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Using Cell Cycle-Restricted Alleles to Study the Chromatin Dynamics and Functions of the Structural Maintenance of Chromosomes (SMC) Complexes In Vivo

  • Demis MenolfiEmail author
  • Dana BranzeiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

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.

Key words

SMC complexes Cell cycle tags Genetic crosses Tetrad dissection and analysis Protein expression 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute for Cancer Genetics, Department of Pathology and Cell Biology, College of Physicians and SurgeonsColumbia UniversityNew YorkUSA
  2. 2.IFOM, the FIRC Institute of Molecular OncologyMilanItaly
  3. 3.Istituto di Genetica MolecolareConsiglio Nazionale delle Ricerche (IGM-CNR)PaviaItaly

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