Wednesday, July 23, 2008

J. Am. Chem. Soc., 130 (29), 9332–9341, 2008.

Characterization of Mg2+ Binding to the DNA Repair Protein Apurinic/Apyrimidic Endonuclease 1 via Solid-State 25Mg NMR Spectroscopy

A. S. Lipton,† R. W. Heck,† S. Primak,† D. R. McNeill,‡ D. M. Wilson, III,*‡ and P. D. Ellis*†

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered α/β-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 and various mutants of APE1, we demonstrate that Mg2+ binds to APE1 and a functional APE1−substrate DNA complex with an overall stoichiometry of one Mg2+ 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 Mg2+ site is disordered. We discuss the probable reasons for the disorder at the Mg2+ binding site. The most likely source of this disorder is arrangement of the protein−ligands about the Mg2+ (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.

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