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The genomes of cellular organisms are organized as double-stranded DNA, a structure that must be unwound to provide DNA replication, recombination, and repair machinery access to genomic information. However, DNA unwinding comes with inherent risks to genome stability. To help mediate these risks, bacterial, archael, and eukaryotic cells have evolved protective ssDNA-binding proteins (SSBs) that bind ssDNA with high affinity and specificity. SSBs also aid genome metabolic processes through direct interactions with key proteins in genome maintenance enzymes. Single-Stranded DNA Binding Proteins: Methods and Protocols assembles methods developed for examining the fundamental properties of SSBs and for exploiting the biochemical functions of SSBs for their use as in vitro and in vivo reagents. Clearly and concisely organized, the volume opens with an introduction to the structures and functions of SSBs, followed protocols for studying SSB/DNA complexes, methods for studying SSB/heterologous protein complexes, protocols for interrogating post-translational modifications of SSBs, and concludes with uses of fluorescently-labeled SSBs for in vitro and in vivo studies of genome maintenance processes. Written in the successful Methods in Molecular Biology(TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible, Single-Stranded DNA Binding Proteins: Methods and Protocols provides a rich introduction for investigators who are interested in this fascinating family of DNA-binding proteins.
Table des matières
Functions of Single-Strand DNA-Binding Proteins in DNA Replication, Recombination and Repair.-Structural Diversity Based on Variability in Quaternary Association. A Case Study Involving Eubacterial and Related SSBs.- SB Binding to ssDNA Using Isothermal Titration Calorimetry.- SSB-DNA Binding Monitored by Fluorescence Intensity and Anisotropy.- Single Molecule Analysis of SSB Dynamics on Single Stranded DNA.- Sample Preparation Methods to Analyze DNA-induced Structural Changes in Replication Protein A (RPA).- Structural Studies of SSB Interaction with RecO.- Investigation of Protein-Protein Interactions of Single-Stranded DNA-Binding Proteins by Analytical Ultracentrifugation.- Ammonium Sulfate Co-Precipitation of SSB and Interacting Proteins.- Analyzing Interactions Between SSB and Proteins by the Use of Fluorescence Anisotropy.- Far Western Blotting as a Rapid and Efficient Method for Detecting Interactions Between DNA Replication and DNA Repair Proteins.- Methods for Analysis of SSB/Protein Interactions by SPR.- Use of Native Gels to Measure Protein Binding to SSB.- Identification of Small Molecules That Disrupt SSB/Protein Interactions Using a High-Throughput Screen.- Detection of Post-translational Modifications of Replication Protein A.- Detecting Posttranslational Modification of Bacterial SSB Proteins.- Fluorescent SSB as a Reagentless Biosensor for Single-Stranded DNA.- Fluorescent Single-Stranded DNA-Binding Proteins Enable in vitro and in vivo Studies<.- Use of Fluorescently Tagged SSB Proteins in in vivo Localization Experiments.
Résumé
The genomes of cellular organisms are organized as double-stranded DNA, a structure that must be unwound to provide DNA replication, recombination, and repair machinery access to genomic information. However, DNA unwinding comes with inherent risks to genome stability. To help mediate these risks, bacterial, archael, and eukaryotic cells have evolved protective ssDNA-binding proteins (SSBs) that bind ssDNA with high affinity and specificity. SSBs also aid genome metabolic processes through direct interactions with key proteins in genome maintenance enzymes. Single-Stranded DNA Binding Proteins: Methods and Protocols assembles methods developed for examining the fundamental properties of SSBs and for exploiting the biochemical functions of SSBs for their use as in vitro and in vivo reagents. Clearly and concisely organized, the volume opens with an introduction to the structures and functions of SSBs, followed protocols for studying SSB/DNA complexes, methods for studying SSB/heterologous protein complexes, protocols for interrogating post-translational modifications of SSBs, and concludes with uses of fluorescently-labeled SSBs for in vitro and in vivo studies of genome maintenance processes. Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible, Single-Stranded DNA Binding Proteins: Methods and Protocols provides a rich introduction for investigators who are interested in this fascinating family of DNA-binding proteins.
