Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats

Nature Structural & Molecular Biology volume 24, pages 55–60 (2017)Cite this article

Subjects

Abstract

Expansions of (CAG)n/(CTG)n trinucleotide repeats are responsible for over a dozen neuromuscular and neurodegenerative disorders. Large-scale expansions are commonly observed in human pedigrees and may be explained by iterative small-scale events such as strand slippage during replication or repair DNA synthesis. Alternatively, a distinct mechanism may lead to a large-scale repeat expansion as a single step. To distinguish between these possibilities, we developed a novel experimental system specifically tuned to analyze large-scale expansions of (CAG)n/(CTG)n repeats in Saccharomyces cerevisiae. The median size of repeat expansions was ∼60 triplets, although we also observed additions of more than 150 triplets. Genetic analysis revealed that Rad51, Rad52, Mre11, Pol32, Pif1, and Mus81 and/or Yen1 proteins are required for large-scale expansions, whereas proteins previously implicated in small-scale expansions are not involved. From these results, we propose a new model for large-scale expansions, which is based on the recovery of replication forks broken at (CAG)n/(CTG)n repeats via break-induced replication.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Large-scale CAG-repeat expansions can be recovered and analyzed in budding yeast.
Figure 2: Genetic control of large-scale CAG-repeat expansions.
Figure 3: Large-scale CAG-repeat expansion is a replication-dependent event associated with replication-fork stalling and collapse.
Figure 4: Model of large-scale CAG-repeat expansions.

Similar content being viewed by others

References

  1. La Spada, A.R. & Taylor, J.P. Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat. Rev. Genet. 11, 247–258 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. McMurray, C.T. Mechanisms of trinucleotide repeat instability during human development. Nat. Rev. Genet. 11, 786–799 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Nance, M.A. & Myers, R.H. Juvenile onset Huntington's disease: clinical and research perspectives. Ment. Retard. Dev. Disabil. Res. Rev. 7, 153–157 (2001).

    CAS  PubMed  Google Scholar 

  4. Thornton, C.A. Myotonic dystrophy. Neurol. Clin. 32, 705–719 (2014).

    PubMed  PubMed Central  Google Scholar 

  5. Kim, J.C. & Mirkin, S.M. The balancing act of DNA repeat expansions. Curr. Opin. Genet. Dev. 23, 280–288 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Usdin, K., House, N.C. & Freudenreich, C.H. Repeat instability during DNA repair: insights from model systems. Crit. Rev. Biochem. Mol. Biol. 50, 142–167 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Gacy, A.M., Goellner, G., Juranić, N., Macura, S. & McMurray, C.T. Trinucleotide repeats that expand in human disease form hairpin structures in vitro. Cell 81, 533–540 (1995).

    CAS  PubMed  Google Scholar 

  8. Kerrest, A. et al. SRS2 and SGS1 prevent chromosomal breaks and stabilize triplet repeats by restraining recombination. Nat. Struct. Mol. Biol. 16, 159–167 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  12. Liu, Y. & Wilson, S.H. DNA base excision repair: a mechanism of trinucleotide repeat expansion. Trends Biochem. Sci. 37, 162–172 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kovtun, I.V. et al. OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature 447, 447–452 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Concannon, C. & Lahue, R.S. Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions. DNA Repair (Amst.) 13, 42–49 (2014).

    CAS  Google Scholar 

  15. Lin, Y. & Wilson, J.H. Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair. Mol. Cell. Biol. 27, 6209–6217 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Miret, J.J., Pessoa-Brandão, 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Callahan, J.L., Andrews, K.J., Zakian, V.A. & Freudenreich, C.H. Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility. Mol. Cell. Biol. 23, 7849–7860 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Jung, J., van Jaarsveld, M.T., Shieh, S.Y., Xu, K. & Bonini, N.M. Defining genetic factors that modulate intergenerational CAG repeat instability in Drosophila melanogaster. Genetics 187, 61–71 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Kovtun, I.V. & McMurray, C.T. Trinucleotide expansion in haploid germ cells by gap repair. Nat. Genet. 27, 407–411 (2001).

