Identification of the methyl phosphotriester of thymidylyl (3',5')thymidine as a product from reaction of DNA with the carcinogen N-methyl-N-nitrosourea.

The methyl phosphotriester of thymidylyl(3'-5')thymidine, Tp(Me)T, was obtained as a product of enzymic digestion of N-[14C]methyl-N-nitrosourea-methylated DNA or of N-methyl-N-nitrosourea-methylated [14C]thymine-labelled DNA. The identity of the 14C-labelled Tp(Me)T products was shown by co-chromatography of the 14C-labelled enzymic digests with synthetic Tp(Me)T on Dowex 50 (NH4+ form, eluted at pH 8), and by co-chromatography, on silica gel in 3 solvent systems, of the Tp(Me)T-containing fractions from the Dowex 50 column. This identity was confirmed by showing that the 14C-labelled DNA-derived products hydrolysed in 0.1 M sodium hydroxide at 37 degrees C at a rate identical with that of synthetic Tp(Me)T, and gave the four expected UV-absorbing products (thymidine, thymidylyl(3'-5')thymidine, and the methyl esters of 3'- and 5'-TMP) in the same ratios as the authentic triester.