Investigation of the role of Mg2+ in DNA repair proteins APE1, Pol ?, and FEN1
EMSL Project ID
10600a
Abstract
Nuclear DNA is continuously damaged by endogenous sources, such as the reactive free radicals produced during oxidative metabolism, or by exogenous sources, such as ionizing radiation. It has been estimated that about 10,000 DNA lesions are produced in each cell every day due to endogenous sources. The bulk of DNA lesions produced by both oxidative stress and ionizing radiation in cells are oxidized bases (e.g. 8 oxoguanosine, 8OG), single-strand breaks, and sites of base loss, that are in turn, substrates for base excision repair (BER) pathway. Because genomic preservation is critical to an organism?s health and survival, a variety of DNA repair pathways, such as BER, nucleotide excision repair (NER), and DNA mismatch repair (DMR) just to mention a few have evolved to recognize and repair DNA damage. While the overwhelming majority of DNA lesions are repaired, the cumulative effects of lesions that go unrepaired lead to carcinogenesis, aging and genetic diseases. DNA repair is essential for life. In particular, we will focus on three proteins that are central to the BER pathway; apurinic/apyrimidinic endonuclease 1 (APE1), polymerase ? (Pol ?), and flap endonuclease 1 (FEN1).The chemistry central to the function of these DNA repair proteins APE1, Pol ? 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 ?. 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 ?. Moreover, these same structural and dynamic questions are key to understanding the mechanism of action of Pol ?. In order to understand the role of the Mg2+ in native proteins 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 partial 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 (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 ? 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.
Project Details
Project type
Capability Research
Start Date
2005-04-11
End Date
2006-04-17
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