Project 1. Efficient DNA repair is dependent on the intranuclear assembly of damage-dependent repair complexes, and thus requires the necessary DNA repair polymerases to be present in the cell nucleus. DNA polymerase beta (pol beta) plays a key role in base excision repair, as well as participating in other repair pathways and in lesion bypass. The strong relationship between functional mutations in pol beta and the development and progression of cancer is increasingly substantiated. Variations in the expression level of pol beta and other components of the base excision repair complexes have been reported to be associated with various pathologies, particularly cancer. In addition to dysregulated expression levels, altered subcellular distribution provides another increasingly appreciated mechanism for perturbing nuclear protein concentrations, resulting in cellular dysfunction and disease. Consistent with this mechanism, a variant form of Xeroderma Pigmentosum was recently determined to result from a mutation in the nuclear localization signal (NLS) of the translesion repair polymerase DNA polymerase Eta. Although generally not considered to contain a classical nuclear localization signal (NLS), sequence and structural analysis suggested that one might be present in pol beta. Significant binding affinity of pol beta for Importin alpha 1 was confirmed by gel filtration and NMR studies. Binding affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide. These studies indicate high affinity binding characterized by low micromolar Kd values. We have confirmed the role of the pol beta NLS in transporting pol beta into the nucleus using fluorescent imaging of wild-type and NLS mutated pol beta in mouse embryonic fibroblasts lacking the endogenous pol enzyme. The functional importance of this import mechanism was further demonstrated by a reduced survival of cells expressing the NLS-mutated form of the enzyme that were exposed to the alkylating agent methyl methanesulfonate. Together these data demonstrate that pol beta contains a specific NLS sequence that promotes transport of the protein independent of its interaction partners. Project 2. Aprataxin and PNKP-like factor (APLF) was recently was identified as a novel component of non-homologous end-joining that promotes intracellular re-joining of transfected linear plasmid DNA molecules and accelerates the repair of chromosomal double strand breaks following gamma irradiation. The enzyme contains a forkhead associated (FHA) domain that interacts with phosphorylated peptide motifs on the repair scaffold proteins XRCC1 and XRCC4. Interestingly, other repair enzymes including aprataxin and polynucleotide kinase-phosphatase (PNKP) also contain homologous FHA domains, all of which appear to interact with the same phosphorylated peptide motifs. This introduces questions of what factors determine the relative binding affinities of each enzyme, and why the interactions apparently rely on competition for common binding motifs, so that only a single binding partner can be present at any one time. Interestingly, it is thought that recruitment of aprataxin and PNKP to sites of DNA strand breaks is mediated by prior recruitment of XRCC1/XRCC4 which in turn are recruited to damage sites by an initial, poly(ADP-ribosylation). Alternatively, APLF can be recruited directly by poly(ADP-ribose) and is thus not dependent on an indirect recruitment pathway. The functional significance of these alternate recruitment pathways is currently unclear. During the past year, we have investigated the FHA-phosphopeptide interactions in order to better understand them physically and biochemically. We have utilized a combination of NMR spectroscopy and crystallography to investigate the binding motifs and modes of interaction. We also have identified unusual structural characteristics common to the peptide recognition sites of the phosphorylating enzyme CK2, which is involved in phosphorylating both the XRCC1 and XRCC4 substrates. Project 3. Dynamic behavior of DNA polymerase beta. DNA polymerase beta (pol beta) is an important DNA repair protein that catalyzes both DNA synthesis (nucleotidytransferase) and deoxyribose phosphate (dRP) lyase reactions in the base excision repair pathway. In spite of the large amount of information on the structure and function of pol beta very limited information has been available characterizing the dynamic behavior of the protein in solution. Recently, 15N CPMG relaxation dispersion experiments have been used to study the backbone motion of apo pol beta, a binary complex of pol beta and DNA, and ternary complexes with nonhydrolyzable nucleotides. These studies found evidence of millisecond backbone motion in the apo enzyme, but much less backbone motion in the binary and ternary complexes. To determine how side-chain motion contributes to nucleotide binding and the effect of nucleotide binding on side-chain motion, methyl TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of isoleucine C-delta and methionine methyl groups were conducted on a binary complex of pol beta containing a 1-nucleotide gapped DNA substrate and ternary complexes with matched and mismatched nonhydrolyzable nucleotides. A large number of the side-chains were found to exhibit millisecond dynamics and line broadening throughout the binary complex, whereas the ternary complexes were substantially less dynamic, with the side-chain motion located primarily in the catalytic sub-domain as well as one residue in the DNA binding sub-domain and in the Nucleotide binding sub-domain. The average magnitude of the side-chain motion in the ternary complex was also considerably smaller than in the binary complex as evidenced by the low exchange rate contributions to the transverse relaxation rates of the methyl groups. These results seem to indicate that the side-chain motion in the binary complex is important to facilitate sampling of the incoming nucleotide, but that once the correct nucleotide is selected a stable closed ternary complex is formed with reduced millisecond side chain motion. 4. NMR studies of the interaction of AP endonuclease 2 with DNA. AP Endonuclease 2 (APE2) is a minor apurinic (AP) endonuclease that exhibits PCNA-stimulated 3-5 exonuclease and 3-phosphodiesterase activities. It plays an essential role in activation the ATR-Chk1 checkpoint that arrests the cell cycle in response to unresolved DNA damage. It contains a highly conserved Zinc finger-GRF domain of unknown function. We have collaborated with the Williams group to investigate the interaction of this domain with DNA in solution.

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Project End
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Budget End
Support Year
28
Fiscal Year
2016
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Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
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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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