This project utilizes state-of-the-art NMR spectroscopy to characterize DNA damage and to understand the molecular mechanisms involved in DNA repair. The primary emphasis during the recent review period includes 1) structural characterization of the repair complex formed from the XRCC1 N-terminal domain and DNA pol beta;2) characterization of the interaction of XRCC1 BRCT domains with cognate BRCT domains on Ligase IIIa and on Poly(ADP-ribose) polymerase-1 (PARP-1); 3) characterization of how the nucleotide excision repair protein UvrB interacts with various DNA structures;3) characterization of the structure of DNA containing isolated ribonucleotides;4) analysis of methionine NMR parameters to facilitate interpretation of studies of methionine-labeled polymerases. Project 1. DNA repair is a complex process requiring the coordinated activities of multiple enzymes that interpret the damage, excise the damaged components, and resynthesize the DNA. X-ray cross complementing group 1 protein (XRCC1) is a scaffold that has been identified to play a central role in the formation of DNA repair complexes. Although a substantial amount of structural data is available for the individual proteins involved in DNA repair, little structural data is currently available for the protein interactions formed during the repair process. We utilized a combination of approaches: X-ray crystallography, NMR spectroscopy, and small angle X-ray scattering, to determine the structure of the complex formed between the N-terminal domain (NTD) of XRCC1 and DNA polymerase . Although several structural features of the XRCC1-Pol complex were anticipated from previous studies, one significant unanticipated result was the observation of two structurally distinct complexes in which the XRCC1 NTD was found to adopt either of two alternative folds that are distinguished by the presence/absence of a disulfide bond, dramatically altered secondary structure and electrostatic surface. Although most of the structural changes occur distal to the XRCC1-Pol interface, the oxidized form is characterized by shorter hydrogen bond lengths and additional inter-protein interactions. The prediction of a stronger binding interaction with the oxidized form of XRCC1 was verified with a binding assay that revealed a 10-fold enhancement in the affinity of XRCC1ox for Pol . This indicates that a redox signal may play an important role in the formation of the XRCC1-Pol complex. Subsequent efforts directed toward the characterization of the redox-induced structural transition have indicated that it is more complex than analogous transitions that have been characterized for other disulfide-switch proteins. We have found that, in addition to oxidizing conditions, the XRCC1 transition is facilitated by two additional factors: protein disulfide isomerase and bicarbonate, with the latter playing a structural role. Thus, the XRCC1 disulfide switch may function as a transducer of oxidative stress, and may also regulate the sequential steps of the base excision repair process. Further studies are in progress to understand the full biological implications of the XRCC1 redox switch and the role it plays in cellular DNA transactions. Project 2. A second project involves the structural characterization of additional interactions between the two BRCT domains of XRCC1 and their cognate binding domains on PARP1 and Ligase IIIa. The solution structure of the BRCT domain of Ligase IIIa was previously determined using NMR spectroscopy. Although data indicated that it exists as a dimer, there was insufficient NOE information available for characterization of the dimer structure, so that the structure of the monomer was determined. Unfortunately, analysis of NMR data using an incorrect assumption about dimeerization has in some cases been found to lead to very significant errors. We therefore have determined the structure of the Ligase IIIa dimer, and are currently working to understand the implications for the formation of a heterodimer with XRCC1. Project 3. Effect of 2 hydroxylation on DNA structure. It has recently been demonstrated that the extent of incorporation of ribonucleotides by replicative polymerases is sufficient to make 2-hydroxylation the most common mutation in newly synthesized DNA. We have used the Dickerson dodecamer as a model for the analysis of the effect of an isolated ribonucleotide on DNA structure. The self-complementary sequence investigated, d(GCG)rCd(TTAAGCGC), contains two symmetrically positioned cytidine residues paired with deoxyguanosine residues. Consistent with previous studies of mixed ribo/deoxyribo nucleic acids, the absence of an observable H1-H2 scalar coupling interaction is indicative of a C3-endo conformation for the cytidine residue. However, longer range structural perturbations resulting from the presence of the ribonucleotide appear to be quite modest, so that the B-form DNA structure is maintained. The ribose-3-phosphate resonance exhibits a unique shift, presumably as a consequence of the interaction with the additional 2-OH substituent. We conclude that the structural perturbation introduced by the isolated ribonucleotide is strongly localized. Thus, enzymatic recognition of this lesion is expected to be characterized by overall affinity for B-form DNA combined with localized ribonucleotide interactions. Project 4. Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of 3JCSCC, 3JCSCH, and the isotropic shielding, σiso. Results have been compared with experimental data reported in the literature, as well as data obtained on methyl-13Cmethionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, 3JCSCC /3JCOCC .7. It is further demonstrated that the 3JCSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of δ13Cε. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large 100 χ3 values. The utility of the δ13Cεas a basis for estimating the gauche/trans ratio for χ3 is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.
| 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 : |
| 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: |
| 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 |
| 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 |
| 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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