Abstract
SMC complexes play fundamental functions in chromosome architecture and organization as well as in DNA replication and repair throughout the cell cycle. The essential nature of the SMC components makes the study of their specific functions challenging. In this chapter, we describe the application of cell cycle tags to S. cerevisiae SMC genes. The cell cycle tags regulate both gene expression and protein degradation, allowing for restriction of the gene of interest to either the S or the G2/M phase. In case of SMC genes, the tags lead to valuable mutants that can bring insights into cell cycle specific essential functions, chromatin binding pattern and functional interactions. Here, we describe the generation of the cell cycle-restricted mutants in diploid and haploid cells and the validation of their functionality with several approaches.
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References
Jeppsson K, Kanno T, Shirahige K, Sjogren C (2014) The maintenance of chromosome structure: positioning and functioning of SMC complexes. Nat Rev Mol Cell Biol 15(9):601–614. https://doi.org/10.1038/nrm3857
Uhlmann F (2016) SMC complexes: from DNA to chromosomes. Nat Rev Mol Cell Biol 17(7):399–412. https://doi.org/10.1038/nrm.2016.30
Varejao N, Ibars E, Lascorz J, Colomina N, Torres-Rosell J, Reverter D (2018) DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex. EMBO J 37(12):pii: e98306. https://doi.org/10.15252/embj.201798306
Zhao X, Blobel G (2005) A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc Natl Acad Sci U S A 102(13):4777–4782. https://doi.org/10.1073/pnas.0500537102
Branzei D, Sollier J, Liberi G, Zhao X, Maeda D, Seki M, Enomoto T, Ohta K, Foiani M (2006) Ubc9- and mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks. Cell 127(3):509–522. https://doi.org/10.1016/j.cell.2006.08.050
Bustard DE, Menolfi D, Jeppsson K, Ball LG, Dewey SC, Shirahige K, Sjogren C, Branzei D, Cobb JA (2012) During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks. J Biol Chem 287(14):11374–11383. https://doi.org/10.1074/jbc.M111.336263
Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430(6999):573–578. https://doi.org/10.1038/nature02742
Lindroos HB, Strom L, Itoh T, Katou Y, Shirahige K, Sjogren C (2006) Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. Mol Cell 22(6):755–767. https://doi.org/10.1016/j.molcel.2006.05.014
D’Ambrosio C, Schmidt CK, Katou Y, Kelly G, Itoh T, Shirahige K, Uhlmann F (2008) Identification of cis-acting sites for condensin loading onto budding yeast chromosomes. Genes Dev 22(16):2215–2227. https://doi.org/10.1101/gad.1675708
Kegel A, Betts-Lindroos H, Kanno T, Jeppsson K, Strom L, Katou Y, Itoh T, Shirahige K, Sjogren C (2011) Chromosome length influences replication-induced topological stress. Nature 471(7338):392–396. https://doi.org/10.1038/nature09791
Menolfi D, Delamarre A, Lengronne A, Pasero P, Branzei D (2015) Essential roles of the Smc5/6 complex in replication through natural pausing sites and endogenous DNA damage tolerance. Mol Cell 60(6):835–846. https://doi.org/10.1016/j.molcel.2015.10.023
Hombauer H, Srivatsan A, Putnam CD, Kolodner RD (2011) Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication. Science 334(6063):1713–1716. https://doi.org/10.1126/science.1210770
Karras GI, Jentsch S (2010) The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase. Cell 141(2):255–267. https://doi.org/10.1016/j.cell.2010.02.028
Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21(11):947–962. https://doi.org/10.1002/yea.1142
Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, Andrews B, Tyers M, Boone C (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294(5550):2364–2368. https://doi.org/10.1126/science.1065810
Acknowledgments
We thank all the Branzei lab members for discussion. The work in the Branzei laboratory is supported by the Italian Association for Cancer Research (IG 18976), and European Research Council (Consolidator Grant 682190) grants to D.B. D.M. was supported by an FIRC/AIRC fellowship. The authors declare no conflict of interest.
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Menolfi, D., Branzei, D. (2019). Using Cell Cycle-Restricted Alleles to Study the Chromatin Dynamics and Functions of the Structural Maintenance of Chromosomes (SMC) Complexes In Vivo. 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_1
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DOI: https://doi.org/10.1007/978-1-4939-9520-2_1
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