Advertisement

Genetic Screens to Study GAA/TTC and Inverted Repeat Instability in Saccharomyces cerevisiae

  • Wenying Guo
  • Kirill S. LobachevEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2056)

Abstract

Instability of trinucleotide and inverted repeats is a causative factor in the development of a number of neurological diseases, hereditary syndromes, and cancer. To understand the mechanisms that lead to repeat-induced genome destabilization it is important to identify factors that affect repeat metabolism. Here we present an approach that utilizes systematic and unbiased genome-wide screen in yeast Saccharomyces cerevisiae aimed to find genes that govern GAA/TTC and inverted repeat instability. These screens allowed for the identification of more than 30 mutants with increased fragility of both repeat motifs.

Keywords

Trinucleotide repeats Inverted repeats Genome instability Double-strand breaks Gross chromosomal rearrangements Genome-wide screen 

Notes

Acknowledgments

We thank Dr. N. Degtyareva for critical reading of the manuscript and Dr. Y. Zhang for carrying out the screens. This work was supported by NIH grant R01GM129119 to KSL.

References

  1. 1.
    Saini N, Zhang Y, Usdin K, Lobachev KS (2013) When secondary comes first--the importance of non-canonical DNA structures. Biochimie 95:117–123CrossRefGoogle Scholar
  2. 2.
    Lobachev KS, Gordenin DA, Resnick MA (2002) The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell 108:183–193CrossRefGoogle Scholar
  3. 3.
    Kim HM, Narayanan V, Mieczkowski PA, Petes TD, Krasilnikova MM, Mirkin SM, Lobachev KS (2008) Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair. EMBO J 27:2896–2906CrossRefGoogle Scholar
  4. 4.
    Zhang Y, Saini N, Sheng Z, Lobachev KS (2013) Genome-wide screen reveals replication pathway for quasi-palindrome fragility dependent on homologous recombination. PLoS Genet 9:e1003979CrossRefGoogle Scholar
  5. 5.
    Zhang Y, Shishkin AA, Nishida Y, Marcinkowski-Desmond D, Saini N, Volkov KV, Mirkin SM, Lobachev KS (2012) Genome-wide screen identifies pathways that govern GAA/TTC repeat fragility and expansions in dividing and nondividing yeast cells. Mol Cell 48:254–265CrossRefGoogle Scholar
  6. 6.
    Chen C, Kolodner RD (1999) Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Nat Genet 23:81–85CrossRefGoogle Scholar
  7. 7.
    Narayanan V, Mieczkowski PA, Kim HM, Petes TD, Lobachev KS (2006) The pattern of gene amplification is determined by the chromosomal location of hairpin-capped breaks. Cell 125:1283–1296CrossRefGoogle Scholar
  8. 8.
    Tong AHY, Evangelista M, Tyers M, Boone C, Parsons AB, Xu H, Bader GD, Pagé N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, Andrews B (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294:2364–2368CrossRefGoogle Scholar
  9. 9.
    Shishkin AA, Voineagu I, Matera R, Cherng N, Chernet BT, Krasilnikova MM, Narayanan V, Lobachev KS, Mirkin SM (2009) Large-scale expansions of Friedreich’s ataxia GAA repeats in yeast. Mol Cell 35:82–92CrossRefGoogle Scholar
  10. 10.
    Li Z, Vizeacoumar FJ, Bahr S, Li J, Warringer J, Vizeacoumar FS, Min R, VanderSluis B, Bellay J, DeVit M, Fleming JA (2011) Systematic exploration of essential yeast gene function with temperature-sensitive mutants. Nat Biotechnol 29:361–367CrossRefGoogle Scholar
  11. 11.
    Lobachev KS, Stenger JE, Kozyreva OG, Jurka J, Gordenin DA, Resnick MA (2000) Inverted Alu repeats unstable in yeast are excluded from the human genome. EMBO J 19:3822–3830CrossRefGoogle Scholar
  12. 12.
    Smith S, Hwang JY, Banerjee S, Majeed A, Gupta A, Myung K (2004) Mutator genes for suppression of gross chromosomal rearrangements identified by a genome-wide screening in Saccharomyces cerevisiae. Proc Natl Acad Sci 101:9039–9044CrossRefGoogle Scholar
  13. 13.
    Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541–1553CrossRefGoogle Scholar
  14. 14.
    Kaufer NF, Fried HM, Schwindinger WF, Jasin M, Warner JR (1983) Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res 11:3123–3135CrossRefGoogle Scholar
  15. 15.
    Storici F, Lewis LK, Resnick MA (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol 19:773–776CrossRefGoogle Scholar
  16. 16.
    Krasilnikova MM, Mirkin SM (2004) Replication stalling at Friedreich’s ataxia (GAA)n repeats in vivo. Mol Cell Biol 24:2286–2295CrossRefGoogle Scholar
  17. 17.
    Costanzo M, Boone C (2009) SGAM: an array-based approach for high-resolution genetic mapping in Saccharomyces cerevisiae. Methods Mol Biol 548:37–53Google Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Institute for Bioengineering and Bioscience, School of Biological SciencesGeorgia Institute of TechnologyAtlantaUSA

Personalised recommendations