PNNL

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered a/ß-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 ²5Mg NMR spectroscopy, we demonstrate that Mg²? binds to APE1 in the absence of DNA and that there are two Mg²? resonances per APE1 molecule. In addition, the ²5Mg NMR spectra in the presence of damaged DNA (either before or after incision) can be rationalized in terms of two sites being present within the complex. Subtraction of the ²5Mg NMR spectra obtained for the E96Q APE1 mutant in the presence of damaged DNA, however, indicates that the overall stoichiometry with respect to Mg²? is one per mole of APE1 as predicted by the x-ray work of Tainer and coworkers (Mol, C. D., Kuo, C. F., Thayer, M. M., Cunningham, R. P. & Tainer, J. A. (1995) Nature 374, 381-386.) Our results therefore argue that there is a single specific binding site for Mg²? in a functional APE1-substrate DNA complex, with the two observed resonances representing disorder at the metal binding site, and that the two metal mechanism or the “moving metal mechanism” recently proposed are not compatible with current evidence.