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Friedreich's ataxia–associated GAA repeats induce replication-fork reversal and unusual molecular junctions

Nature Structural & Molecular Biology volume 20, pages 486–494 (2013)Cite this article

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Abstract

Expansion of GAA/TTC repeats is the causative event in Friedreich's ataxia. GAA repeats have been shown to hinder replication in model systems, but the mechanisms of replication interference and expansion in human cells remained elusive. To study in vivo replication structures at GAA repeats, we designed a new plasmid-based system that permits the analysis of human replication intermediates by two-dimensional gel electrophoresis and EM. We found that replication forks transiently pause and reverse at long GAA/TTC tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions, located between GAA/TTC repeats and other homopurine-homopyrimidine tracts, that were associated with breakage of the plasmid fork not traversing the repeats. Finally, we detected postreplicative, sister-chromatid hemicatenanes on control plasmids, which were replaced by persistent homology-driven junctions at GAA/TTC repeats. These data prove that GAA/TTC tracts interfere with replication in humans and implicate postreplicative mechanisms in trinucleotide repeat expansion.

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Figure 1: Expanded GAA/TTC repeats induce unusual replication intermediates in human cells.
Figure 2: GAA/TTC-specific intermediates are largely homologous-recombination independent and display biochemical properties of triplex DNA.
Figure 3: Replication-associated DNA looping at expanded GAA/TTC repeats.
Figure 4: GAA/TTC repeats induce replication-fork reversal and affect the integrity of the sister fork.
Figure 5: GAA repeats induce unusual postreplicative junctions involving the repetitive sequences.
Figure 6: Model of replication interference by expanded GAA/TTC repeats and implications for repeat stability.

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Acknowledgements

We thank M. Pandolfo (Campus ERASME, Université Libre de Bruxelles, Brussels, Belgium) for providing plasmids and L. Pelkmans (Institute of Molecular Life Science, University of Zürich, Zürich, Switzerland) for the SV40 strain. We thank also the Center for Microscopy and Image Analysis of the University of Zürich for technical assistance with EM. We thank J. Jiricny for critical reading of the manuscript. We are grateful to S. Mirkin, V. Zakian and all members of the Lopes lab for helpful discussions and valuable input on this project. This work was supported by the Swiss National Science Foundation grants PP0033-114922 and PP00P3-135292 to M.L.

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  1. Judith Oehler

    Present address: Present address: Institute of Pharmacology and Toxicology, University of Zürich, Zürich, Switzerland.,

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  1. Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland

    Cindy Follonier, Judith Oehler, Raquel Herrador & Massimo Lopes

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Contributions

C.F. established and optimized the plasmid-based system used in this study and performed the 2D-gel and EM experiments. J.O. performed the 2D gel in Figure 1b. R.H. contributed to the RAD51-depletion experiment and performed the western blot. M.L. planned and supervised the project and wrote the paper.

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Correspondence to Massimo Lopes.

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Follonier, C., Oehler, J., Herrador, R. et al. Friedreich's ataxia–associated GAA repeats induce replication-fork reversal and unusual molecular junctions. Nat Struct Mol Biol 20, 486–494 (2013). https://doi.org/10.1038/nsmb.2520

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