Invesitgation of the role of Mg2+ in DNA repair proteins
EMSL Project ID
7797a
Abstract
The chemistry central to the function of the DNA repair proteins APE1, Pol b and FEN1 is the chemistry of water activated by a magnesium ion. Further, Mg2+ also facilitates the organization of the substrates at the active site of Pol b. The precise role the Mg2+?s play in substrate anchoring and catalysis in APE1 and FEN1 is not clear. The detailed organization (structure and dynamics) associated with the ligands for the magnesium and their immediate environment is crucial to questions concerning the synchronization of substrate binding, product release, and the intermolecular signaling by APE1 to Pol b. Moreover, these same structural and dynamic questions are key to understanding the mechanism of action of Pol b. In order to understand the role of the Mg2+ in native proteins or to engineer a Mg2+ catalyst with predictable structure and function one must first be able to characterize the structure and bonding at the metal site. Historically, the only reliable method for such a characterization of a magnesium site has been through X-ray crystallography. The X-ray crystallography of magnesium containing proteins has a complication in that it is difficult to distinguish Mg2+ and O2- in modest resolution protein structures due to the isoelectronic nature of the two species. Further, positional disorder and incomplete site occupancy can lead to confusion between disordered water and the presence of Mg2+. However, X-ray methods are still dominant because of the unfavorable spectroscopic properties (Ne closed shell electron configuration) associated with Mg2+. NMR spectroscopy of the quadrupolar nuclide, 25Mg, has been regarded as difficult due to its inherent low sensitivity. This difficulty is exacerbated by dilution of the spin in the rare environment of a protein (mass of 25 Da out of ~39 kDa for Pol b in the absence of further diluting ligands such as DNA and dNTP). To be able to address this specific point, we have employed low temperature (10K) solid-state NMR , utilizing cross polarization (CP) from protons to magnesium. We now discuss each of the three proteins, which importantly are key members of independent superfamilies of proteins, in more detail.
Project Details
Project type
Capability Research
Start Date
2007-01-22
End Date
2007-07-18
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Lipton AS, RW Heck, SV Primak, DR McNeill, DM Wilson Iii, and PD Ellis. 2008. "Characterization of Mg2+ Binding to the DNA Repair Protein Apurinic/Apyrimidic Endonuclease 1 via Solid-State 25Mg NMR Spectroscopy." Journal of the American Chemical Society 130(29):9332-9341. doi:10.1021/ja0776881