Advertisement

Detection of RNA-Templated Double-Strand Break Repair in Yeast

  • Ying Shen
  • Francesca StoriciEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

Abstract

The discovery of RNA-templated DNA repair has revealed a novel case where genetic information can flow directly from RNA to genomic DNA without passing through a reverse transcript intermediate. As initially demonstrated in the yeast Saccharomyces cerevisiae via transformation by RNA-containing oligonucleotides (oligos), RNA sequences can serve as templates for chromosomal double-strand break (DSB) repair. Synthetic oligos containing embedded RNA tracts of various sizes, or even RNA-only molecules, although with lower efficiency, can guide DNA repair synthesis at sites of broken DNA. Mechanisms and circumstances in which cells can use RNA to repair DNA damage such as a DSB are yet to be identified. Here we show the approach we utilize to detect repair of a chromosomal DSB by RNA-containing oligos in yeast cells.

Key words

RNA-containing oligonucleotides double-strand break (DSB) repair transformation yeast Saccharomyces cerevisiae single-strand annealing 

References

  1. 1.
    Storici, F., Bebenek, K., Kunkel, T.A., Gordenin, D.A., and Resnick, M.A. (2007) RNA-templated DNA repair. Nature 447, 338–341.PubMedCrossRefGoogle Scholar
  2. 2.
    Paques, F., Leung, W.Y., and Haber, J.E. (1998) Expansions and contractions in a tandem repeat induced by double-strand break repair. Mol Cell Biol 18, 2045–2054.PubMedGoogle Scholar
  3. 3.
    Baltimore, D. (1985) Retroviruses and retrotransposons: the role of reverse transcription in shaping the eukaryotic genome. Cell 40, 481–482.PubMedCrossRefGoogle Scholar
  4. 4.
    Autexier, C., and Lue, N.F. (2006) The structure and function of telomerase reverse transcriptase. Annu Rev Biochem 75, 493–517.PubMedCrossRefGoogle Scholar
  5. 5.
    Moore, J.K., and Haber, J.E. (1996) Capture of retrotransposon DNA at the sites of chromosomal double-strand breaks. Nature 383, 644–646.PubMedCrossRefGoogle Scholar
  6. 6.
    Teng, S.C., Kim, B., and Gabriel, A. (1996) Retrotransposon reverse-transcriptase-mediated repair of chromosomal breaks. Nature 383, 641–644.PubMedCrossRefGoogle Scholar
  7. 7.
    Derr, L.K., and Strathern, J.N. (1993) A role for reverse transcripts in gene conversion. Nature 361, 170–173.PubMedCrossRefGoogle Scholar
  8. 8.
    Lesage, P., and Todeschini, A.L. (2005) Happy together: the life and times of Ty retrotransposons and their hosts. Cytogenet Genome Res 110, 70–90.PubMedCrossRefGoogle Scholar
  9. 9.
    Morrish, T.A., Gilbert, N., Myers, J.S., Vincent, B.J., Stamato, T.D., Taccioli, G.E., Batzer, M.A., and Moran, J.V. (2002) DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat Genet 31, 159–165.PubMedCrossRefGoogle Scholar
  10. 10.
    Storici, F., Snipe, J.R., Chan, G.K., Gordenin, D.A., and Resnick, M.A. (2006) Conservative repair of a chromosomal double-strand break by single-strand DNA through two steps of annealing. Mol Cell Biol 26, 7645–7657.PubMedCrossRefGoogle Scholar
  11. 11.
    Houseley, J., LaCava, J., and Tollervey, D. (2006) RNA-quality control by the exosome. Nat Rev Mol Cell Biol 7, 529–539.PubMedCrossRefGoogle Scholar
  12. 12.
    Storici, F., and Resnick, M.A. (2006) The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol 409, 329–345.PubMedCrossRefGoogle Scholar
  13. 13.
    Harrison, J.C., and Haber, J.E. (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40, 209–235.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.School of Biology, Georgia Institute of TechnologyAtlantaUSA

Personalised recommendations