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The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates

Nature volume 443, pages 713–716 (2006)Cite this article

Abstract

Ataxia oculomotor apraxia-1 (AOA1) is a neurological disorder caused by mutations in the gene (APTX) encoding aprataxin1,2. Aprataxin is a member of the histidine triad (HIT) family of nucleotide hydrolases and transferases3, and inactivating mutations are largely confined to this HIT domain. Aprataxin associates with the DNA repair proteins XRCC1 and XRCC4, which are partners of DNA ligase III and ligase IV, respectively4,5,6,7, suggestive of a role in DNA repair. Consistent with this, APTX-defective cell lines are sensitive to agents that cause single-strand breaks and exhibit an increased incidence of induced chromosomal aberrations4,5,8. It is not, however, known whether aprataxin has a direct or indirect role in DNA repair, or what the physiological substrate of aprataxin might be. Here we show, using purified aprataxin protein and extracts derived from either APTX-defective chicken DT40 cells or Aptx-/- mouse primary neural cells, that aprataxin resolves abortive DNA ligation intermediates. Specifically, aprataxin catalyses the nucleophilic release of adenylate groups covalently linked to 5′-phosphate termini at single-strand nicks and gaps, resulting in the production of 5′-phosphate termini that can be efficiently rejoined. These data indicate that neurological disorders associated with APTX mutations may be caused by the gradual accumulation of unrepaired DNA strand breaks resulting from abortive DNA ligation events.

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Figure 1: Removal of AMP from abortive DNA ligation intermediates by aprataxin.
Figure 2: Aprataxin facilitates the ligation of DNA–adenylate intermediates.
Figure 3: DNA–adenylate hydrolysis activity is unique to aprataxin.
Figure 4: Analysis of aprataxin activity in vertebrate extracts.

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Acknowledgements

We thank D. Barnes and T. Lindahl for discussions and the gift of DNA ligase III–XRCC1 complex, and A. Ciccia for advice and comments. We thank S. Rulten for the gift of recombinant His-tagged aprataxin, and M. Taylor for provision of AOA1 lymphoblastoid cells. This work was supported by Cancer Research UK (S.C.W.), the EU DNA Repair Consortium (S.C.W.), the Medical Research Council (K.W.C.) and the NIH (P.J.M.). I.A. is supported by a fellowship from the European Molecular Biology Organisation. Author Contributions I.A. made the initial discovery of DNA–adenylate activity, and I.A. and U.R. were responsible for experimental design and the generation of most of the experimental data. S.F.E.-K., S.K. and P.M.C. were responsible for the development of vertebrate models and performed some of the experiments. P.J.M. and K.W.C. provided critical resources and expertise, and contributed to writing the manuscript. S.C.W. was the project leader and produced the final version of the manuscript.

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Authors and Affiliations

  1. Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, UK

    Ivan Ahel, Ulrich Rass & Stephen C. West

  2. Genome Damage and Stability Centre, University of Sussex, BN1 9RQ, Falmer, Brighton, UK

    Sherif F. El-Khamisy, Paula M. Clements & Keith W. Caldecott

  3. Biochemistry Department, Faculty of Pharmacy, Ain Shams University, PO Box 11566, Cairo, Egypt

    Sherif F. El-Khamisy

  4. Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, 332N Lauderdale, Tennessee, 38105, Memphis, USA

    Sachin Katyal & Peter J. McKinnon

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  1. Ivan Ahel
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Correspondence to Stephen C. West.

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Supplementary information

Supplementary Notes

This file contains Supplementary Methods and Supplementary Figure Legends. (DOC 74 kb)

Supplementary Figure 1

Reaction mechanism of DNA ligases. (PDF 114 kb)

Supplementary Figure 2

Purification of recombinant human Aprataxin. (PDF 257 kb)

Supplementary Figure 3

Direct measurement of AMP release from the DNA-adenylate intermediate. (PDF 209 kb)

Supplementary Figure 4

Analysis of Aprataxin on the DNA ligase-adenylate complex. (PDF 133 kb)

Supplementary Figure 5

Phylogenetic tree showing that Aprataxin (APTX) forms a distinct branch of the HIT superfamily. (PDF 111 kb)

Supplementary Figure 6

Extracts from Aptx-disrupted DT40 cells exhibit reduced ligation activity with the DNA adenylate substrate. (PDF 355 kb)

Supplementary Figure 7

Generation and characterization of Aptx-/- mice. (PDF 210 kb)

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Ahel, I., Rass, U., El-Khamisy, S. et al. The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates. Nature 443, 713–716 (2006). https://doi.org/10.1038/nature05164

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