OXIDATION OF THYMINE TO 5-FORMYLURACIL IN DNA - MECHANISMS OF FORMATION, STRUCTURAL IMPLICATIONS, AND BASE EXCISION BY HUMAN CELL-FREE-EXTRACTS

Oxidative agents produce several different types of base modifications in DNA, and only a few of these have been properly characterized with respect to mechanisms of formation and biological implications. We have established a procedure using neutral thermal hydrolysis and reverse phase highperformance liquid chromatography to determine the content of the oxidation product 5-formyluracil (5-foU) in DNA. With this method, it is shown that 5-foU residues are formed with high frequency from thymine by quinone-sensitized UV-A photooxidation. Since 5-foU is also induced by ionizing radiation, it appears to be formed under conditions where thymidine radical cations are generated and react with molecular oxygen. It was previously shown that 5-foU is formed directly from [methyl-H-3]thymine residues in radioactively labeled DNA by two consecutive transmutations of H-3 to He-3. The theoretical basis for the kinetics of such conversion is presented in this paper, and the calculated yields are confirmed experimentally by measuring the content of 5-foU in [methyl-H-3] thymine-labeled DNA aged for different time periods. Such DNA contains virtually only 5-(hydroxymethyl)uracil and 5-foU, apart from normal bases, and is therefore very useful for the investigation of repair enzyme activities involved in the repair of 5-foU-containing, DNA. Using this substrate, a DNA glycosylase activity was identified in human cell extracts for the removal of 5-foU. Molecular modeling of DNA containing 5-foU suggests that the formyl group will be kept in plane with the pyrimidine ring because of electrostatic interactions between formyl O and H-6 and formyl H and O-4, with a potential weakening effect on the base-pairing properties of 5-foU relative to thymine.