This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the area of environmental health. The primary emphasis during the recent review period involves several different research projects: 1) structural characterization of the repair complex formed from the XRCC1 N-terminal domain and DNA pol beta; 2) characterization of how the nucleotide excision repair protein UvrB interacts with various DNA structures;3) characterization of a putative acetylcholine binding receptor from a marine annelid; 4) structural work on dust mite allergens. Project 1. The repair of damaged DNA involves multiple enzymatic steps, the activities of which must be spatially and temporally organized in order to optimize the repair process and to minimize the possibility of increased damage that could result from the release of repair intermediates such as nicked or gapped DNA. For the base excision repair (BER) and single strand break repair (SSBR) pathways, a major organizational role has been assigned to the protein XRCC1 acting as a scaffold for other base excision repair enzymes including DNA polymerase beta, DNA ligase III, PARP1 and 2, polynucleotide kinase (PNK) and AP endonuclease 1. Nevertheless, limited structural information corresponding to these putative repair complexes is currently available. We recently have utilized a combination of small angle X-ray scattering (SAXS), X-ray crystallography, and NMR spectroscopy to determine the structure of the complex formed from the XRCC1 N-terminal domain (XNTD) with DNA polymerase beta. The structure differs from several previously proposed models that involved simultaneous interactions with the Pol beta catalytic (palm) and nascent base pair (thumb) domains, as well as with the gapped DNA substrate, forming a protective """"""""sandwich"""""""" around the damaged area. The present structural results indicate that Pol beta binds directly only with the thumb domain of Pol beta and is also not positioned to allow direct interactions with the gapped DNA repair intermediate. The interaction with Pol beta occurs primarily through the """"""""V303 loop"""""""" and involves salt bridges, hydrophobic contacts, as well as multiple water-mediated hydrogen bonds. Areas of secondary structure agree well with the previously reported XRCC1 N-terminal domain structure, while there is substantial difference in many of the looop regions. Project 2. NMR characterization of the interaction of UvrB with hairpin DNA. UvrB, an essential component of the prokaryotic nucleotide excision repair (NER) pathway, plays a critical role in DNA damage recognition. Recent crystallographic structures of several UvrB-DNA complexes provide intriguing but incomplete insight into the damage recognition process. In order to further explore this interaction, we have performed NMR studies of the interaction of methyl-13Cmethionine UvrB from B. caldotenax with a compact hairpin DNA. The studies utilized a 14 nucleotide hairpin incorporating the highly stable seven nucleotide Hirao sequence and having a three nucleotide overhang at the 3'-terminus. Titration of the labeled UvrB with the hairpin perturbs the resonance of M350, which comes in direct contact with the DNA in a reported crystal structure, and also produces significant shifts for four methionine residues grouped together on helices F and G. The hypothesis that these perturbations are mediated primarily by the interaction of the 3'-nucleotide with F302 was confirmed by studies of the interaction of the DNA with the F302A mutant. In order to more specifically investigate the details of the UvrB conformational response, two additional methionine probes were introduced into the enzyme on the beta-hairpin (residues S91-N116) that has been proposed to play a central role in damage recognition. The I115M mutant, places a methionine residue at the base of the hairpin, while I109M places the methionine at the center of the hairpin. Based on the similarity of the response of the remaining methionine resonances to the addition of the DNA, it was concluded that the UvrB-DNA interaction was not significantly perturbed by either of these substitutions. Both the M109 and M115 resonances provided well resolved and sensitive probes for DNA binding. In particular, M115 exhibits a large upfield 1H shift that can be attributed to the altered position of the methionine methyl group relative to tyrosine residues Y92 and Y93. Tight binding of the DNA hairpin (KD
| Tumbale, Percy; Schellenberg, Matthew J; Mueller, Geoffrey A et al. (2018) Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease. EMBO J 37: |
| DeRose, Eugene F; Kirby, Thomas W; Mueller, Geoffrey A et al. (2018) Transitions in DNA polymerase ? ?s-ms dynamics related to substrate binding and catalysis. Nucleic Acids Res 46:7309-7322 |
| Kirby, Thomas W; Pedersen, Lars C; Gabel, Scott A et al. (2018) Variations in nuclear localization strategies among pol X family enzymes. Traffic : |
| Schellenberg, Matthew J; Lieberman, Jenna Ariel; Herrero-Ruiz, Andrés et al. (2017) ZATT (ZNF451)-mediated resolution of topoisomerase 2 DNA-protein cross-links. Science 357:1412-1416 |
| Kim, Kyungmin; Pedersen, Lars C; Kirby, Thomas W et al. (2017) Characterization of the APLF FHA-XRCC1 phosphopeptide interaction and its structural and functional implications. Nucleic Acids Res 45:12374-12387 |
| 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 |
| Kirby, Thomas W; Gassman, Natalie R; Smith, Cassandra E et al. (2017) DNA polymerase ? contains a functional nuclear localization signal at its N-terminus. Nucleic Acids Res 45:1958-1970 |
| Gabel, Scott A; Duff, Michael R; Pedersen, Lars C et al. (2017) A Structural Basis for Biguanide Activity. Biochemistry 56:4786-4798 |
| London, Robert E (2015) The structural basis of XRCC1-mediated DNA repair. DNA Repair (Amst) 30:90-103 |
| Kirby, Thomas W; Gassman, Natalie R; Smith, Cassandra E et al. (2015) Nuclear Localization of the DNA Repair Scaffold XRCC1: Uncovering the Functional Role of a Bipartite NLS. Sci Rep 5:13405 |
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