SMC Complexes pp 197-208 | Cite as

A Protocol for Assaying the ATPase Activity of Recombinant Cohesin Holocomplexes

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


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 


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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

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