Structure of human apurinic/apyrimidinic endonuclease 1 with the essential Mg ²⁺ cofactor

Abstract

Human AP endonuclease 1 (APE1) belongs to the DNase I-like superfamily of enzymes that require divalent cation(s) to catalyze phosphoryl-transfer reactions. A new 1.92 Å resolution crystal structure of APE1 reveals ideal octahedral coordination of a single Mg ²⁺ ion and informs on the role of this essential cofactor.

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5′-deoxyribose phosphate and the 3′-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3′-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg ²⁺, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm ³⁺ or Pb ²⁺ in the active site. However, these are poor surrogates for Mg ²⁺ because Sm ³⁺ is not a cofactor and Pb ²⁺ inhibits APE1, and their coordination geometry is expected to differ from that of Mg ²⁺. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg ²⁺ ion in the active site. The structure reveals ideal octahedral coordination of Mg ²⁺ via two carboxylate groups and four water molecules. One residue that coordinates Mg ²⁺ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme–product complex, inform on the stoichiometry and the role of Mg ²⁺ in APE1-catalyzed reactions.