    CAS  PubMed  Google Scholar 

  20. Wheeler, V.C. et al. Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse. Hum. Mol. Genet. 8, 115–122 (1999).

    CAS  PubMed  Google Scholar 

  21. Bhattacharyya, S. & Lahue, R.S. Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats. Mol. Cell. Biol. 24, 7324–7330 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Su, X.A., Dion, V., Gasser, S.M. & Freudenreich, C.H. Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability. Genes Dev. 29, 1006–1017 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Debacker, K. et al. Histone deacetylase complexes promote trinucleotide repeat expansions. PLoS Biol. 10, e1001257 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Viterbo, D., Michoud, G., Mosbach, V., Dujon, B. & Richard, G.F. Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair. DNA Repair (Amst.) 42, 94–106 (2016).

    CAS  Google Scholar 

  25. Samadashwily, G.M., Raca, G. & Mirkin, S.M. Trinucleotide repeats affect DNA replication in vivo. Nat. Genet. 17, 298–304 (1997).

    CAS  PubMed  Google Scholar 

  26. Møllersen, L., Rowe, A.D., Larsen, E., Rognes, T. & Klungland, A. Continuous and periodic expansion of CAG repeats in Huntington's disease R6/1 mice. PLoS Genet. 6, e1001242 (2010).

    PubMed  PubMed Central  Google Scholar 

  27. Gomes-Pereira, M. et al. CTG trinucleotide repeat “big jumps”: large expansions, small mice. PLoS Genet. 3, e52 (2007).

    PubMed  PubMed Central  Google Scholar 

  28. Liu, G. et al. Altered replication in human cells promotes DMPK (CTG)n · (CAG)n repeat instability. Mol. Cell. Biol. 32, 1618–1632 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Liu, G., Chen, X., Bissler, J.J., Sinden, R.R. & Leffak, M. Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells. Nat. Chem. Biol. 6, 652–659 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Nakatani, R., Nakamori, M., Fujimura, H., Mochizuki, H. & Takahashi, M.P. Large expansion of CTG•CAG repeats is exacerbated by MutSβ in human cells. Sci. Rep. 5, 11020 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Cherng, N. et al. Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast. Proc. Natl. Acad. Sci. USA 108, 2843–2848 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Shah, K.A., McGinty, R.J., Egorova, V.I. & Mirkin, S.M. Coupling transcriptional state to large-scale repeat expansions in yeast. Cell Rep. 9, 1594–1602 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Dobi, K.C. & Winston, F. Analysis of transcriptional activation at a distance in Saccharomyces cerevisiae. Mol. Cell. Biol. 27, 5575–5586 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kang, S., Jaworski, A., Ohshima, K. & Wells, R.D. Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli. Nat. Genet. 10, 213–218 (1995).

    CAS  PubMed  Google Scholar 

  36. Freudenreich, C.H., Stavenhagen, J.B. & Zakian, V.A. Stability of a CTG/CAG trinucleotide repeat in yeast is dependent on its orientation in the genome. Mol. Cell. Biol. 17, 2090–2098 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Bhattacharyya, S. & Lahue, R.S. Srs2 helicase of Saccharomyces cerevisiae selectively unwinds triplet repeat DNA. J. Biol. Chem. 280, 33311–33317 (2005).

    CAS  PubMed  Google Scholar 

  38. Schmidt, M.H. & Pearson, C.E. Disease-associated repeat instability and mismatch repair. DNA Repair (Amst.) 38, 117–126 (2016).

    CAS  Google Scholar 

  39. Kantartzis, A. et al. Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions. Cell Rep. 2, 216–222 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Sundararajan, R., Gellon, L., Zunder, R.M. & Freudenreich, C.H. Double-strand break repair pathways protect against CAG/CTG repeat expansions, contractions and repeat-mediated chromosomal fragility in Saccharomyces cerevisiae. Genetics 184, 65–77 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Symington, L.S., Rothstein, R. & Lisby, M. Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics 198, 795–835 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Lydeard, J.R., Jain, S., Yamaguchi, M. & Haber, J.E. Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature 448, 820–823 (2007).

