The primary focus of this project has evolved over the past year to cover studies of DNA polymerase proteins and the mechanisms by which they achieve a high degree of replication fidelity. Current studies involve the E. coli DNA polymerase III subunits epsilon and theta, which are components of the polymerase core. The epsilon subunit contains the 3'-5' proofreading exonuclease activity of the polymerase holoenzyme, while the function of theta, a small protein which binds to the epsilon subunit, is at present unclear. We have completed the NMR assignments of the backbone resonances of the epsilon catalytic domain, corresonding to the N-terminal 186 amino acid residues. During the past year, we used a selective protonation scheme to introduce protons into both the exchangable positions as well as the methyl groups of the aliphatic amino acids, while the remaining non-exchangeable protons are replaced by deuterons. It was then possible to assign and analyze long range methyl-methyl, methyl-amide, and amide-amide NOEs. These results have been combined with molecular modeling approaches to yield a three dimensional structure for the epsilon catalytic subunit. We also have continued our studies of protein radical formation using the protein spin trap, [methyl-13C3]-MNP. Using this isotopically labeled trap, we observed radical adduct formation in myoglobin which had been exposed to hydrogen peroxide. NMR studies demonstrated formation of an adduct at the C-3 position of Tyr-103. HMQC-NOESY studies revealed the proximity of the labeled methyl groups to the tyrosyl ring protons, and to additional aromatic protons proposed to correspond to Phe-106, which is located next to Tyr-103.
| 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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