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.
Lipton
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The Biological Sciences Directorate, Pacific Northwest National Laboratory, K8-98, 902 Battelle Boulevard, Richland, Washington 99352, USA.
Heck
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Primak
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McNeill
D R
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Wilson
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Ellis
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D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium