DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the AlkA enzyme in Escherichia coli

• Published in: The Journal of biological chemistry Vol. 269; no. 48; pp. 30489 - 30495
• Main Authors: Bjelland, S, Birkeland, N K, Benneche, T, Volden, G, Seeberg, E
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-12-1994; American Society for Biochemistry and Molecular Biology
• Subjects: Adenine - analogs & derivatives; Adenine - analysis; Adenine - metabolism; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; DNA Glycosylases; DNA, Bacterial - metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Genes, Bacterial; Kinetics; N-Glycosyl Hydrolases - biosynthesis; N-Glycosyl Hydrolases - isolation & purification; N-Glycosyl Hydrolases - metabolism; Oxidation-Reduction; Thymine - metabolism; Uracil - analogs & derivatives; Uracil - isolation & purification; Uracil - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)43840-9

The alkA gene of Escherichia coli encodes a DNA glycosylase involved in base excision repair of DNA alkylation damage. In an attempt to define the reactions of the AlkA enzyme with methylated DNA, we discovered that the enzyme released substantial amounts of radioactivity from [methyl-3H]thymidine-labeled DNA even without any exposure of the DNA to methylating agents. The excised material was identified by chromatography as two different oxidized derivatives of thymine, 5-hydroxymethyluracil and 5-formyluracil. These products are formed in such DNA by one and two consecutive decays, respectively, of the tritiums of the labeled methyl group. Kinetic analysis showed that both the apparent Km and Vmax values for 5-formyluracil removal are within the same range as found for 3-methyladenine removal, suggesting that this catalytic property of AlkA is also significant under in vivo conditions. Removal of 5-hydroxymethyluracil proceeds at a rate that is 1-3 orders of magnitude 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 oxidative DNA damage. Neither of the two other enzymes involved in the repair of oxidative DNA damage in E. coli, i.e. endonuclease III and formamidopyrimidine DNA glycosylase, has any affinity for oxidized unsaturated pyrimidines in DNA.