Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus

Abstract

A slow conversion of adenine residues to hypoxanthine occurs in single-stranded DNA when heated in neutral aqueous buffers. The rate of this reaction at pH 7.6 and 110.degree. C is k = 4 .times. 10-8 s-1, as determined by base analysis of heat-treated DNA that contains radioactively labeled adenine residues. Adenine deamination is 1 of several forms of hydrolytic damage that may occur as spontaneous premutagenic lesions in DNA in vivo. Cell extracts from calf thymus and human fibroblasts contain a DNA glycoslyase activity which specifically catalyzes the release of free hypoxanthine from DNA or polydeoxyribonucleotides that contain dIMP residues. Several properties of the purified enzyme from calf thymus are described: It has an MW of .apprx. 31,000. No cofactors are required for activity. The enzymatic release of hypoxanthine occurs readily from double-stranded polydeoxyribonucleotides that have either thymine or cytosine residues in the complementary strand. Single-stranded polymers are 10-20-fold more slowly attacked, and there is no detectable cleavage of monomeric dIMP. Hypoxanthine is liberated from DNA directly as a free base. When poly(dI).cntdot.poly(dC) containing both [3H]-dIMP and [32P]dIMP residues was employed as the substrate, 3H-labeled hypoxanthine but no 32P-labeled material was released in ethanol-soluble form. The hypoxanthine-DNA glycoslyase presumably acts in DNA repair by preventing deaminated adenine residues from being expressed as mutations.