Advertisement

Conditional Mutation of SMC5 in Mouse Embryonic Fibroblasts

  • Himaja Gaddipati
  • Marina V. Pryzhkova
  • Philip W. JordanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

Abstract

The structural maintenance of chromosomes (SMC) complex, SMC5/6, is important for genome maintenance in all model eukaryotes. To date, the most extensive studies have focused on the roles of Smc5/6 in lower eukaryotes, such as yeast and fly. In the handful of studies that have used mammalian cells, siRNA was used by most to knockdown SMC5/6 components. RNAi methods have been very important for scientific progression, but they are hindered by incomplete silencing of protein expression and off-target effects. This chapter outlines the use of a conditional knockout approach in mouse embryonic fibroblasts to study the function of the SMC5/6 complex. These cell lines provide an alternative method to study the function and properties of the SMC5/6 complex in mammals.

Key words

Structural maintenance of chromosomes SMC5/6 Mouse embryonic fibroblast Conditional knockout DNA damage Chromosome segregation Micronuclei DNA bridges Mitotic catastrophe 

Notes

Acknowledgements

This work was supported by the National Institutes of Health (NIH) grant K99/R00 HD069458 to P.W.J.

References

  1. 1.
    Murray JM, Carr AM (2008) Smc5/6: a link between DNA repair and unidirectional replication? Nat Rev Mol Cell Biol 9:177–182.  https://doi.org/10.1038/nrm2309 CrossRefPubMedGoogle Scholar
  2. 2.
    Uhlmann F (2016) SMC complexes: from DNA to chromosomes. Nat Rev Mol Cell Biol 17:399–412.  https://doi.org/10.1038/nrm.2016.30 CrossRefPubMedGoogle Scholar
  3. 3.
    Hirano T (2006) At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7:311–322.  https://doi.org/10.1038/nrm1909 CrossRefPubMedGoogle Scholar
  4. 4.
    Piccoli G, Torres-Rosell J, Aragón L (2009) The unnamed complex: what do we know about Smc5-Smc6? Chromosome Res 17:251–263CrossRefGoogle Scholar
  5. 5.
    Verver DE, Hwang GH, Jordan PW, Hamer G (2016) Resolving complex chromosome structures during meiosis: versatile deployment of Smc5/6. Chromosoma 125:15–27.  https://doi.org/10.1007/s00412-015-0518-9 CrossRefPubMedGoogle Scholar
  6. 6.
    Hirano T (2016) Condensin-based chromosome organization from bacteria to vertebrates. Cell 164:847–857.  https://doi.org/10.1016/j.cell.2016.01.033 CrossRefPubMedGoogle Scholar
  7. 7.
    Wu N, Yu H (2012) The Smc complexes in DNA damage response. Cell Biosci 2:5.  https://doi.org/10.1186/2045-3701-2-5 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S et al (2006) Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination. Nat Cell Biol 8:1032–1034.  https://doi.org/10.1038/ncb1466 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Irmisch A, Ampatzidou E, Mizuno K, O’Connell MJ, Murray JM (2009) Smc5/6 maintains stalled replication forks in a recombination-competent conformation. EMBO J 28:144–155.  https://doi.org/10.1038/emboj.2008.273 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N et al (2007) The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol 9:923–931.  https://doi.org/10.1038/ncb1619 CrossRefPubMedGoogle Scholar
  11. 11.
    Wu N, Kong X, Ji Z, Zeng W, Potts PR et al (2012) Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl. Genes Dev 26:1473–1485.  https://doi.org/10.1101/gad.193615.112 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342.  https://doi.org/10.1038/nature10163 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Pryzhkova MV, Jordan PW (2016) Conditional mutation of Smc5 in mouse embryonic stem cells perturbs condensin localization and mitotic progression. J Cell Sci 129:1619–1634.  https://doi.org/10.1242/jcs.179036 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hwang G, Sun F, O’Brien M, Eppig JJ, Handel MA et al (2017) SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes. Development 144:1648–1660.  https://doi.org/10.1242/dev.145607 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17:299–313.  https://doi.org/10.1083/jcb.17.2.299 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Herbert AD, Carr AM, Hoffmann E (2014) FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis. PLoS One 9:e114749.  https://doi.org/10.1371/journal.pone.0114749 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M et al (1998) DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev 12:2831–2841.  https://doi.org/10.1101/gad.12.18.2831 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Himaja Gaddipati
    • 1
  • Marina V. Pryzhkova
    • 1
  • Philip W. Jordan
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA

Personalised recommendations