Abstract
Cohesin is a protein complex with key roles in chromosome biology, from chromatid segregation to DNA repair. Cohesin function is regulated by several posttranslational modifications, including phosphorylation, acetylation, ubiquitylation, and SUMOylation. Recent studies have shown that cohesin SUMOylation is essential for sister chromatid cohesion during normal cell cycle and in response to DNA damage. Posttranslational modification by the small ubiquitin-like modifier (SUMO) is a field in expansion, however, detecting SUMOylation can be challenging because the amount of modified substrates are usually low and de-conjugation during sample preparation often occurs. In this chapter we describe a method that can be adapted to different model organisms, and substrates to detect SUMOylation. We focus on cohesin and show that SUMOylation indeed occurs in most of the subunits of budding yeast cohesin.
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
Nasmyth K (2002) Segregating sister genomes: the molecular biology of chromosome separation. Science 297:559–65
Onn I et al (2008) Sister chromatid cohesion: a simple concept with a complex reality. Annu Rev Dev Biol 24:105–29
Uhlmann F, Nasmyth K (1998) Cohesion between sister chromatids must be established during DNA replication. Curr Biol 8:1095–1101
Uhlmann F, Lottspeich F, Nasmyth K (1999) Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400:37–42
Ström L et al (2004) Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol Cell 16:1003–15
Ünal E et al (2004) DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol Cell 16:991–1002
Ünal E, Heidinger-Pauli JM, Koshland D (2007) DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7). Science 317:245–8
Cortés-Ledesma F, Aguilera A (2006) Double-strand breaks arising by replication through a nick are repaired by cohesin-dependent sister-chromatid exchange. EMBO Rep 7:919–26
Rudra S, Skibbens RV (2013) Cohesin codes—interpreting chromatin architecture and the many facets of cohesin function. J Cell Sci 126:31–41
Almedawar S et al (2012) A SUMO-dependent step during establishment of sister chromatid cohesion. Curr Biol 22:1576–1581
McAleenan, A., et al., SUMOylation of the α-Kleisin Subunit of Cohesin Is Required for DNA Damage-Induced Cohesion. Curr Biol 22(17):1564-75.
Wu N et al (2012) Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl. Genes Dev 26:1473–85
Potts PR, Yu H (2005) Human MMS21/NSE2 is a SUMO ligase required for DNA repair. Mol Cell Biol 25:7021–32
Stephan AK et al (2011) Roles of vertebrate Smc5 in sister chromatid cohesion and homologous recombinational repair. Mol Cell Biol 31:1369–1381
Behlke-Steinert S et al (2009) SMC5 and MMS21 are required for chromosome cohesion and mitotic progression. Cell Cycle 8:2211–2218
Stead K et al (2003) Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion. J Cell Biol 163:729–41
Denison C et al (2005) A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell Proteomics 4:246–254
Hannich JT et al (2005) Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J Biol Chem 280:4102–4110
Panse VG et al (2004) A proteome-wide approach identifies sumoylated substrate proteins in yeast. J Biol Chem 279:41346–41351
Rosas-Acosta G et al (2005) A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers. Mol Cell Proteomics 4:56–72
Wohlschlegel JA et al (2004) Global analysis of protein sumoylation in Saccharomyces cerevisiae. J Biol Chem 279:45662–45668
Wohlschlegel JA et al (2006) Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J Proteome Res 5:761–770
Zhao Y et al (2004) Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins. J Biol Chem 279:20999–21002
Zhou, W., J.J. Ryan, and H. Zhou, Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J. Biol. Chem., 2004. 279: p. 32262-32268.
Hickey CM, Wilson NR, Hochstrasser M (2012) Function and regulation of SUMO proteases. Nat Rev Mol Cell Biol 13:755–766
Sarangi P, Zhao X (2015) SUMO-mediated regulation of DNA damage repair and responses. Trends Biochem Sci 40:233–242
Sacher M, Pfander B, Jentsch S (2005) Identification of SUMO-protein conjugates. Methods Enzymol 399:392–404
Tatham MH et al (2009) Detection of protein SUMOylation in vivo. Nat Protoc 4:1363–1371
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Bermúdez-López, M., Aragón, L. (2017). Detection of Cohesin SUMOylation In Vivo. In: Yokomori, K., Shirahige, K. (eds) Cohesin and Condensin. Methods in Molecular Biology, vol 1515. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6545-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6545-8_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6543-4
Online ISBN: 978-1-4939-6545-8
eBook Packages: Springer Protocols