    CAS  PubMed  Google Scholar 

  43. Schulz, V.P. & Zakian, V.A. The saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation. Cell 76, 145–155 (1994).

    CAS  PubMed  Google Scholar 

  44. Wilson, M.A. et al. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration. Nature 502, 393–396 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Sakofsky, C.J. et al. Translesion polymerases drive microhomology-mediated break-induced replication leading to complex chromosomal rearrangements. Mol. Cell 60, 860–872 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Anand, R.P., Lovett, S.T. & Haber, J.E. Break-induced DNA replication. Cold Spring Harb. Perspect. Biol. 5, a010397 (2013).

    PubMed  PubMed Central  Google Scholar 

  47. Morrow, D.M., Connelly, C. & Hieter, P. “Break copy” duplication: a model for chromosome fragment formation in Saccharomyces cerevisiae. Genetics 147, 371–382 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Deem, A. et al. Break-induced replication is highly inaccurate. PLoS Biol. 9, e1000594 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Mayle, R. et al. DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage. Science 349, 742–747 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Lee, C.S., Lee, K., Legube, G. & Haber, J.E. Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break. Nat. Struct. Mol. Biol. 21, 103–109 (2014).

    CAS  PubMed  Google Scholar 

  51. Szilard, R.K. et al. Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX. Nat. Struct. Mol. Biol. 17, 299–305 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Sogo, J.M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602 (2002).

    CAS  PubMed  Google Scholar 

  53. Follonier, C., Oehler, J., Herrador, R. & Lopes, M. Friedreich's ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions. Nat. Struct. Mol. Biol. 20, 486–494 (2013).

    CAS  PubMed  Google Scholar 

  54. Richard, G.-F., Goellner, G.M., McMurray, C.T. & Haber, J.E. Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11-RAD50-XRS2 complex. EMBO J. 19, 2381–2390 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Jankowski, C., Nasar, F. & Nag, D.K. Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast. Proc. Natl. Acad. Sci. USA 97, 2134–2139 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Kim, H.-M. et al. Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair. EMBO J. 27, 2896–2906 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Hastings, P.J., Ira, G. & Lupski, J.R. A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLoS Genet. 5, e1000327 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Payen, C., Koszul, R., Dujon, B. & Fischer, G. Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication-based mechanisms. PLoS Genet. 4, e1000175 (2008).

    PubMed  PubMed Central  Google Scholar 

  59. Higham, C.F., Morales, F., Cobbold, C.A., Haydon, D.T. & Monckton, D.G. High levels of somatic DNA diversity at the myotonic dystrophy type 1 locus are driven by ultra-frequent expansion and contraction mutations. Hum. Mol. Genet. 21, 2450–2463 (2012).

    CAS  PubMed  Google Scholar 

  60. Raca, G., Siyanova, E.Y., McMurray, C.T. & Mirkin, S.M. Expansion of the (CTG)(n) repeat in the 5′-UTR of a reporter gene impedes translation. Nucleic Acids Res. 28, 3943–3949 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Shah, K.A. et al. Role of DNA polymerases in repeat-mediated genome instability. Cell Rep. 2, 1088–1095 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Goldstein, A.L. & McCusker, J.H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553 (1999).

    CAS  PubMed  Google Scholar 

  63. Rosche, W.A. & Foster, P.L. Determining mutation rates in bacterial populations. Methods 20, 4–17 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Aksenova, A.Y. et al. Genome rearrangements caused by interstitial telomeric sequences in yeast. Proc. Natl. Acad. Sci. USA 110, 19866–19871 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Research in the laboratory of S.M.M. is supported by NIH grants R01GM60987 and P01GM105473 and by a generous contribution from the White family. J.C.K. was supported by the NIH Training in Education and Critical Research Skills postdoctoral program (K12GM074869) and by Tufts University. We thank C. Freudenreich for valuable comments on the manuscript, G. Ira and S. Jinks-Robertson for helpful discussions, K. Spivakovsky-Gonzalez for help analyzing MSH mutants, and D. Padmanhaban for help with strain construction. S.T.H. received support from REU award NSF DBI 1263030. T.D. received support from Tufts Summer Scholars.

