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Incision-dependent and error-free repair of (CAG)n/(CTG)n hairpins in human cell extracts

Nature Structural & Molecular Biology volume 16, pages 869–875 (2009)Cite this article

Abstract

Expansion of CAG/CTG trinucleotide repeats is associated with certain familial neurological disorders, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism contributes to their expansion. However, the molecular mechanism(s) by which cells remove CAG/CTG hairpins remain unknown. Here we demonstrate that human cell extracts can catalyze error-free repair of CAG/CTG hairpins in a nick-directed manner. The repair system specifically targets CAG/CTG tracts for incisions in the nicked DNA strand, followed by DNA resynthesis using the continuous strand as a template, thereby ensuring CAG/CTG stability. Proliferating cell nuclear antigen (PCNA) is required for the incision step of the hairpin removal, which uses distinct endonuclease activities for individual CAG/CTG hairpins depending on their strand locations and/or secondary structures. We discuss the implications of these data for understanding the etiology of neurological diseases and trinucleotide repeat instability.

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Figure 1: Error-free CAG/CTG HPR in human extracts.
Figure 2: Removal of nicked strand CTG hairpin by dual incisions.
Figure 3: Analysis of repair intermediates of substrate C-(CAG)25.
Figure 4: Analysis of repair intermediates of substrates V-(CAG)25 and V-(CTG)25.
Figure 5: PCNA is required for CAG/CTG HPR.
Figure 6: Model of CAG/CTG HPR in human cells.

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Acknowledgements

We thank B. Shen (City of Hope National Medical Center), J. Hurwitz (Memorial Sloan Kettering Cancer Center) and A. Dutta (University of Virginia) for FEN-1, PCNA and p21C expression vectors, respectively, and D. Li (Shanghai Institutes for Biological Sciences) for helpful discussions. The work was supported in part by US National Institute of Helath grants GM072756 (to G.-M.L.) and CA104333 (to L.G.). G.-M.L. is the James-Gardner Endowed Chair in Cancer Research.

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Authors and Affiliations

  1. Graduate Center for Toxicology and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA

    Caixia Hou, Nelson L S Chan, Liya Gu & Guo-Min Li

  2. Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA

    Guo-Min Li

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Contributions

C.H. performed all the experiments; N.L.S.C. helped to determine some incision intermediates; L.G. designed and constructed DNA hairpin substrates, and developed the in vitro HPR assay; G.-M.L. designed and analyzed all the experiments, and wrote the manuscript.

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Correspondence to Guo-Min Li.

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Hou, C., Chan, N., Gu, L. et al. Incision-dependent and error-free repair of (CAG)n/(CTG)n hairpins in human cell extracts. Nat Struct Mol Biol 16, 869–875 (2009). https://doi.org/10.1038/nsmb.1638

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