Kooperativer Bibliotheksverbund

UID:

Format: 1 online resource (453 pages) : , illustrations

ISBN: 0-12-812516-0 , 0-12-812515-2

Note: Intro -- Title page -- Table of Contents -- Copyright -- Contributors -- Preface -- Chapter One: MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes -- Abstract -- 1 Introduction -- 2 Entry Cloning Into MacroBac Series 11 or 438 by LIC -- 3 Building Complexes by Restriction and Ligation: MacroBac Series 11 -- 4 Building Complexes by LIC: MacroBac Series 438 -- 5 Bacmid Preparation and Virus Production -- 6 Protein Expression and Viral Stock Maintenance -- 7 Trouble Shooting -- 8 Future Considerations -- Acknowledgments -- Chapter Two: Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes -- Abstract -- 1 Introduction -- 2 Cloning -- 3 Case Studies -- 4 Nucleosome Sliding and Histone Exchange Kinetic Assays -- 5 Nucleosome Sliding by Recombinant hINO80 Chromatin Remodeling Complex -- 6 Histone Exchange by Recombinant ySWR1 Chromatin Remodeling Complex -- Chapter Three: Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR -- Abstract -- 1 Introduction -- 2 Small-Angle X-Ray Scattering -- 3 Nuclear Magnetic Resonance -- 4 Summary and Conclusion-Using SAX and NMR to Characterize Architectural Remodeling -- Chapter Four: Use of Single-Cysteine Variants for Trapping Transient States in DNA Mismatch Repair -- Abstract -- 1 Introduction -- 2 Generation of Defined Cysteine Variants -- 3 Cross-linking of Defined Cysteine Variants -- 4 Use of Single-Cysteine Variants to Study Conformational Changes in DNA Mismatch Repair -- 5 Concluding Remarks -- Acknowledgments -- Chapter Five: Expression and Structural Analyses of Human DNA Polymerase θ (POLQ) -- Abstract -- 1 Introduction -- 2 Purification and Structure Determination of Human Pol θ -- 3 Crystallization and Structure Determination of Human Pol θ -- 4 Conclusions -- Acknowledgments. , Chapter Six: Structural Studies of RNases H2 as an Example of Crystal Structure Determination of Protein-Nucleic Acid Complexes -- Abstract -- 1 Introduction -- 2 Purification of RNases H2 -- 3 Biochemical Assays for RNases H2 -- 4 General Considerations in the Crystallization of Protein-Nucleic Acid Complexes -- 5 Crystallization and Structure Determination of RNases H2 -- 6 Concluding Remarks -- Acknowledgment -- Chapter Seven: DNA-PKcs, Allostery, and DNA Double-Strand Break Repair: Defining the Structure and Setting the Stage -- Abstract -- 1 Introduction -- 2 Defining the Structure of DNA-PKcs -- 3 Exploiting Multiple Se-Met Substitutions -- 4 Using Se-Met Sites to Check Sequence Registration -- 5 DNA-PKcs as a Stage for the Assembly of the Actors in NHEJ -- Acknowledgments -- Chapter Eight: Single-Particle Electron Microscopy Analysis of DNA Repair Complexes -- Abstract -- 1 Introduction -- 2 Sample Preparation -- 3 Grid Preparation -- 4 Data Collection -- 5 Data Processing -- Acknowledgments -- Chapter Nine: Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair -- Abstract -- 1 Introduction -- 2 Methods to Study DNA Repair Complexes With AFM -- 3 Image Analysis -- 4 DREEM Imaging -- 5 Complementary Techniques -- 6 Conclusions -- Acknowledgments -- Chapter Ten: Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time -- Abstract -- 1 Introduction -- 2 Preparation of Defined Lesion Substrates for AFM and DNA Tightrope Assay -- 3 Atomic Force Microscopy -- 4 Single-Molecule DNA Tightrope Assay -- 5 Conclusions -- Acknowledgments -- Chapter Eleven: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination -- Abstract -- 1 Introduction -- 2 Methods -- 3 Applications -- 4 Notes. , Acknowledgments -- Chapter Twelve: Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography -- Abstract -- 1 Introduction -- 2 DNA Polymerase η -- 3 Ribonuclease H1 -- 4 Endonuclease V -- 5 X-Ray Data Processing -- 6 Applications and Future Prospects -- Acknowledgments -- Chapter Thirteen: Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases -- Abstract -- 1 Introduction -- 2 Purification and Structures of Nonclassical Polymerases -- 3 Catalytic Activities of Nonclassical Polymerases -- 4 Interactions of Nonclassical Polymerases -- 5 Conclusions -- Acknowledgments -- Chapter Fourteen: Kinetic Methods for Studying DNA Glycosylases Functioning in Base Excision Repair -- Abstract -- 1 Introduction -- 2 Kinetic Methods to Study Catalysis by DNA Glycosylases -- 3 Analytical Methods for Monitoring DNA Glycosylase Activity -- 4 Determining Rate Constants From Kinetics Experiments -- Acknowledgment -- Chapter Fifteen: Transient Kinetic Methods for Mechanistic Characterization of DNA Binding and Nucleotide Flipping -- Abstract -- 1 Introduction -- 2 General Considerations for Monitoring Binding -- 3 Association Kinetics -- 4 Comparison of Binding and Flipping Mechanisms for AAG and AlkA -- 5 Use of Berkeley Madonna for Evaluating Kinetic Models -- 6 Useful Equations for Kinetic Analysis -- Acknowledgments -- Chapter Sixteen: What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses -- Abstract -- 1 Introduction -- 2 Metabolic Signaling: NAD and DDR by PARP, PARG, and AIF -- 3 DNA Double-Strand Break Responses -- 4 Taking Structural Mechanisms Into Cells by Advanced Imaging -- 5 What's Ahead -- Acknowledgments -- Author Index -- Subject Index.