Author information

Author notes

  1. Jane C Kim & Kartik A Shah

    Present address: Present addresses: Department of Biological Sciences, California State University San Marcos, San Marcos, California, USA (J.C.K.) and Amgen, Cambridge, Massachusetts, USA (K.A.S.).,

Authors and Affiliations

  1. Department of Biology, Tufts University, Medford, Massachusetts, USA

    Jane C Kim, Samantha T Harris, Teresa Dinter, Kartik A Shah & Sergei M Mirkin

Authors
  1. Jane C Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samantha T Harris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Teresa Dinter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kartik A Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sergei M Mirkin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.C.K. and S.M.M. designed the study; J.C.K., S.T.H., and T.D. performed experiments; J.C.K., S.T.H., T.D., and S.M.M. analyzed data; K.A.S. provided reagents; J.C.K. and S.M.M. wrote the manuscript.

Corresponding author

Correspondence to Sergei M Mirkin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Sequence of the cassette used to generate the (CAG)140 strain YJK146.

Gray letters show chromosome III sequences used to target the construct to chromosome III (SGD coordinates 75,227–75,594 at the beginning of the cassette and coordinates 75,641–75,895 at the end). Sequence from the GAL1 promoter is in orange with the four Gal4 binding sites in yellow (Frolova, E. et al., Nucleic Acids Research. 27, 1350–1358, 1999). Other sequences are from the tetracycline resistance gene (green), CAG repeats (lime), TATA box (magenta), CAN1 (turquoise), and TRP1 (blue). Unmarked sequences are from the vector/plasmid.

Supplementary Figure 2 Construction of the (CAG)140 strain used to analyze large-scale CAG-repeat expansions in Saccharomyces cerevisiae.

Spot assay of strains containing no repeats and (CAG)140 with varying distances from UAS (last Gal4 binding site) to TATA box. Spots show 10-fold serial dilutions. Canavanine plate is shown after 5 days of growth at 30°C. Strain marked with red box (YJK146) was used for expansion rate analysis in various gene knockouts.

Supplementary Figure 3 Large-scale CAG-repeat expansions can be recovered and analyzed in budding yeast.

(A) Experimental system to study large-scale CAG repeat expansions. In the starting strain, the distance between the upstream activating sequence (UASGAL) and TATA box promoter (PGAL) allows transcription of the forward selection marker CAN1, which encodes an arginine permease. Large-scale expansions will result in CanR clones. Bars above the CAG repeats indicate products of single colony PCR used for sequencing (PCR1) and determination of repeat length (PCR2). (B) Agarose gel images showing PCR amplification of CAG repeat length for CanR clones. Arrow points to the initial length of (CAG)140. Only expanded clones are used to calculate expansion rates. Asterisks in lanes 2, 3, 6, and 8 indicate expanded clones. Clone 5 may be a large expansion with secondary contractions or the result of a gross chromosomal rearrangement.

Supplementary Figure 4 Genetic control of large-scale CAG-repeat expansions.

Effect of different gene knockouts and mutations on the large-scale expansion rate of (CAG)140 (A) Non-selective growth on galactose. Selection at 60 μg/mL canavanine concentration (B) Non-selective growth on glucose. Selection at 60 μg/mL canavanine concentration Dashed line designates 3-fold decrease from wild-type. Numbers above the dashed line show fold decrease compared to wild-type. Dotted line in (A) indicates 3-fold increase from wild-type.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–4 (PDF 1023 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, J., Harris, S., Dinter, T. et al. The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats. Nat Struct Mol Biol 24, 55–60 (2017). https://doi.org/10.1038/nsmb.3334

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3334

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing