RAPID DISSOCIATION OF HUMAN APURINIC ENDONUCLEASE (APE1) FROM INCISEDDNA INDUCED BY MAGNESIUM

Repair of apurinic/apyrimidinic (AP) sites is initiated by AP endonucl eases, such as the human Ape1 protein (also called Hap1, Apex, and Ref 1). This and related enzymes show strong dependence on divalent cation s, particularly magnesium, Here we explore the role of this metal in d ifferent stages of the Ape1 reaction: substrate binding, cleavage, and product release. We examined DNA binding using an electrophoretic app roach and DNA cleavage in single-turnover and steady-state reactions. Magnesium at low to moderate concentrations accelerated both substrate and product release by wildtype Ape1 protein. For a mutant Ape1 prote in with an aspartate to alanine substitution at residue 308, substrate in preformed protein-DNA complexes was more efficiently cleaved befor e release in contrast to wildtype Ape1, whereas product release was ac celerated dramatically. The magnesium dependence of steady-state AP en donuclease reactions was sigmoidal for both wild-type and the aspartat e 308 to alanine protein but was not sigmoidal for an aspartate 283 to alanine derivative of Ape1. These results show that magnesium affects both DNA interactions with and phosphodiester cleavage by Ape1 and ca n change the rate-limiting step of the reaction. Structural studies wi ll need to be interpreted in the context of these diverse effects of t he metal.