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. During the past year, we used NMR methods to determine the solution structure of the phage protein, HOT, which is a sequence homolog of the theta subunit of E. coli DNA polymerase III. This enzyme is the main replicative polymerase of E. coli and, among other things, serves as an important model for understanding the factors that determine replicative fidelilty. Recent work by our collaborators has shown that genetically modified E. coli cells which express the HOT protein rather than its theta homolog exhibit subtle and interesting differences in fidelity that are related to altered function of the proofreading exonuclease epsilon that interactions with theta/HOT. Surprisingly, although the secondary structure of HOT was found to be fairly similar to that reported for theta, the folding topology was completely different, so that even a DALI search did not identify HOT as a structural homolog of theta despite a high level of sequence homology. Circular dichroism analysis indicates that both theta and HOT are able to unfold and refold reversibly as a function of temperature, with HOT having a melting temperature that is 6 degrees C below that of theta. 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 prepared U-[13C,15N] R67 DHFR and assigned most of the resonances. We characterized the region and dissociation constant for NADP binding. Unexpectedly, we observed that NADP was slowly hydrolyzed to NAD by the enzyme, an activity which may provide clues into the catalytic mechanism of this enzyme. We also observed inter-ligand Overhauser effects between the nicotinamide protons of NADP and NADPH. Project 3. We have demonstrated that it is possible to introduce a correlation in the labeling of protein amino acid residues by bacterial expression from a medium containing a mixture of U-[2H] and U-[13C,15N] amino acids. This approach facilitates sidechain assignments.
| 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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