Advertisement

Tracking Expansions of Stable and Threshold Length Trinucleotide Repeat Tracts In Vivo and In Vitro Using Saccharomyces cerevisiae

  • Gregory M. Williams
  • Athena K. Petrides
  • Lata Balakrishnan
  • Jennifer A. SurteesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2056)

Abstract

Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CAG)n and (CTG)n repeat sequences that, when expanded, lead to Huntington’s disease (HD) and myotonic dystrophy type 1 (DM1), respectively, as well as a number of other neurodegenerative diseases. TNR tracts in most individuals are relatively small and stable in terms of length. However, TNR tracts become increasingly prone to expansion as tract length increases, eventually leading to very long tracts that disrupt coding (e.g. HD) or noncoding (e.g., DM1) regions of the genome. It is important to understand the early stages in TNR expansions, that is, the transition from small, stable lengths to susceptible threshold lengths. We describe PCR-based in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts in the stable and threshold ranges. We also describe a simple in vitro system to assess tract dynamics during 5′ single-stranded DNA (ssDNA) flap processing and to assess the role of different DNA metabolism proteins in these dynamics. These assays can ultimately be used to determine factors that influence the early stages of TNR tract expansion.

Keywords

Trinucleotide repeat Expansion Contraction Polymerase chain reaction Saccharomyces cerevisiae DNA replication Repeat tract dynamics Microsatellite instability 

Notes

Acknowledgments

We are particularly grateful for the generosity of Dr. Robert Lahue in providing reagents and for many useful discussions and comments in developing this protocol. We are also grateful for discussions with Dr. Catherine Freudenreich, Dr. Robert Bambara, and Dr. Eric Alani. Work in the Balakrishnan laboratory is supported by the National Institutes of Health (GM0938328 to L.B.) Work in the Surtees laboratory is supported by the National Institutes of Health (GM087459 to J.A.S.) and the American Cancer Society (RSG-14-235-01 to J.A.S.). J.A.S. is an ACS Research Scholar.

