This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the NIEHS, the Laboratory of Structural Biology, and the NMR research group. The primary emphasis involves applications in three areas: 1) Understanding how the structural and dynamic behavior of DNA polymerases relates to the fidelity of nucleotide incorporation, 2) studies of ligand-macromolecule interactions, and 3) development and evaluation of new methodologies for the structural and dynamic characterization of proteins and other biological macromolecules in support of the research goals. Progress during the past year is summarized below:? Project 1. A principal objective of this research has involved the structural characterization of the E. coli DNA polymerase III, an important model system for understanding the factors that influence reproductive fidelity. During the past year, we completed our work on determination of the solution structure of the theta subunit of the polymerase core. This work was facilitated by our recent determination of the structure of a theta homolog coded by the phage P1 gene called """"""""HOT"""""""". In addition, we now have obtained structural data for the complex that forms between HOT and the catalytic domain of the epsilon proofreading endonuclease subunit of DNA polymerase III. This work relied heavily on our previous NMR studies of the behavior of epsilon in solution. The structure provides insight into how interactions between HOT or its homolog theta and the proofreader stabilize the latter and enhance its ability to function as an antimutator. Interestingly, the N-terminal retion of HOT, which is shown by NMR studies to be disordered in solution, becomes ordered upon binding epsilon, and terminates the central beta sheet. ? ? Project 2. Studies of ligand-macromolecule interactions have continued to focus on the Type II dihydrdofolate reductase, R67 DHFR, a plasmid encoded enzyme which confers resistance to anti-folate drugs on the bacteria containing the plasmid. During the past year, we further characterized the binding of numerous analogs, inhibitors and fragments of NADPH and/or folate by isothermal titration calorimetry, nuclear magentic resonance, and X-ray crystallography. A buffer dependence for binding of folate was also observed, which correlates with perturbation of the N3 pKa, such that binding of a neutral pteridine ring is preferred. In addition to these calorimetric studies, we have obtained preliminary data on the structure of the ternary R67 DHFR?NADP?dihydrofolate complex. These studies suggest that the type II DHFR is able to recognize the substrate and cofactor by a parallel mode of binding that involves interaction of Ile68 carbonyl and amide groups with the carboxamide group of NADP and with the N3-O4 amide group of the pteridine ring system. Crystallography and inter-ligand Overhauser effect studies show that the two ring systems adopt a relative endo geometry, characterized by tilted aromatic rings which approach each other most closely at nicotinamide C4-pteridine C6, corresponding to the reactive positions on the corresponding substrates. This relative orientation differs from that observed in Type I DHFR, but is more similar to that recently observed in pteridine reductase. This study provides a basis for understanding trimethoprim resistance conferred by the enzyme and for the development of Type II DHFR-targeted inhibitors.? ? Project 3. The development of NMR methods for the analysis of protein structure has been intimately linked with isotopic labeling strategies. Although uniform labeling with 13C, 15N and occasionally 2H is extremely useful for smaller proteins, the structural analysis of proteins with MW > 25,000 has often required specific labeling approaches. We have recently investigated the photochemical H/D exchange reaction in tyrosine in order to obtain useful labeling patterns for structural NMR studies. This reaction proceeds via the formation of a tyrosyl radical anion, leading to a cyclohexadienyl radical intermediate.
| Wallace, Bret D; Berman, Zachary; Mueller, Geoffrey A et al. (2017) APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress. Proc Natl Acad Sci U S A 114:304-309 |
| Gabel, Scott A; Smith, Cassandra E; Cuneo, Matthew J et al. (2014) Characterization of the redox transition of the XRCC1 N-terminal domain. Structure 22:1754-1763 |
| Gabel, Scott A; DeRose, Eugene F; London, Robert E (2013) XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair. DNA Repair (Amst) 12:1105-13 |
| Loeffler, Paul A; Cuneo, Matthew J; Mueller, Geoffrey A et al. (2011) Structural studies of the PARP-1 BRCT domain. BMC Struct Biol 11:37 |
| Butterfoss, Glenn L; DeRose, Eugene F; Gabel, Scott A et al. (2010) Conformational dependence of 13C shielding and coupling constants for methionine methyl groups. J Biomol NMR 48:31-47 |
| London, Robert E; Wingad, Brett D; Mueller, Geoffrey A (2008) Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis. J Am Chem Soc 130:11097-105 |
| DeRose, Eugene F; Clarkson, Michael W; Gilmore, Steven A et al. (2007) Solution structure of polymerase mu's BRCT Domain reveals an element essential for its role in nonhomologous end joining. Biochemistry 46:12100-10 |
| DellaVecchia, Matthew J; Merritt, W Keither; Peng, Ye et al. (2007) NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution. J Mol Biol 373:282-95 |
| Krahn, Joseph M; Jackson, Michael R; DeRose, Eugene F et al. (2007) Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. Biochemistry 46:14878-88 |
| London, Robert E; Gabel, Scott A (2006) Photoactivated h/d exchange in tyrosine: involvement of a radical anion intermediate. J Am Chem Soc 128:2268-75 |
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