Currently, we are focused on examining structure/function of DNA end processing factor Aprataxin (Aptx). 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.
| Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C et al. (2018) Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support. Protein Sci 27:1083-1092 |
| 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: |
| Kaminski, Andrea M; Tumbale, Percy P; Schellenberg, Matthew J et al. (2018) Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis. Nat Commun 9:2642 |
| 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 |
| Andres, Sara N; Williams, R Scott (2017) CtIP/Ctp1/Sae2, molecular form fit for function. DNA Repair (Amst) 56:109-117 |
| 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 |
| 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 |
| 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 |
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