Smc Complexes: Methods and Protocols

ISBN-13: 9781493995196 / Angielski / Twarda / 2019 / 336 str.

Anjana Badrinarayanan

Spis treści

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 Paleček, Lucie Vondrová, Kateřina Zábrady, and Jakub Otočka

 

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

            Rupesh Kumar, Soon Bahng, and Kenneth J. Marians

 

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

            Logan R. Myler, Michael M. Soniat, Xiaoming Zhang, Rajashree A. Deshpande, Tanya T. Paull, and Ilya J. Finkelstein

 

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

            Yuji Sakai, Tatsuya Hirano, and Masashi Tachikawa

Blurb

This book brings together recent methods and theoretical approaches developed to dissect the activity and function of bacterial and eukaryotic Structural Maintenance of Chromosome (SMC) proteins. The protocols explore numerous subjects such as depletion systems to assess SMC function, genetic manipulation of SMC proteins, chromosomal, biochemical, and microscopy-based assays of SMC activity, as well as theoretical modeling of SMC activity. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Authoritative and practical, SMC Complexes: Methods and Protocols will greatly aid researchers looking into the mechanism of action of SMC complexes on DNA.

Chapters 3, 10 and 19 are available Open Access under a CC BY 4.0 license via link.springer.com.