Language: English

UID:

Format: 1 online resource (453 pages) : , illustrations

ISBN: 0-12-812516-0 , 0-12-812515-2

Note: Intro -- Title page -- Table of Contents -- Copyright -- Contributors -- Preface -- Chapter One: MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes -- Abstract -- 1 Introduction -- 2 Entry Cloning Into MacroBac Series 11 or 438 by LIC -- 3 Building Complexes by Restriction and Ligation: MacroBac Series 11 -- 4 Building Complexes by LIC: MacroBac Series 438 -- 5 Bacmid Preparation and Virus Production -- 6 Protein Expression and Viral Stock Maintenance -- 7 Trouble Shooting -- 8 Future Considerations -- Acknowledgments -- Chapter Two: Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes -- Abstract -- 1 Introduction -- 2 Cloning -- 3 Case Studies -- 4 Nucleosome Sliding and Histone Exchange Kinetic Assays -- 5 Nucleosome Sliding by Recombinant hINO80 Chromatin Remodeling Complex -- 6 Histone Exchange by Recombinant ySWR1 Chromatin Remodeling Complex -- Chapter Three: Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR -- Abstract -- 1 Introduction -- 2 Small-Angle X-Ray Scattering -- 3 Nuclear Magnetic Resonance -- 4 Summary and Conclusion-Using SAX and NMR to Characterize Architectural Remodeling -- Chapter Four: Use of Single-Cysteine Variants for Trapping Transient States in DNA Mismatch Repair -- Abstract -- 1 Introduction -- 2 Generation of Defined Cysteine Variants -- 3 Cross-linking of Defined Cysteine Variants -- 4 Use of Single-Cysteine Variants to Study Conformational Changes in DNA Mismatch Repair -- 5 Concluding Remarks -- Acknowledgments -- Chapter Five: Expression and Structural Analyses of Human DNA Polymerase θ (POLQ) -- Abstract -- 1 Introduction -- 2 Purification and Structure Determination of Human Pol θ -- 3 Crystallization and Structure Determination of Human Pol θ -- 4 Conclusions -- Acknowledgments. , Chapter Six: Structural Studies of RNases H2 as an Example of Crystal Structure Determination of Protein-Nucleic Acid Complexes -- Abstract -- 1 Introduction -- 2 Purification of RNases H2 -- 3 Biochemical Assays for RNases H2 -- 4 General Considerations in the Crystallization of Protein-Nucleic Acid Complexes -- 5 Crystallization and Structure Determination of RNases H2 -- 6 Concluding Remarks -- Acknowledgment -- Chapter Seven: DNA-PKcs, Allostery, and DNA Double-Strand Break Repair: Defining the Structure and Setting the Stage -- Abstract -- 1 Introduction -- 2 Defining the Structure of DNA-PKcs -- 3 Exploiting Multiple Se-Met Substitutions -- 4 Using Se-Met Sites to Check Sequence Registration -- 5 DNA-PKcs as a Stage for the Assembly of the Actors in NHEJ -- Acknowledgments -- Chapter Eight: Single-Particle Electron Microscopy Analysis of DNA Repair Complexes -- Abstract -- 1 Introduction -- 2 Sample Preparation -- 3 Grid Preparation -- 4 Data Collection -- 5 Data Processing -- Acknowledgments -- Chapter Nine: Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair -- Abstract -- 1 Introduction -- 2 Methods to Study DNA Repair Complexes With AFM -- 3 Image Analysis -- 4 DREEM Imaging -- 5 Complementary Techniques -- 6 Conclusions -- Acknowledgments -- Chapter Ten: Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time -- Abstract -- 1 Introduction -- 2 Preparation of Defined Lesion Substrates for AFM and DNA Tightrope Assay -- 3 Atomic Force Microscopy -- 4 Single-Molecule DNA Tightrope Assay -- 5 Conclusions -- Acknowledgments -- Chapter Eleven: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination -- Abstract -- 1 Introduction -- 2 Methods -- 3 Applications -- 4 Notes. , Acknowledgments -- Chapter Twelve: Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography -- Abstract -- 1 Introduction -- 2 DNA Polymerase η -- 3 Ribonuclease H1 -- 4 Endonuclease V -- 5 X-Ray Data Processing -- 6 Applications and Future Prospects -- Acknowledgments -- Chapter Thirteen: Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases -- Abstract -- 1 Introduction -- 2 Purification and Structures of Nonclassical Polymerases -- 3 Catalytic Activities of Nonclassical Polymerases -- 4 Interactions of Nonclassical Polymerases -- 5 Conclusions -- Acknowledgments -- Chapter Fourteen: Kinetic Methods for Studying DNA Glycosylases Functioning in Base Excision Repair -- Abstract -- 1 Introduction -- 2 Kinetic Methods to Study Catalysis by DNA Glycosylases -- 3 Analytical Methods for Monitoring DNA Glycosylase Activity -- 4 Determining Rate Constants From Kinetics Experiments -- Acknowledgment -- Chapter Fifteen: Transient Kinetic Methods for Mechanistic Characterization of DNA Binding and Nucleotide Flipping -- Abstract -- 1 Introduction -- 2 General Considerations for Monitoring Binding -- 3 Association Kinetics -- 4 Comparison of Binding and Flipping Mechanisms for AAG and AlkA -- 5 Use of Berkeley Madonna for Evaluating Kinetic Models -- 6 Useful Equations for Kinetic Analysis -- Acknowledgments -- Chapter Sixteen: What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses -- Abstract -- 1 Introduction -- 2 Metabolic Signaling: NAD and DDR by PARP, PARG, and AIF -- 3 DNA Double-Strand Break Responses -- 4 Taking Structural Mechanisms Into Cells by Advanced Imaging -- 5 What's Ahead -- Acknowledgments -- Author Index -- Subject Index.