References

  1. 1.
    Ireland MJ, Reinke SS, Livingston DM (2000) The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast. Genetics 155(4):1657–1665PubMedPubMedCentralGoogle Scholar
  2. 2.
    Schweitzer JK, Livingston DM (1997) Destabilization of CAG trinucleotide repeat tracts by mismatch repair mutations in yeast. Hum Mol Genet 6(3):349–355PubMedCrossRefGoogle Scholar
  3. 3.
    Gordenin DA, Kunkel TA, Resnick MA (1997) Repeat expansion [mdash] all in flap? Nat Genet 16(2):116–118PubMedCrossRefGoogle Scholar
  4. 4.
    Balakrishnan L, Bambara RA (2013) Flap endonuclease 1. Annu Rev Biochem 82(1):119–138PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Kovtun IV et al (2007) OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature 447:447PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Zhao X-N, Usdin K (2015) The repeat expansion diseases: the dark side of DNA repair. DNA Repair 32:96–105PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Tishkoff DX et al (1997) A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. Cell 88:253–263PubMedCrossRefGoogle Scholar
  8. 8.
    Yang J, Freudenreich CH (2007) Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner. Gene 393(1):110–115PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Liu Y et al (2004) Saccharomyces cerevisiae flap endonuclease 1 uses flap equilibration to maintain triplet repeat stability. Mol Cell Biol 24(9):4049–4064PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Callahan JL et al (2003) Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility. Mol Cell Biol 23(21):7849–7860PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Daee DL, Mertz T, Lahue RS (2007) Postreplication repair inhibits CAG {middle dot} CTG repeat expansions in Saccharomyces cerevisiae. Mol Cell Biol 27(1):102–110.  https://doi.org/10.1128/MCB.01167-06PubMedCrossRefGoogle Scholar
  12. 12.
    Arana ME, Kunkel TA (2010) Mutator phenotypes due to DNA replication infidelity. Semin Cancer Biol 20(5):304–311PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5(6):435–445PubMedCrossRefGoogle Scholar
  14. 14.
    Umar A, Kunkel TA (1996) DNA-replication fidelity, mismatch repair and genome instability in cancer cells. Eur J Biochem 238(2):297–307PubMedCrossRefGoogle Scholar
  15. 15.
    Strand M et al (1993) Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365(6443):274–276PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Kantartzis A et al (2012) Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions. Cell Rep 2(2):216–222PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Sia E et al (1997) Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes. Mol Cell Biol 17(5):2851–2858PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Romanova NV, Crouse GF (2013) Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast. PLoS Genet 9(10):e1003920PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Kunkel TA, Erie DA (2015) Eukaryotic mismatch repair in relation to DNA replication. Annu Rev Genet 49(1):291–313PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Crouse GF (2016) Non-canonical actions of mismatch repair. DNA Repair 38:102–109PubMedCrossRefGoogle Scholar
  21. 21.
    Owen BAL et al (2005) (CAG)n-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition. Nat Struct Mol Biol 12(8):663–670PubMedCrossRefGoogle Scholar
  22. 22.
    Manley K, Pugh J, Messer A (1999) Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington’s disease transgenic mice. Brain Res 835(1):74–79PubMedCrossRefGoogle Scholar
  23. 23.
    van den Broek WJAA et al (2002) Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch–repair proteins. Hum Mol Genet 11(2):191–198PubMedCrossRefGoogle Scholar
  24. 24.
    Foiry L et al (2006) Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice. Hum Genetics 119(5):520–526CrossRefGoogle Scholar
  25. 25.
    Gannon A-MM et al (2012) MutSβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells. Nucleic Acids Res 40(20):10324–10333PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Halabi A et al (2012) DNA mismatch repair complex MutSβ promotes GAA·TTC repeat expansion in human cells. J Biol Chem 287(35):29958–29967PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Williams GM, Surtees JA (2015) MSH3 promotes dynamic behavior of Trinucleotide repeat tracts in vivo. Genetics 200(3):737–754PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Schmidt MHM, Pearson CE (2016) Disease-associated repeat instability and mismatch repair. DNA Repair 38:117–126PubMedCrossRefGoogle Scholar
  29. 29.
