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Full Description
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 10 and 19 areavailable Open Access under a CC BY 4.0 license via link.springer.com.
Contents
Using Cell Cycle Restricted Alleles to Study the Chromatin Dynamics and Functions of the Structural Maintenance of Chromosome (SMC) Complexes In Vivo.- Degradation of S. cerevisiae Cohesin with the Auxin-Inducible Degron System.- Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation.- Conditional Mutation of SMC5 in Mouse Embryonic Fibroblasts.- High-Throughput Allelic Replacement Screening in Bacillus subtilis.- Identifying Functional Domains in Subunits of Structural Maintenance of Chromosome (SMC) Complexes by Transposon Mutagenesis Screen in Yeast.- Multicomponent Yeast Two-Hybrid System: Applications to Study Protein-Protein Interactions in SMC Complexes.- Knocking in Multi-Functional Gene Tags into SMC Complex Subunits Using Gene Editing.- Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the Structural Maintenance of Chromosomes Complex In Vivo.- Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation.- Analysis of Cohesin Association to Newly Replicated DNA through Nascent Strand Binding Assay (NSBA).- Preparation of Cell Cycle Synchronized Saccharomyces cerevisiae Cells for Hi-C.- Dissecting DNA Compaction by the Bacterial Condensin MukB.- In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA.- A Protocol for Assaying the ATPase Activity of Recombinant Cohesin Holocomplexes.- In Vitro Detection of Long Noncoding RNA Generated from DNA Double-Strand Breaks.- Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging.- Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus under DNA Damage.- Microinjection Techniques in Fly Embryos to Study the Function and Dynamics of SMC Complexes.- Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex.- Three-Dimensional Thermodynamic Simulation of Condensin as a DNA-Based Translocase.- Molecular Dynamics Simulations of Condensin-Mediated Mitotic Chromosome Assembly.