Language: English

UID:

Format: 1 online resource (461 pages).

Edition: First edition.

ISBN: 0-12-811847-4 , 0-12-811846-6

Series Statement: Methods in Enzymology ; Volume 591

Note: Front Cover -- DNA Repair Enzymes: Cell, Molecular, and Chemical Biology -- Copyright -- Contents -- Contributors -- Preface -- Chapter One: Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells -- 1. Introduction -- 2. Suspension Cell Growth and Handling for iPOND and aniPOND -- 2.1. Optimizing Growth Conditions to Obtain iPOND/aniPOND Cell Numbers -- 2.2. Handling of Suspension Cells During Pulse and Chase -- 3. Optimizations to the aniPOND Protocol to Increase Functionality -- 3.1. Optimized Sonication Regimen -- 3.2. Preventing Chromatin Precipitation in Sonicated Chromatin -- 3.3. Eliminating Sources of Background -- 3.4. Optimization Limitations -- 4. Comparison of iPOND and aniPOND in Suspension Cells -- 5. Protocol for Optimized aniPOND in Suspension Cells -- 5.1. Suspension Cell Growth to aniPOND Experimental Numbers -- 5.1.1. Equipment -- 5.1.2. Buffers and Reagents -- 5.1.3. Procedure -- 5.1.4. Notes -- 5.2. EdU Pulse, Thymidine Chase, and Click Reaction -- 5.2.1. Equipment -- 5.2.2. Buffers and Reagents -- 5.2.3. Procedure -- 5.2.4. Notes -- 5.3. Solubilization and Pulldown of Biotin-Labeled Chromatin -- 5.3.1. Equipment -- 5.3.2. Buffers and Reagents -- 5.3.3. Procedure -- 5.3.4. Notes -- 5.4. Protein Elution and Quantitative Western Analysis -- 5.4.1. Equipment -- 5.4.2. Buffers and Reagents -- 5.4.3. Procedure -- 5.4.4. Notes -- 5.5. Troubleshooting -- Acknowledgments -- References -- Chapter Two: Proteomic Analyses of the Eukaryotic Replication Machinery -- 1. Introduction -- 2. Replisome Purification Approaches -- 2.1. Isolation of Proteins on Nascent DNA -- 2.1.1. iPOND-Label-Free MS -- 2.1.2. iPOND-iTRAQ-MS -- 2.1.3. iPOND-SILAC-MS -- 2.1.4. Variations on iPOND -- 2.2. Nascent Chromatin Capture -- 2.2.1. Comparison of iPOND and NCC -- 3. Additional Applications. , 3.1. Purifying Replication Stress Response Proteins -- 3.2. Analysis of Chromatin Deposition and Maturation -- 3.3. Analysis of Viral Replication and Other Opportunities -- 4. Summary and Conclusions -- Acknowledgments -- References -- Chapter Three: DNA Fiber Analysis: Mind the Gap! -- 1. Introduction -- 2. Preparation of DNA Fibers -- 2.1. Pulse-Labeling Replication Forks In Vivo -- 2.2. Preparation of DNA Fibers Using DNA Combing and maRTA -- 2.2.1. Preparation