Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy.

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered alpha/beta-sandwich structural core, is an essential protein that functions as part of base excision repair to remove mutagenic and cytotoxic abasic sites from DNA. Using low-temperature solid-state (25)Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg(2+) binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg(2+) per mole of APE1 as predicted by the X-ray work of Tainer and co-workers (Mol, C. D.; Kuo, C. F.; Thayer, M. M.; Cunningham, R. P.; Tainer, J. A. Nature 1995, 374 , 381-386). However, the NMR spectra show that the single Mg(2+) site is disordered. We discuss the probable reasons for the disorder at the Mg(2+) binding site. The most likely source of this disorder is arrangement of the protein-ligands about the Mg(2+) (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.