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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kimura K, Hirano T (1997) ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation. Cell 90(4):625–634
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):581–594
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):1941–1953
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
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):1249–1256
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
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
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):951–964
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
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
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
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
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
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):4884–4887
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
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Voulgaris, M., Gligoris, T.G. (2019). A Protocol for Assaying the ATPase Activity of Recombinant Cohesin Holocomplexes. In: Badrinarayanan, A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9520-2_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9519-6
Online ISBN: 978-1-4939-9520-2
eBook Packages: Springer Protocols