    Kim JC, Mirkin SM (2013) The balancing act of DNA repeat expansions. Curr Opin Genet Dev 23(3):280–288PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Neil AJ, Kim JC, Mirkin SM (2017) Precarious maintenance of simple DNA repeats in eukaryotes. BioEssays 39(9):1700077CrossRefGoogle Scholar
  31. 31.
    Kang S et al (1995) Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes. J Biol Chem 270(45):27014–27021PubMedCrossRefGoogle Scholar
  32. 32.
    Usdin K, House NCM, Freudenreich CH (2015) Repeat instability during DNA repair: insights from model systems. Crit Rev Biochem Mol Biol:1–26Google Scholar
  33. 33.
    McGinty RJ, Mirkin SM (2018) Cis- and trans-modifiers of repeat expansions: blending model systems with human genetics. Trends Genet 34(6):448–465PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Polyzos AA, McMurray CT (2017) Close encounters: moving along bumps, breaks, and bubbles on expanded trinucleotide tracts. DNA Repair 56:144–155PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Mirkin SM (2007) Expandable DNA repeats and human disease. Nature 447(7147):932–940PubMedCrossRefGoogle Scholar
  36. 36.
    Kovtun IV, McMurray CT (2008) Features of trinucleotide repeat instability in vivo. Cell Res 18(1):198–213PubMedCrossRefGoogle Scholar
  37. 37.
    Paulson HL, Fischbeck KH (1996) Trinucleotide repeats in neurogenetic disorders. Annu Rev Neurosci 19(1):79–107PubMedCrossRefGoogle Scholar
  38. 38.
    Veitch NJ et al (2007) Inherited CAG·CTG allele length is a major modifier of somatic mutation length variability in Huntington disease. DNA Repair 6(6):789–796PubMedCrossRefGoogle Scholar
  39. 39.
    Groh WJ et al (2011) Survival and CTG repeat expansion in adults with myotonic dystrophy type 1. Muscle Nerve 43(5):648–651PubMedCrossRefGoogle Scholar
  40. 40.
    Kay C et al (2016) Huntington disease reduced penetrance alleles occur at high frequency in the general population. Neurology 87(3):282–288PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Langbehn DR, Hayden MR, Paulsen JS (2010) CAG-repeat length and the age of onset in Huntington disease (HD): a review and validation study of statistical approaches. Am J Med Genet B Neuropsychiatr Genet 153B(2):397–408PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Keum JW et al (2016) The HTT CAG-expansion mutation determines age at death but not disease duration in Huntington disease. Am J Hum Genet 98(2):287–298PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Chao T-K, Hu J, Pringsheim T (2017) Risk factors for the onset and progression of Huntington disease. Neurotoxicology 61:79–99PubMedCrossRefGoogle Scholar
  44. 44.
    Long JD et al (2018) Genetic modification of Huntington disease acts early in the prediagnosis phase. Am J Hum Genet 103(3):349–357PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Cumming SA et al (2018) De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1. Eur J Hum Genet 26(11):1635–1647PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Zhao X-N, Usdin K (2018) Timing of expansion of fragile X premutation alleles during intergenerational transmission in a mouse model of the fragile X-related disorders. Front Genet 9:314PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Braida C et al (2010) Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients. Hum Mol Genet 19(8):1399–1412PubMedCrossRefGoogle Scholar
  48. 48.
    Concannon C, Lahue RS (2014) Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions. DNA Repair 13(0):42–49PubMedCrossRefGoogle Scholar
  49. 49.
    Zhang L et al (1994) Studying human mutations by sperm typing: instability of CAG trinucleotide repeats in the human androgen receptor gene. Nat Genet 7(4):531–535PubMedCrossRefGoogle Scholar
  50. 50.
    Leeflang EP et al (1995) Single sperm analysis of the trinucleotide repeats in the Huntington’s disease gene: quantification of the mutation frequency spectrum. Hum Mol Genet 4(9):1519–1526PubMedCrossRefGoogle Scholar
  51. 51.
    Leeflang EP et al (1999) Analysis of Germline mutation spectra at the Huntington’s disease locus supports a mitotic mutation mechanism. Hum Mol Genet 8(2):173–183PubMedCrossRefGoogle Scholar
  52. 52.
    Martorell L et al (2004) Germline mutational dynamics in myotonic dystrophy type 1 males: allele length and age effects. Neurology 62(2):269–274PubMedCrossRefGoogle Scholar
  53. 53.
    Castel AL, Cleary JD, Pearson CE (2010) Repeat instability as the basis for human diseases and as a potential target for therapy. Nat Rev Mol Cell Biol 11(3):165–170CrossRefGoogle Scholar
  54. 54.
    Du J et al (2013) Length-dependent CTG·CAG triplet-repeat expansion in myotonic dystrophy patient-derived induced pluripotent stem cells. Hum Mol Genet 22(25):5276–5287PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    Higham CF et al (2012) 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(11):2450–2463PubMedCrossRefGoogle Scholar
  56. 56.
    Morales F et al (2012) Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity. Hum Mol Genet 21(16):3558–3567PubMedCrossRefGoogle Scholar
  57. 57.
    Higham CF, Monckton DG (2013) Modelling and inference reveal nonlinear length-dependent suppression of somatic instability for small disease associated alleles in myotonic dystrophy type 1 and Huntington disease. J R Soc Interface 10(88)PubMedCentralCrossRefGoogle Scholar
  58. 58.
    Lee J-M et al (2015) Identification of genetic factors that modify clinical onset of Huntington’s disease. Cell 162(3):516–526CrossRefGoogle Scholar
  59. 59.
    Moss DJH et al (2017) Identification of genetic variants associated with Huntington’s disease progression: a genome-wide association study. Lancet Neurol 16(9):701–711PubMedCrossRefGoogle Scholar
  60. 60.
    Morales F et al (2016) A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients. DNA Repair 40:57–66PubMedCrossRefGoogle Scholar
  61. 61.
    Miret JJ, Pessoa-Brandão L, Lahue RS (1997) Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae. Mol Cell Biol 17(6):3382–3387PubMedPubMedCentralCrossRefGoogle Scholar
  62. 62.
    Miret JJ, Pessoa-Brandão L, Lahue RS (1998) Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae. Proc Natl Acad Sci 95(21):12438–12443PubMedCrossRefGoogle Scholar
  63. 63.
    Dixon M, Bhattacharyya S, Lahue R (2004) Genetic assays for triplet repeat instability in yeast. In: Kohwi Y (ed) Trinucleotide repeat protocols. Humana Press, pp 29–45Google Scholar
  64. 64.
    Williams GM, Surtees JA (2018) Measuring dynamic behavior of Trinucleotide repeat tracts in vivo in Saccharomyces cerevisiae. In: Muzi-Falconi M, Brown GW (eds) Genome instability: methods and protocols. Springer, New York, NY, pp 439–470CrossRefGoogle Scholar
  65. 65.
    Gietz D et al (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20(6):1425PubMedPubMedCentralCrossRefGoogle Scholar
  66. 66.
    Drake JW (1991) A constant rate of spontaneous mutation in DNA-based microbes. Proc Natl Acad Sci U S A 88(16):7160–7164PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Dixon W, Massey F (1969) Introduction to statistical analysis. McGraw Hill, New YorkGoogle Scholar
  68. 68.
    Nair KR (1940) Table of confidence interval for the median in samples from any continuous population. SankhyÄ: The Indian Journal of Statistics (1933–1960) 4(4):551–558Google Scholar
  69. 69.
    Foster PL, Judith LC, Paul M (2006) Methods for determining spontaneous mutation rates. In: Methods in enzymology. Academic Press, pp 195–213Google Scholar
  70. 70.
    Zar JH (1999) Biostatistical analysis, fourth edition. Prentice Hall, Upper Saddle River, NJGoogle Scholar
  71. 71.
    Surtees JA, Alani E (2006) Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination. J Mol Biol 360(3):523–536PubMedCrossRefGoogle Scholar
  72. 72.
    Kumar C et al (2014) ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2–Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates. DNA Repair 18(0):18–30PubMedPubMedCentralCrossRefGoogle Scholar
  73. 73.
    Xie Y et al (2001) Identification of rad27 mutations that confer differential defects in mutation avoidance, repeat tract instability, and flap cleavage. Mol Cell Biol 21(15):4889–4899PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Ayyagari R et al (2003) Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J Biol Chem 278(3):1618–1625PubMedCrossRefGoogle Scholar
  75. 75.
    Langston LD, O’Donnell M (2008) DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA. J Biol Chem 283(43):29522–29531PubMedPubMedCentralCrossRefGoogle Scholar
  76. 76.
    Rolfsmeier ML et al (2001) Cis-elements governing Trinucleotide repeat instability in Saccharomyces cerevisiae. Genetics 157(4):1569–1579PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Gregory M. Williams
    • 1
    • 2
  • Athena K. Petrides
    • 3
  • Lata Balakrishnan
    • 4
  • Jennifer A. Surtees
    • 5
    • 6
    Email author
  1. 1.Centre for Chromosome BiologyNational University of IrelandGalwayIreland
  2. 2.Galway Neuroscience CentreNational Universityof IrelandGalwayIreland
  3. 3.Department of PathologyHarvard Medical SchoolBostonUSA
  4. 4.Department of BiologyIndiana University Purdue University IndianapolisIndianapolisUSA
  5. 5.Department of Biochemistry, JacobsSchool of Medicine and BiomedicalSciencesState University of New York atBuffaloBuffaloUSA
  6. 6.Genetics, Genomics and Bioinformatics Program, Jacobs School of Medicine and Biomedical SciencesState University of New York at BuffaloBuffaloUSA

Personalised recommendations