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Replication-dependent instability at (CTG)•(CAG) repeat hairpins in human cells

Nature Chemical Biology volume 6, pages 652–659 (2010)Cite this article

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Abstract

Instability of (CTG)•(CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG)•(CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG)•(CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.

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Figure 1: Hairpin-induced trinucleotide repeat instability.
Figure 2: DNA replication affects TNR instability.
Figure 3: ZFN cleave specifically in vitro.
Figure 4: (CTG)102•(CAG)102 TNRs form hairpins in vivo.
Figure 5: (CTG)102•(CAG)102 hairpin formation is suppressed by serum starvation.
Figure 6: (CTG)12•(CAG)12 TNRs are stable in vivo.

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References

  1. Pearson, C.E., Edamura, K.N. & Cleary, J.D. Repeat instability: mechanisms of dynamic mutations. Nat. Rev. Genet. 6, 729–742 (2005).

    Article  CAS  Google Scholar 

  2. Lia, A.S. et al. Somatic instability of the CTG repeat in mice transgenic for the myotonic dystrophy region is age dependent but not correlated to the relative intertissue transcription levels and proliferative capacities. Hum. Mol. Genet. 7, 1285–1291 (1998).

    Article  CAS  Google Scholar 

  3. Fortune, M.T., Vassilopoulos, C., Coolbaugh, M.I., Siciliano, M.J. & Monckton, D.G. Dramatic, expansion-biased, age-dependent, tissue-specific somatic mosaicism in a transgenic mouse model of triplet repeat instability. Hum. Mol. Genet. 9, 439–445 (2000).

    Article  CAS  Google Scholar 

  4. Miret, J.J., Pessoa-Brandao, L. & Lahue, R.S. Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 95, 12438–12443 (1998).

    Article  CAS  Google Scholar 

  5. Petruska, J., Arnheim, N. & Goodman, M.F. Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases. Nucleic Acids Res. 24, 1992–1998 (1996).

    Article  CAS  Google Scholar 

  6. Pearson, C.E., Wang, Y.H., Griffith, J.D. & Sinden, R.R. Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n(CAG)n repeats from the myotonic dystrophy locus. Nucleic Acids Res. 26, 816–823 (1998).

    Article  CAS  Google Scholar 

  7. Pelletier, R., Krasilnikova, M.M., Samadashwily, G.M., Lahue, R. & Mirkin, S.M. Replication and expansion of trinucleotide repeats in yeast. Mol. Cell. Biol. 23, 1349–1357 (2003).

    Article  CAS  Google Scholar 

  8. McMurray, C.T. DNA secondary structure: a common and causative factor for expansion in human disease. Proc. Natl. Acad. Sci. USA 96, 1823–1825 (1999).

    Article  CAS  Google Scholar 

  9. Mirkin, S.M. Expandable DNA repeats and human disease. Nature 447, 932–940 (2007).

    Article  CAS  Google Scholar 

  10. Sinden, R.R., Pytlos, M.J. & Potaman, V. Mechanisms of DNA repeat expansion. in Human Nucleotide Expansion Disorders (eds. Fry, M. & Usdin, K.) 3–53 (Springer, Berlin; New York, 2006).

  11. Hashem, V.I. et al. Chemotherapeutic deletion of CTG repeats in lymphoblast cells from DM1 patients. Nucleic Acids Res. 32, 6334–6346 (2004).

    Article  CAS  Google Scholar 

  12. Yang, Z., Lau, R., Marcadier, J.L., Chitayat, D. & Pearson, C.E. Replication inhibitors modulate instability of an expanded trinucleotide repeat at the myotonic dystrophy type 1 disease locus in human cells. Am. J. Hum. Genet. 73, 1092–1105 (2003).

    Article  CAS  Google Scholar 

  13. Cleary, J.D. & Pearson, C.E. Replication fork dynamics and dynamic mutations: the fork-shift model of repeat instability. Trends Genet. 21, 272–280 (2005).

    Article  CAS  Google Scholar 

  14. Trinh, T.Q. & Sinden, R.R. Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E. coli. Nature 352, 544–547 (1991).

    Article  CAS  Google Scholar 

  15. Yang, J. & Freudenreich, C.H. Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner. Gene 393, 110–115 (2007).

    Article  CAS  Google Scholar 

  16. Spiro, C. & McMurray, C.T. Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability. Mol. Cell. Biol. 23, 6063–6074 (2003).

    Article  CAS  Google Scholar 

  17. van den Broek, W.J., Nelen, M.R., van der Heijden, G.W., Wansink, D.G. & Wieringa, B. Fen1 does not control somatic hypermutability of the (CTG)(n)•(CAG)(n) repeat in a knock-in mouse model for DM1. FEBS Lett. 580, 5208–5214 (2006).

    Article  CAS  Google Scholar 

  18. Mirkin, E.V. & Mirkin, S.M. Replication fork stalling at natural impediments. Microbiol. Mol. Biol. Rev. 71, 13–35 (2007).

    Article  CAS  Google Scholar 

  19. Delagoutte, E., Goellner, G.M., Guo, J., Baldacci, G. & McMurray, C.T. Single-stranded DNA-binding protein in vitro eliminates the orientation-dependent impediment to polymerase passage on CAG/CTG repeats. J. Biol. Chem. 283, 13341–13356 (2008).

    Article  CAS  Google Scholar 

  20. Malott, M. & Leffak, M. Activity of the c-myc replicator at an ectopic chromosomal location. Mol. Cell. Biol. 19, 5685–5695 (1999).

    Article  CAS  Google Scholar 

  21. Liu, G., Malott, M. & Leffak, M. Multiple functional elements comprise a mammalian chromosomal replicator. Mol. Cell. Biol. 23, 1832–1842 (2003).

    Article  CAS  Google Scholar 

  22. Liu, G., Bissler, J.J., Sinden, R.R. & Leffak, M. Unstable spinocerebellar ataxia Type 10 (ATTCT)•(AGAAT) repeats are associated with aberrant replication at the ATX10 locus and replication origin-dependent expansion at an ectopic site in human cells. Mol. Cell. Biol. 27, 7828–7838 (2007).

    Article  CAS  Google Scholar 

  23. Burhans, W.C. et al. Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. EMBO J. 10, 4351–4360 (1991).

    Article  CAS  Google Scholar 

  24. Gacy, A.M. & McMurray, C.T. Influence of hairpins on template reannealing at trinucleotide repeat duplexes: a model for slipped DNA. Biochemistry 37, 9426–9434 (1998).

    Article  CAS  Google Scholar 

  25. Bitinaite, J., Wah, D.A., Aggarwal, A.K. & Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10570–10575 (1998).

    Article  CAS  Google Scholar 

  26. Mani, M., Smith, J., Kandavelou, K., Berg, J.M. & Chandrasegaran, S. Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem. Biophys. Res. Commun. 334, 1191–1197 (2005).

    Article  CAS  Google Scholar 

  27. Segal, D.J., Dreier, B., Beerli, R.R. & Barbas, C.F. III. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc. Natl. Acad. Sci. USA 96, 2758–2763 (1999).

    Article  CAS  Google Scholar 

  28. Mittelman, D. et al. Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells. Proc. Natl. Acad. Sci. USA 106, 9607–9612 (2009).

    Article  CAS  Google Scholar 

  29. Carroll, D., Morton, J.J., Beumer, K.J. & Segal, D.J. Design, construction and in vitro testing of zinc finger nucleases. Nat. Protoc. 1, 1329–1341 (2006).

    Article  CAS  Google Scholar 

  30. Usdin, K. & Woodford, K.J. CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro. Nucleic Acids Res. 23, 4202–4209 (1995).

    Article  CAS  Google Scholar 

  31. Freudenreich, C.H., Kantrow, S.M. & Zakian, V.A. Expansion and length-dependent fragility of CTG repeats in yeast. Science 279, 853–856 (1998).

    Article  CAS  Google Scholar 

  32. Carbajal Rivera, M.L., Ordaz Tellez, M.G., Castaneda Sortibran, A. & Rodriguez-Arnaiz, R. Emetine and/or its metabolites are genotoxic in somatic cells of Drosophila melanogaster. J. Toxicol. Environ. Health A 70, 1713–1716 (2007).

    Article  Google Scholar 

  33. Hashem, V.I., Rosche, W.A. & Sinden, R.R. Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E. coli. Mutat. Res. 554, 95–109 (2004).

    Article  CAS  Google Scholar 

  34. Mirkin, S.M. DNA structures, repeat expansions and human hereditary disorders. Curr. Opin. Struct. Biol. 16, 351–358 (2006).

    Article  CAS  Google Scholar 

  35. Gordenin, D.A., Kunkel, T.A. & Resnick, M.A. Repeat expansion–all in a flap? Nat. Genet. 16, 116–118 (1997).

    Article  CAS  Google Scholar 

  36. Lin, Y., Dion, V. & Wilson, J.H. Transcription promotes contraction of CAG repeat tracts in human cells. Nat. Struct. Mol. Biol. 13, 179–180 (2006).

    Article  CAS  Google Scholar 

  37. Jackson, S.M. et al. A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage. Gene 347, 35–41 (2005).

    Article  CAS  Google Scholar 

  38. Libby, R.T. et al. Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice. Hum. Mol. Genet. 12, 41–50 (2003).

    Article  CAS  Google Scholar 

  39. Monckton, D.G., Wong, L.J., Ashizawa, T. & Caskey, C.T. Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum. Mol. Genet. 4, 1–8 (1995).

    Article  CAS  Google Scholar 

  40. Seznec, H. et al. Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability. Hum. Mol. Genet. 9, 1185–1194 (2000).

    Article  CAS  Google Scholar 

  41. Khajavi, M. et al. “Mitotic drive” of expanded CTG repeats in myotonic dystrophy type 1 (DM1). Hum. Mol. Genet. 10, 855–863 (2001).

    Article  CAS  Google Scholar 

  42. Hashem, V.I., Rosche, W.A. & Sinden, R.R. Genetic assays for measuring rates of (CAG).(CTG) repeat instability in Escherichia coli. Mutat. Res. 502, 25–37 (2002).

    Article  CAS  Google Scholar 

  43. Shishkin, A.A. et al. Large-scale expansions of Friedreich's ataxia GAA repeats in yeast. Mol. Cell 35, 82–92 (2009).

    Article  CAS  Google Scholar 

  44. Bidichandani, S.I., Ashizawa, T. & Patel, P.I. The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure. Am. J. Hum. Genet. 62, 111–121 (1998).

    Article  CAS  Google Scholar 

  45. Potaman, V.N. et al. Unpaired Structures in SCA10 (ATTCT)(n)•(AGAAT)(n) Repeats. J. Mol. Biol. 326, 1095–1111 (2003).

    Article  CAS  Google Scholar 

  46. Sinden, R.R., Pytlos-Sinden, M.J. & Potaman, V.N. Slipped strand DNA structures. Front. Biosci. 12, 4788–4799 (2007).

    Article  CAS  Google Scholar 

  47. Voineagu, I., Surka, C.F., Shishkin, A.A., Krasilnikova, M.M. & Mirkin, S.M. Replisome stalling and stabilization at CGG repeats, which are responsible for chromosomal fragility. Nat. Struct. Mol. Biol. 16, 226–228 (2009).

    Article  CAS  Google Scholar 

  48. Sander, J.D., Zaback, P., Joung, J.K., Voytas, D.F. & Dobbs, D. Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res. 35, W599–W605 (2007).

    Article  Google Scholar 

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Acknowledgements

The authors thank C. Pearson and D.G. Monckton for their comments on this work. This work was supported by a grant from the Wright State University Boonshoft School of Medicine to G.L. and by grants from the US National Institutes of Health to M.L. (GM53819) and to J.J.B. (DK61458).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA

    Guoqi Liu, Xiaomi Chen & Michael Leffak

  2. Division of Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    John J Bissler

  3. Department of Biological Sciences, Florida Institute of Technology, Melbourne, Florida, USA

    Richard R Sinden

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Contributions

Experiments were conceived and designed by G.L., M.L., J.J.B. and R.R.S. and performed by G.L., X.C. and M.L. The manuscript was drafted by G.L. and M.L. and revised by all authors.

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Correspondence to Michael Leffak.

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Liu, G., Chen, X., Bissler, J. et al. Replication-dependent instability at (CTG)•(CAG) repeat hairpins in human cells. Nat Chem Biol 6, 652–659 (2010). https://doi.org/10.1038/nchembio.416

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