Currently, we are focused on examining structure/function of the DNA end processing factors, Tyrosyl DNA phosphodiesterase 2 (Tdp2) (project 1) and Aprataxin (Aptx) (project2). Project1 (Tdp2) summary and progress: Topoisomerase II (topo II) DNA incision/ligation reactions can be poisoned (e.g following treatment with chemotherapeutics) to generate DNA double strand breaks (DSBs) with topo II covalently bound to DNA. Left un-processed, such protein-adducted DNA ends can impair DSB repair, thereby contributing to accumulation of clastogenic DSBs, genomic instability, mutagenesis, and cell death. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5′-phosphotyrosyl (5′-Y) linked topo II protein-DNA adducts. Tdp2 functions in cellular topo II drug resistance, and also mediates mutant p53 gain of function phenotypes. However, the molecular basis underlying Tdp2 topo II-DNA adduct repair activities remains unclear in the absence of protein structural information for any Tdp2 homolog. To understand Tdp2 functions we determined X-ray crystal structures of mammalian Tdp2 in three DNA bound states, and studied Tdp2 activities using mutational and functional studies that define determinants of Tdp2 DNA-protein covalent adduct recognition and reversal. Overall, these results support a testable structure-based single magnesium ion mediated catalytic mechanism whereby Tdp2 interacts with a DNA-protein conjugate using two binding pockets, one for the DNA, and a second for the Topoisomerase derived protein adduct. The Tdp2 DNA binding groove is composed of a helical cap and novel beta-2 helix-beta DNA damage binding "grasp" that together envelop an exposed 5′-adducted ssDNA terminus. Tailored DNA and 5′-adduct recognition elements make Tdp2 distinct from the related DNA repair endonucleases such as Apurinic endonuclease 1 (Ape1), and Tdp2 excludes an intact phosphodiester backbone from its active site to ensure selectivity, and restrict endo- or exonucleolytic processing. Together, our results provide insights to the mechanism of Tdp2-linked cancer chemotherapeutic resistance, and establish a framework for the development of Tdp2 inhibitors that could be employed as adjuvants for commonly employed topoisomerase II poisons (e.g Etoposide). Project 2 (Aptx) summary and progress: (Aptx) is a conserved eukaryotic DNA repair enzyme that is important for protection of cells from oxidative DNA damage, and APTX mutations cause the hereditary neurodegenerative disorder Ataxia with Oculomotor Apraxia 1 (AOA1). In the ultimate step of DNA replication and repair processes, DNA ligases seal DNA nicks through with a mechanism that can abort when the ligase encounters DNA termini harboring the products of oxidative or DNA-alkylation damage. Such "abortive ligation" generates a secondary form of damage, 5'-adenylated DNA-termini, which is corrected by Aptx to protect genomic integrity. However, due to a lack of protein structural information, the molecular basis for APTX catalytic reversal of 5'adenylation damage remains largely unknown. Furthermore, how Aptx is inactivated in disease is unknown. To understand APTX function, we determined the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn complex revealing active site and DNA interaction clefts formed by fusing a HIT (histidine triad) nucleotide hydrolase with an unprecedented DNA minor groove binding C2HE Zn-finger (Znf). This work highlights how an Aptx alpha-helical wedge interrogates the DNA base stack for DNA end/nick sensing. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5′-AMP adduct recognition and removal, and suggest mutations impacting protein folding, the active site pocket, and the pivot underlie Aptx dysfunction in the neurodegenerative disorder Ataxia Oculomotor Apraxia 1 (AOA1).
We aim to further define molecular determinants of APTX DNA repair, and how APTX integrates into damage repair pathways through interactions with DNA break repair pathways through binding Xrcc1 (DNA single strand break repair, SSBR) and Xrcc4 (DNA double strand break repair, DSBR). We are testing hypotheses that: 1) APTX Histidine triad (HIT) and Zinc finger (Znf) domains form a composite fused catalytic domain for DNA structure specific nick-binding, 5'-AMP recognition, and DNA-deadenylation processing, 2) AOA1 patient mutations disrupt APTX protein folding and/or directly impair APTX catalytic activities through active site distortion, and 3) The FHA domain and FHA-HIT linker provides a flexible leash targeting APTX DNA deadenylation activity to phosphorylated XRCC4 and XRCC1 DNA repair scaffolds.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2012
Total Cost
$1,341,546
Indirect Cost
City
State
Country
Zip Code
Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver et al. (2016) ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J 35:791
Schellenberg, Matthew J; Perera, Lalith; Strom, Christina N et al. (2016) Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2. Nucleic Acids Res 44:3829-44
Appel, C Denise; Feld, Geoffrey K; Wallace, Bret D et al. (2016) Structure of the sirtuin-linked macrodomain SAV0325 from Staphylococcus aureus. Protein Sci 25:1682-91
Schellenberg, Matthew J; Tumbale, Percy P; Williams, R Scott (2015) Molecular underpinnings of Aprataxin RNA/DNA deadenylase function and dysfunction in neurological disease. Prog Biophys Mol Biol 117:157-65
Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D et al. (2015) Recognition and repair of chemically heterogeneous structures at DNA ends. Environ Mol Mutagen 56:1-21
Andres, Sara N; Appel, C Denise; Westmoreland, James W et al. (2015) Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair. Nat Struct Mol Biol 22:158-66
Tumbale, Percy; Williams, Jessica S; Schellenberg, Matthew J et al. (2014) Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity. Nature 506:111-5
Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver et al. (2014) ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J 33:482-500
Gao, Rui; Schellenberg, Matthew J; Huang, Shar-Yin N et al. (2014) Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2·DNA and Top2·RNA covalent complexes by tyrosyl-DNA-phosphodiesterase 2 (TDP2). J Biol Chem 289:17960-9
Wallace, Bret D; Williams, R Scott (2014) Ribonucleotide triggered DNA damage and RNA-DNA damage responses. RNA Biol 11:1340-6

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