France.

³ To whom requests for reprints should be addressed.

Received 1/14/85. Revised 7/17/85. Accepted 8/28/85.