Identification of thymidine-5'-aldehyde at DNA strand breaks induced by neocarzinostatin chromophore

Snake venom phosphodiesterase or endonuclease S1 digestion of neocarzinostatin chromophore-treated DNA, labeled in its thymidine residues, liberates an unusual labeled nucleoside from the 5' end of a drug-induced break. This substance, isolated by reverse-phase HPLC, possesses carbons from both the thymine and the deoxyribose moieties of thymidine in the DNA but, unlike thymidine, is readily degraded at pH 12 to thymine and a sugar fragment. The altered nucleoside was shown to contain a carbonyl group by its reduction with NaBH(4) to form a substance that has the chromatographic properties of thymidine and by its reaction with various hydrazines to form the respective hydrazone derivatives; the carbonyl exists as the 5' aldehyde as shown by its mild chemical oxidation to the carboxylic acid with simultaneous loss of the 5' (3)H. Mass spectral analysis showed a fragmentation pattern compatible with the structure thymidine-5'-aldehyde. These data indicate that the nonprotein chromophore of neocarzinostatin, in the presence of a reducing substance (2-mercaptoethanol) and molecular oxygen, selectively oxidizes the 5' carbon of nucleosides in DNA to the aldehyde, resulting in a strand break and a DNA fragment bearing nucleoside-5'-aldehyde at its 5' end.