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) identification and characterization of a REV1-interacting region (RIR) motif in XRCC1 that mediates binding to the REV1 C-terminal domain;2) evaluation of the structural and functional effects of a cancer-associated mutation, L22P, in DNA pol beta; 3) characterization of factors that determine the redox transition in the XRCC1 N-terminal domain. Project 1. 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 (X)3FF(Y)4 motif that has been demonstrated to mediate an interaction with the repair protein REV1. REV1 exhibits both a deoxycytidyl transferase activity and also functions as a scaffold that binds to other DNA polymerases that are involved in translesion synthesis. We found that peptide fragments derived from the first linker region of XRCC1 that contain the above, Phe191-Phe192 motif, bind to the REV1 C-terminal domain with dissociation constants in the low micromolar range, similar to values obtained with the RIR fragments from pol eta and pol iota, but weaker than the binding of pol kappa. The structure of a REV1-XRCC1 peptide complex was determined by using NOE restraints to dock the unlabeled XRCC1 peptide with a labeled REV1 C-terminal domain. The structure is generally homologous with previously determined complexes with the pol kappa and pol eta RIR peptides, although the helical segment in XRCC1 is shorter than was observed in these cases. The RIR motif in XRCC1 is located in the first linker, just after the N-terminal domain which functions to bind pol beta. Since the REV1 C-terminal domain appears to recruit polymerases capable of translesion synthesis, and since DNA pol beta has been demonstrated to be capable of translesion synthesis for cisplatin adducts and psoralen adducts, among others, it is likely that this interaction provides an additional example of recruitment of a polymerase capable of translesion synthesis. Project 2. DNA polymerase (pol) beta is a multi-domain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single strand break repair pathways. The high frequency of pol βvariants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making it of interest to determine the functional consequences of these variants. Pol beta containing a proline substitution for leucine 22 in the lyase domain, identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. NMR spectroscopic evaluations of both pol beta and the isolated lyase domain containing the Leu22Pro mutation demonstrate destabilization sufficient to result in selective unfolding of the lyase domain with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca2+ and a complementary dNTP indicates that the fraction of pol beta(L22P) containing the folded lyase domain undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol beta indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. Thus, the substate is able to partially """"""""rescue"""""""" the mutated enzyme. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded lyase domain containing the L22P mutation. These studies illustrate that substrate-induced catalytic activation of pol beta provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the inactive variant interferes with DNA repair. Project 3. 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. 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 CO2 adduct with the N-terminal proline residue. This """"""""carbimate"""""""" adduct is selected by interactions with Arg7, Ser44, and Lys129 in the oxidized form of the N-terminal domain, and in turn helps to stabilize the oxidized structure. We found that both NMR and 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 H2O2 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. The rate constant could be considerably accelerated by addition of protein disulfide isomerase. A manuscript describing this work has recently been submitted.
| 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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