SMC Complexes: Methods and Protocols

Part I: Depletion Systems to Assess SMC Function

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

            Demis Menolfi and Dana Branzei

2. Degradation of S. cerevisiae Cohesin with the Auxin-Inducible Degron System

            Clémentine Brocas, Cécile Ducrot, and Karine Dubrana

3. Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation

            Yasutaka Kakui and Frank Uhlmann

4. Conditional Mutation of SMC5 in Mouse Embryonic Fibroblasts

            Himaja Gaddipati, Marina V. Pryzhkova, and Philip W. Jordan

Part II: Genetic Manipulation of SMC Function

5. High-Throughput Allelic Replacement Screening in Bacillus subtilis

            Marie-Laure Diebold-Durand, Frank Bürmann, and Stephan Gruber

6. Identifying Functional Domains in Subunits of Structural Maintenance of Chromosome (SMC) Complexes by Transposon Mutagenesis Screen in Yeast

            Avi Matityahu, Michal Shwartz, and Itay Onn

7. Multicomponent Yeast Two-Hybrid System: Applications to Study Protein-Protein Interactions in SMC Complexes

            Jan Josef Palecek, Lucie Vondrová, Katerina Zábrady, and Jakub Otocka

8. Knocking in Multi-Functional Gene Tags into SMC Complex Subunits Using Gene Editing

            Paul Kalitsis, Tao Zhang, Ji Hun Kim, Christian F. Nielsen, Kathryn M. Marshall, and Damien F. Hudson

Part III: Chromosomal Assays of SMC Activity

9. Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the Structural Maintenance of Chromosomes Complex In Vivo

            Tung B.K. Le

10. Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation

            Vasso Makrantoni, Daniel Robertson, and Adele L. Marston

11. Analysis of Cohesin Association to Newly Replicated DNA through Nascent Strand Binding Assay (NSBA)

            Camilla Frattini and Rodrigo Bermejo

12. Preparation of Cell Cycle Synchronized Saccharomyces cerevisiae Cells for Hi-C

            Stephanie A. Schalbetter and Jonathan Baxter

Part IV: Biochemical Assays of SMC Activity

13. Dissecting DNA Compaction by the Bacterial Condensin MukB

14. In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA

            Koichi Yano, Koichiro Akiyama, and Hironori Niki

15. A Protocol for Assaying the ATPase Activity of Recombinant Cohesin Holocomplexes

            Menelaos Voulgaris and Thomas G. Gligoris

16. In Vitro Detection of Long Noncoding RNA Generated from DNA Double-Strand Breaks

            Sheetal Sharma and Fabrizio d'Adda di Fagagna

Part V: Microscopy-Based Assays of SMC Activity

17. Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging

            Suchitha Raghunathan and Anjana Badrinarayanan

18. Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus under DNA Damage

            Afroze Chimthanawala and Anjana Badrinarayanan

19. Microinjection Techniques in Fly Embryos to Study the Function and Dynamics of SMC Complexes

            Catarina Carmo, Margarida Araújo, and Raquel A. Oliveira

20. Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex

Part VI: Theoretical Modeling of SMC Activity

21. Three-Dimensional Thermodynamic Simulation of Condensin as a DNA-Based Translocase

            Josh Lawrimore, Yunyan He, M. Gregory Forest, and Kerry Bloom

22. Molecular Dynamics Simulations of Condensin-Mediated Mitotic Chromosome Assembly