EXCISION OF HYPOXANTHINE FROM DNA CONTAINING DIMP RESIDUES BY THE ESCHERICHIA-COLI, YEAST, RAT, AND HUMAN ALKYLPURINE DNA GLYCOSYLASES

The deamination of adenine residues in DNA generates hypoxanthine, whi ch is mutagenic since it gives rise to an A.T to G.C transition, Hypox anthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residu es, we show that this activity in E. coli is associated with the 3-met hyladenine DNA glycosylase II coded for by the alkA gene. This conclus ion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they ha ve the same molecular weight, (ii) both are induced during the adaptiv e response, (iii) a multicopy plasmid bearing the alkA gene overproduc es both activities, (iv) homogeneous preparation of AlkA has both enzy matic activities, (v) the E. coli alkA(-) mutant does not show any det ectable hypoxanthine DNA glycosylase activity. Under the same experime ntal conditions, but using different substrates, the same amount of Al kA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA, The K-m for the lat ter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkyla ted DNA. Hypoxanthine is released as a free base during the reaction, Duplex oligodeoxynucleotides containing hypoxanthine positioned opposi te T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein , and MAG protein-the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively-were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian en zyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.