Abstract
Replication forks engage chromatin-bound cohesin complexes during chromosome replication. Interfacing between cohesin and replication forks influences both cohesion establishment and fork functionality. However, the mechanisms mediating this process are scarcely understood. Here we describe the nascent strand binding assay (NSBA) methodology, developed in budding yeast to discriminate the association of cohesin to either parental unreplicated or nascent DNA in the environment of replication forks. NSBA quantitatively estimates the association of a protein of interest to newly replicated DNA. For this, nascent strands are in vivo labeled with the thymine analogue bromodeoxyuridine and chromatin is immunoprecipitated to isolate a fraction enriched in DNA associated to the target protein. The abundance of nascent DNA is then assessed through BrdU immunoprecipitation followed by quantitative PCR, allowing for the parallel analysis of diverse genomic regions. While originally employed to characterize the association of cohesin to nascent sister chromatids, NSBA can be applied to study other factors dynamically associating to nascent DNA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ström L, Lindroos H, Shirahige K, Sjögren C (2004) Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol Cell 16:1003–1015
Fumasoni M, Zwicky K, Vanoli F, Lopes M, Branzei D (2015) Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the polα/primase/Ctf4 complex. Mol Cell 57:812–823
Losada A, Hirano M, Hirano T (1998) Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev 12:1986–1997
Uhlmann F, Wernic D, Poupart MA, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103:375–386
Makrantoni V, Marston AL (2018) Cohesin and chromosome segregation. Curr Biol 28:R688–R693
Toth A, Ciosk R, Uhlmann F, Galova M, Schleiffer A, Nasmyth K (1999) Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev 13:320–333
Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner KP, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23:787–799
Zhang J, Shi X, Li Y, Kim BJ, Jia J, Huang Z, Yang T, Fu X, Jung SY, Wang Y, Zhang P, Kim ST, Pan X, Qin J (2008) Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast. Mol Cell 31:143–151
Frattini C, Villa-Hernandez S, Pellicano G, Jossen R, Katou YM, Shirahige K, Bermejo R (2017) Cohesin ubiquitylation and mobilization facilitate stalled replication fork dynamics. Mol Cell 68:1–15
Bermejo R, Katou YM, Shirahige K, Foiani M (2009) ChIP-on-Chip analysis of DNA topoisomerases. Methods Mol Biol 582:103–118
Bermejo R, Doksani Y, Capra T, Katou YM, Tanaka H, Shirahige K, Foiani M (2007) Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21:1921–1936
Fachinetti D, Bermejo R, Cocito A, Minardi S, Katou YM, Kanoh Y, Shirahige K, Azvolinsky A, Zakian V, Foiani M (2010) Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Mol Cell 39:595–605
Villa-Hernandez S, Bermejo R (2018) Cohesin dynamic association to chromatin and interfacing with replication forks in genome integrity maintenance. Curr Genet 64(5):1005–1013. https://doi.org/10.1007/s00294-018-0824-x
Acknowledgments
We are thankful to current and past members of our laboratory for insightful discussions. This work was supported by the Ministry of Science, Innovation and Universities—MCINN (BFU2017-87013-R to R.B) and the Castilla y León regional government (to C.F.).
Author information
Authors and Affiliations
Corresponding author
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
Frattini, C., Bermejo, R. (2019). Analysis of Cohesin Association to Newly Replicated DNA Through Nascent Strand Binding Assay (NSBA). 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9520-2_11
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