This project utilizes state-of-the-art NMR spectroscopy to characterize DNA damage and to determine the structure of enzymes and enzyme complexes involved in DNA excision repair. The primary emphasis during the recent review period includes 1) characterization of factors that determine the redox transition in the XRCC1 N-terminal domain, and 2) identification and characterization of the XRCC1 nuclear localization sequence (NLS) that is reqired in order to transport XRCC1 and several associated repair enzymes into the nucleus. Project 1. Solution characterization of the XRCC1 N-terminal domain. We previously found that the N-terminal domain of XRCC1, which interacts specifically with DNA pol beta, is subject to a redox-dependent structural transition involving formation of a disulfide bond between Cys12 and Cys20. In the reduced protein, Cys12 is buried within the protein, while Cys20 has a high degree of solvent exposure. Since the oxidized form of the protein was initially identified in a crystallographic study, we have been investigating the solution behavior of the N-terminal domain in order to characterize the conditions that determine the oxidized and reduced fractions of the protein, as well as the rate of interconversion. We recently determined that the stability of the oxidized form of the N-terminal domain is also dependent on formation of a carbon dioxide adduct with the N-terminal proline residue. In this carbimate adduct the carbimate carboxyl group interacts with Arg7, Ser44, and Lys129 providing significant stabilization of the N-terminal domain in the oxidized form. We found that both the NMR spectrum as well as the intrinsic tryptophan fluorescence are sensitive to the redox transition and can be used to characterize the kinetics. In contrast with proteins that act as redox sensors, the kinetics of XRCC1 oxidation was found to be extremely slow, requiring hours after addition of hydrogen peroxide to become fully oxidized. This very slow time constant is a consequence of the fact that Cys12 is buried in the reduced structure, and hence is kinetically trapped. However, the rate constant could be considerably accelerated by addition of protein disulfide isomerase. NMR analysis of a sample containing the pol beta polymerase domain, and both the reduced and oxidized forms of X1NTD, indicates that the oxidized form binds to the enzyme 25-fold more tightly than the reduced form. The enhanced affinity of the oxidized form for DNA pol beta suggests that a more oxidizing intracellular environment is associated with an upregulation of XRCC1-pol beta dependent DNA repair pathways. Project 2. The localization, complexity and variability of DNA damage requires a complex cellular response that often utilizes scaffold proteins to recruit and coordinate the activities of the individual repair enzymes required to correct the damage. The X-ray cross complementing group 1 protein (XRCC1) is a scaffold that plays important roles in the overlapping single strand break repair (SSBR) and base excision repair (BER) pathways, and participates in other repair pathways as well. In order to fulfill its DNA repair function, XRCC1 must be targeted to the nuclear compartment and to sites of DNA damage. Nuclear import most frequently utilizes the classical nuclear import machinery that involves formation of a complex between a nuclear localization signal (NLS) on the XRCC1 and importin alpha. Although XRCC1 is believed to contain such as sequence in the first linker domain, identifications of the NLS have been inconsistent. In order to resolve these inconsistencies and to determine the nature of the XRCC1 NLS, we performed a series of binding and crystallization studies. A peptide sequence from the first linker domain of XRCC1 was found to interact with importin alpha as a bipartite sequence in which the major and minor site binding motifs are separated by more than 20 residues, a separation that is unusual but not unprecedented. Binding studies of peptides corresponding to the bipartite XRCC1 NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of importin alpha revealed pronounced cooperative binding behavior that results from the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity thus stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. Based on a cooperative binding model, the stepwise dissociation of the NLS from importin alpha is expected to facilitate unloading of the XRCC1 cargo by providing a partially complexed intermediate that is available for competitive binding by Nup50. These characteristics provide a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.
| 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: |
| 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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