Advertisement

SMC Complexes pp 197-208 | Cite as

A Protocol for Assaying the ATPase Activity of Recombinant Cohesin Holocomplexes

  • Menelaos Voulgaris
  • Thomas G. Gligoris
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

Abstract

Cohesin and other members of the structural maintenance of chromosomes (SMC)-kleisin family such as condensin and Smc5-6, as well as central players in genome function and structure such as topoisomerases, DNA and RNA polymerases, and DNA repair enzymes contain nucleotide binding domains (NBD) which bind and eventually cleave ATP. The released energy is harnessed in various ways by these enzymes in order to fulfill their essential functions. However, unlike other enzymes, Smc-kleisin complexes—well sized, elongated and multisubunit in nature—have only recently been purified as holocomplexes. This progress offers both the opportunity and the challenge to determine in detail the potency of the ATPase activity of these large protein assemblies—typically exceeding 0.5 MDa in molecular weight—and examine its mechanistic features. We describe here in further detail a combined comprehensive protocol which we have successfully employed before for assaying the ATPase activity of recombinant budding yeast cohesin holocomplexes. We believe that with small and appropriate modifications the methods described here should be applicable to other ATPase complexes.

Key words

Cohesin ATPase Spectrophotometric Hydrolysis Smc 

References

  1. 1.
    Kimura K, Hirano T (1997) ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation. Cell 90(4):625–634CrossRefGoogle Scholar
  2. 2.
    Kamada K, Miyata M, Hirano T (2013) Molecular basis of SMC ATPase activation: role of internal structural changes of the regulatory subcomplex ScpAB. Structure 21(4):581–594CrossRefGoogle Scholar
  3. 3.
    Arumugam P, Gruber S, Tanaka K, Haering CH, Mechtler K, Nasmyth K (2003) ATP hydrolysis is required for cohesin’s association with chromosomes. Curr Biol 13(22):1941–1953CrossRefGoogle Scholar
  4. 4.
    Hu B, Itoh T, Mishra A, Katoh Y, Chan KL, Upcher W, Godlee C, Roig MB, Shirahige K, Nasmyth K (2011) ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex. Curr Biol 21(1):12–24.  https://doi.org/10.1016/j.cub.2010.12.004 CrossRefPubMedGoogle Scholar
  5. 5.
    Heidinger-Pauli J-M, Onn I, Koshland D (2010) Genetic evidence that the acetylation of the Smc3p subunit of cohesin modulates its ATP-bound state to promote cohesion establishment in Saccharomyces cerevisiae. Genetics 185(4):1249–1256CrossRefGoogle Scholar
  6. 6.
    Ladurner R, Bhaskara V, Huis in ’t Veld PJ, Davidson IF, Kreidl E, Petzold G, Peters JM (2014) Cohesin’s ATPase activity couples cohesin loading onto DNA with Smc3 acetylation. Curr Biol 24(19):2228–2237.  https://doi.org/10.1016/j.cub.2014.08.011 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Gligoris TG, Scheinost JC, Bürmann F, Petela N, Chan KL, Uluocak P, Beckouët F, Gruber S, Nasmyth K, Löwe J (2014) Closing the cohesin ring: structure and function of its Smc3-kleisin interface. Science 346(6212):963–967.  https://doi.org/10.1126/science.1256917 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Haering CH, Schoffnegger D, Nishino T, Helmhart W, Nasmyth K, Löwe J (2004) Structure and stability of cohesin’s Smc1-kleisin interaction. Mol Cell 15(6):951–964CrossRefGoogle Scholar
  9. 9.
    Terakawa T, Bisht S, Eeftens JM, Dekker C, Haering CH, Greene EC (2017) The condensin complex is a mechanochemical motor that translocates along DNA. Science 358(6363):672–676.  https://doi.org/10.1126/science.aan6516 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ganji M, Shaltiel IA, Bisht S, Kim E, Kalichava A, Haering CH, Dekker C (2018) Real-time imaging of DNA loop extrusion by condensin. Science 360(6384):102–105.  https://doi.org/10.1126/science.aar7831 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Petela NJ, Gligoris TG, Metson J, Lee BG, Voulgaris M, Hu B, Kikuchi S, Chapard C, Chen W, Rajendra E, Srinivisan M, Yu H, Löwe J, Nasmyth KA (2018) Scc2 is a potent activator of cohesin’s ATPase that promotes loading by binding Scc1 without Pds5. Mol Cell 70(6):1134–1148.e7.  https://doi.org/10.1016/j.molcel.2018.05.022 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wells JN, Gligoris TG, Nasmyth K, Marsh JA (2017) Evolution of condensin and cohesin complexes driven by replacement of Kite by Hawk proteins. Curr Biol 27:R17–R18.  https://doi.org/10.1016/j.cub.2016.11.050 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zawadzka K, Zawadzki P, Baker R, Rajasekar KV, Wagner F, Sherratt DJ, Arciszewska LK (2018) MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin. eLife 7.  https://doi.org/10.7554/eLife.31522
  14. 14.
    Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89(11):4884–4887CrossRefGoogle Scholar
  15. 15.
    Murayama Y, Uhlmann F (2014) Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505(7483):367–371.  https://doi.org/10.1038/nature12867 CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Menelaos Voulgaris
    • 1
  • Thomas G. Gligoris
    • 1
  1. 1.Department of BiochemistryUniversity of OxfordOxfordUK

Personalised recommendations