DNA GLYCOSYLASE ACTIVITIES FOR THYMINE RESIDUES OXIDIZED IN THE METHYL-GROUP ARE FUNCTIONS OF THE ALKA ENZYME IN ESCHERICHIA-COLI

The alkA gene of Escherichia coil encodes a DNA glycosylase involved i n base excision repair of DNA alkylation damage. In an attempt to defi ne the reactions of the AIkA enzyme with methylated DNA we discovered that the enzyme released substantial amounts of radioactivity from [me thyl-H-3]thymine-labeled DNA even without any exposure of the DNA to m ethylating agents. The excised material was identified by chromatograp hy as two different oxidized derivatives of thymine, 5-hydroxymethylur acil and 5-formyluracil. These products are formed in such DNA by one and two consecutive decays, respectively, of the tritiums of the label ed methyl group. Kinetic analysis showed that both the apparent K-m an d V-max values for 5-formyluracil removal are within the same range as found for 3-methyladenine removal, suggesting that this catalytic pro perty of AlkA is also significant under in vivo conditions. Removal of 5-hydroxy- methyluracil proceeds at a rate that is 1-3 orders of magn itude slower. Since both 5-formyluracil and 5-hydroxymethyluracil are major products formed in DNA by exposure to ionizing radiation, these results implicate the alkA gene function also in the repair of oxidati ve DNA damage. Neither of the two other enzymes involved in the repair of oxidative DNA damage in E. coil, i.e. endonuclease III and formami dopyrimidine DNA glycosylase, has any affinity for oxidized unsaturate d pyrimidines in DNA.