Abstract
DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5′-adenylation DNA damage. Aprataxin (Aptx) catalyzes direct reversal of 5′-adenylate adducts to protect genome integrity. Here the structure of a Schizosaccharomyces pombe Aptx–DNA–AMP–Zn2+ complex reveals active site and DNA interaction clefts formed by fusing a histidine triad (HIT) nucleotide hydrolase with a DNA minor groove–binding C2HE zinc finger (Znf). An Aptx helical 'wedge' interrogates the base stack for sensing DNA ends or DNA nicks. The HIT-Znf, the wedge and an '[F/Y]PK' pivot motif cooperate to distort terminal DNA base-pairing and direct 5′-adenylate into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5′-adenylate adduct recognition and removal and suggest that mutations affecting protein folding, the active site pocket and the pivot motif underlie Aptx dysfunction in the neurodegenerative disorder ataxia with oculomotor apraxia 1 (AOA1).
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16 October 2011
In the HTML version of this article initially published online, the corresponding author was given as Jessica S. Williams, instead of R. Scott Willliams. The error has been corrected in the HTML version of this article.
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Acknowledgements
This research was supported by the intramural research program of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) (1Z01ES102765-01 to R.S.W.). We thank L. Pedersen, T. Kunkel and S. Wilson for discussions and critical reading of the manuscript, the Advanced Photon Source (APS) Southeast Regional Collaborative Access Team (SER-CAT) staff for assistance with crystallographic data collection, and we thank J. Williams of the NIEHS Protein Microcharacterization Core Facility for mass spectrometry analysis.
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P.T. conducted and analyzed experiments and helped write the manuscript. C.D.A., R.K. and J.S.W. conducted experiments. P.D.R. and J.K. analyzed results. I.A. designed experiments and analyzed results. R.S.W. designed research, did experiments, analyzed results and wrote the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 3010 kb)
Supplementary Movie 1
Aptx engagement of a nicked or gapped DNA-adenylate. Aptx employs the helical wedge with Phe34 (Blue) to displace a stacked 5′-adenylate (orange). The DNA (green) morphs between a model B-DNA conformation and the backbone conformation observed in the Aptx–DNA–AMP–Zn complex structure. A slight under-winding of the duplex is observed upon binding. Grey DNA is a modeled conformation of the predicted positioning of the upstream region of nick or gapped duplex bearing a 5′-AMP. Intercalation of the wedge helix into the base stack necessitates displacement of the upstream DNA. HIT domain is shown in purple and Znf in gold/brown. (MOV 21034 kb)
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Tumbale, P., Appel, C., Kraehenbuehl, R. et al. Structure of an aprataxin–DNA complex with insights into AOA1 neurodegenerative disease. Nat Struct Mol Biol 18, 1189–1195 (2011). https://doi.org/10.1038/nsmb.2146
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DOI: https://doi.org/10.1038/nsmb.2146
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