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Measuring Chromosome Pairing During Homologous Recombination in Yeast

  • Fraulin Joseph
  • So Jung Lee
  • Eric Edward Bryant
  • Rodney RothsteinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2153)

Abstract

The precise organization of the genome inside the cell nucleus is vital to many cell functions including gene expression, cell division, and DNA repair. Here we describe a method to measure pairing of DNA loci during homologous recombination (HR) at a site-specific double-strand break (DSB) in Saccharomyces cerevisiae. This method utilizes a chromosome tagging system in diploid yeast cells to visualize both the DNA at the break site and the homologous DNA that serves as a repair template. DNA repair products are confirmed in parallel by genomic blot. This visualization method provides insight into the physical contact that occurs between homologous loci during HR and correlates physical interaction with the timing of DNA repair.

Key words

DNA repair Homologous recombination Homolog pairing Homology search Repair foci Rad52 Fluorescence microscopy Yeast 

Notes

Acknowledgments

We especially thank Robert Reid as well as the entire Rothstein laboratory, Michael Smith, Ivana Sunjevaric, Olga Marte, Gaël Fortin, and Keerthana Chetlapalli for experimental feedback and suggestions. We also thank Judith Miné-Hattab and Michael Lisby for their work in the initial construction of the yeast strains used in this methods chapter. Lastly, we thank Lorraine Symington and Roberto Donnianni for suggestions and help with genomic blotting. This work was supported by National Institutes of Health grants T32 GM007088 (to F.J.J.), T32 CA009503 (to F.J.J. and E.E.B.), R35 GM118180-S1 (to F.J.J.), T32 GM008798 (to E.E.B.), TL1 TR001875 (to E.E.B.), and R35 GM118180 (to R.R.).

References

  1. 1.
    Lisby M, Rothstein R (2009) Choreography of recombination proteins during the DNA damage response. DNA Repair 8:1068–1076.  https://doi.org/10.1016/j.dnarep.2009.04.007CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Symington LS, Rothstein R, Lisby M (2014) Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics 198:795–835CrossRefGoogle Scholar
  3. 3.
    Smith MJ, Rothstein R (2017) Poetry in motion: increased chromosomal mobility after DNA damage. DNA Repair (Amst) 56:102–108CrossRefGoogle Scholar
  4. 4.
    Mine-Hattab J, Rothstein R (2012) Increased chromosome mobility facilitates homology search during recombination. Nat Cell Biol 14:510–517.  https://doi.org/10.1038/ncb2472CrossRefPubMedGoogle Scholar
  5. 5.
    Smith MJ, Bryant EE, Rothstein R (2018) Increased chromosomal mobility after DNA damage is controlled by interactions between the recombination machinery and the checkpoint. Genes Dev 32:1242–1251CrossRefGoogle Scholar
  6. 6.
    Dion V, Kalck V, Horigome C, Towbin BD, Gasser SM (2012) Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat Cell Biol 14:502–509.  https://doi.org/10.1038/ncb2465CrossRefPubMedGoogle Scholar
  7. 7.
    Amaral N, Ryu T, Li X, Chiolo I (2017) Nuclear dynamics of heterochromatin repair. Trends Genet 33:86–100CrossRefGoogle Scholar
  8. 8.
    Tsouroula K, Furst A, Rogier M, Heyer V, Maglott-Roth A, Ferrand A, Reina-San-Martin B, Soutoglou E (2016) Temporal and spatial uncoupling of DNA double strand break repair pathways within mammalian heterochromatin. Mol Cell 63:293–305CrossRefGoogle Scholar
  9. 9.
    Chiolo I, Minoda A, Colmenares SU, Polyzos A, Costes SV, Karpen GH (2011) Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144:732–744CrossRefGoogle Scholar
  10. 10.
    Robinett CC et al (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135:1685–1700CrossRefGoogle Scholar
  11. 11.
    Michaelis C, Ciosk R, Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91:35–45CrossRefGoogle Scholar
  12. 12.
    Plessis A, Perrin A, Haber JE, Dujon B (1992) Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics 130(3):451–460PubMedPubMedCentralGoogle Scholar
  13. 13.
    Lisby M, Rothstein R, Mortensen UH (2001) Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci U S A 98:8276–8282.  https://doi.org/10.1073/pnas.121006298CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118:699–713.  https://doi.org/10.1016/j.cell.2004.08.015CrossRefPubMedGoogle Scholar
  15. 15.
    Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56:619–630CrossRefGoogle Scholar
  16. 16.
    Zhao X, Muller EG, Rothstein R (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2:329–340CrossRefGoogle Scholar
  17. 17.
    Unal E, Heidinger-Pauli JM, Kim W, Guacci V, Onn I, Gygi SP, Koshland DE (2008) A molecular determinant for the establishment of sister chromatid cohesion. Science 321:566–569CrossRefGoogle Scholar
  18. 18.
    Sherman F, Fink G, Hicks J (1987) Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. ISBN 9780879691974Google Scholar
  19. 19.
    Lim CR, Kimata Y, Nomaguchi K, Kohno K (1995) Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1. J Biochem 118:13–17CrossRefGoogle Scholar
  20. 20.
    Ormo M et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395CrossRefGoogle Scholar
  21. 21.
    Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182CrossRefGoogle Scholar
  22. 22.
    Campbell RE et al (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99:7877–7882CrossRefGoogle Scholar
  23. 23.
    Lisby M, Mortensen U, Rothstein R (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 6:572–577CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Fraulin Joseph
    • 1
  • So Jung Lee
    • 1
  • Eric Edward Bryant
    • 2
  • Rodney Rothstein
    • 1
    Email author
  1. 1.Department of Genetics and DevelopmentColumbia University Irving Medical CenterNew YorkUSA
  2. 2.Department of Biological SciencesColumbia UniversityNew YorkUSA

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