Skip to main content

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

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Storici, F., Bebenek, K., Kunkel, T.A., Gordenin, D.A., and Resnick, M.A. (2007) RNA-templated DNA repair. Nature 447, 338–341.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  3. Baltimore, D. (1985) Retroviruses and retrotransposons: the role of reverse transcription in shaping the eukaryotic genome. Cell 40, 481–482.

    Article  PubMed  CAS  Google Scholar 

  4. Autexier, C., and Lue, N.F. (2006) The structure and function of telomerase reverse transcriptase. Annu Rev Biochem 75, 493–517.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  6. Teng, S.C., Kim, B., and Gabriel, A. (1996) Retrotransposon reverse-transcriptase-mediated repair of chromosomal breaks. Nature 383, 641–644.

    Article  PubMed  Google Scholar 

  7. Derr, L.K., and Strathern, J.N. (1993) A role for reverse transcripts in gene conversion. Nature 361, 170–173.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  11. Houseley, J., LaCava, J., and Tollervey, D. (2006) RNA-quality control by the exosome. Nat Rev Mol Cell Biol 7, 529–539.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  13. Harrison, J.C., and Haber, J.E. (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40, 209–235.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Francesca Storici .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Shen, Y., Storici, F. (2011). Detection of RNA-Templated Double-Strand Break Repair in Yeast. In: Tsubouchi, H. (eds) DNA Recombination. Methods in Molecular Biology, vol 745. Humana Press. https://doi.org/10.1007/978-1-61779-129-1_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-129-1_12

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-128-4

  • Online ISBN: 978-1-61779-129-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics