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; 2) evaluation of the functional consequences of blocking the previously characterized redox transition in the N-terminal domain of XRCC1 that binds to DNA polymerase beta; 3) evaluation of the possible role of the XRCC1 complex in post replication repair. Project 1. Solution characterization of the XRCC1 N-terminal domain. We previously determined 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. 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. The addition of hydrogen peroxide promotes the transition to the oxidized form, but typically leads to some precipitation. The degree of precipitation is dependent on the concentration, temperature, and pH of the buffer, indicating that at least some of the precipitation results from the formation of intermolecular disulfide bonds. Further work is currently in progress to characterize the fluorescence characteristics of the protein, in order to determine whether the intrinsic fluorescence can be used to monitor the redox transition. We are also working on development of improved methods to evaluate the binding affinity for both forms of the XRCC1 N-terminal domain with DNA pol beta. Previous studies of this interaction have used approximate methods, or relied on the introduction of a fluorophore that directly perturbs the binding interaction. NMR may provide a more direct, less perturbing method for quantitative evaluation of this interaction. Project 2. Effects of XRCC1 oxidation on PARP1 activity. Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR-modification of various DNA repair enzymes is required for recruitment to the site of DNA damage, and functions of the key BER factors XRCC1 and DNA polymerase beta (pol beta) that in turn regulate PAR modification. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol beta in regulating the level of PAR modification are poorly understood. A complex of PARP-1, XRCC1 and pol beta is found in vivo, and it is known that pol beta and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further evaluate the importance of the C12C20 disulfide form of XRCC1 and the interaction with pol beta, we characterized cell lines representing stable transfectants in Xrcc1−/−mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol beta (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. Our results suggest a role for the oxidized form of XRCC1 in the interaction with pol beta in (1) controlling the PAR level after MMS exposure and (2) mediating the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response. Project 3. Interaction of XRCC1 with REV1. The scaffold protein XRCC1 plays a central role in the overlapping base excision and single strand break DNA repair pathways. It consists of three globular domains separated by two unstructured linker segments of approximately 140 residues. The first linker contains a nuclear localization sequence and has also been implicated in PCNA binding. We have investigated the interaction of peptide segments derived from this linker with PCNA, but found the interaction to be very weak. However, the first linker segment also contains an (x3)FF(y4) motif that has been demonstrated to mediate an interaction with the repair protein REV1. REV1 exhibits both a deoxycytidyl transferase activity and a scaffold function that allows it to interact with the other DNA polymerases that are involved in translesion synthesis. We found that peptides derived from XRCC1 do bind to REV1. Although the dissociation constant describing the interaction of REV1 with the XRCC1 pepitde initially appeared to be similar to that reported for the interaction with DNA polymerase kappa, this result was not supported by ligand competition studies. Further studies demonstrated that the affinity of REV1 for pol kappa is considerably higher than indicated by the reported Kd value. The affinity of XRCC1 for REV1 is similar to the affinity for the more weakly associated polymerase, pol iota. The interaction of XRCC1 with REV1 may be related to a role in post replication DNA repair. In general, the XRCC1 complex appears to play an important role in the repair of lesions resulting from oxidative stress, which can require the action of polynucleotide kinase, one of the enzymes that binds to XRCC1.
| 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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