of Fibers Using DNA Combing Machine -- 2.2.2. Preparation of Fibers Using Capillary Force (or maRTA) -- 2.3. Preparation of DNA Fibers Using DNA Spreading -- 2.4. Data Acquisition and Analysis -- 3. Biological Questions and Labeling Schemes -- 3.1. Replication Fork Velocity and Symmetry -- 3.2. New Origin Firing and Interorigin Distance -- 3.3. Replication Fork Restart and Progression Upon Treatment With Genotoxic Agents -- 3.3.1. Global Replication-Stalling Drugs -- 3.3.2. DNA-Damaging Agents -- 3.4. Nucleolytic Degradation of Nascent DNA (or Resection) -- 4. Application to Study Postreplication Repair/Gap-Filling Mechanism -- 4.1. Detection of ssDNA Gaps on Ongoing Forks -- 4.1.1. Material, Solutions, and Reagents -- 4.1.2. Procedure -- 4.1.3. Data Analysis -- 4.2. Detection of Postreplication Repair Tracts (or Gap Filling) -- 4.2.1. Reagents -- 4.2.2. Protocol -- 4.2.3. Data Analysis -- 5. Concluding Remarks and Future Directions -- Acknowledgments -- References -- Chapter Four: Comet-FISH for Ultrasensitive Strand-Specific Detection of DNA Damage in Single Cells -- 1. Introduction -- 2. Design and Synthesis of Strand-Specific FISH Probes -- 2.1. Gene or Sequence Selection -- 2.2. Synthesis of Probes by PCR -- 2.3. Purification of Single-Stranded FISH Probes -- 2.4. Labeling of Single-Stranded Probes With Fluorophores -- 3. Comet Assay -- 4. Hybridization. , 5. Counterstaining and Imaging -- 6. Analysis of Strand-Specific Repair -- 7. Analysis of GGR -- 8. Conclusions -- Acknowledgments -- References -- Chapter Five: Examining DNA Double-Strand Break Repair in a Cell Cycle-Dependent Manner -- 1. Introduction -- 2. Dynamics of Repair Proteins to Laser-Generated DSBs -- 2.1. Transient Expression of YFP-Tagged Ku80 and DsRed-Tagged PCNA -- 2.1.1. Equipment -- 2.1.2. Buffers and Reagents -- 2.1.3. Procedure -- 2.1.4. Notes -- 2.2. Microscope and Laser-Irradiation Setup -- 2.2.1. Equipment -- 2.2.2. Procedure -- 2.3. DSB Repair Kinetics With Laser Microirradiation -- 2.3.1. Procedure -- 2.4. Calculation of Relative Fluorescent Intensity for Protein Recruitment Kinetics -- 2.4.1. Procedure -- 2.5. Enhancement of Laser Microirradiation With DNA Photosensitizer -- 3. Cell Cycle-Specific Immunofluorescence Assays to Examine NHEJ, DNA End Resection, and Ongoing HR -- 3.1. Pulse-Labeling Cells With EdU to Allow Differentiation of Cell Cycle Stages -- 3.1.1. Buffers and Reagents -- 3.1.2. Procedure -- 3.2. Monitoring NHEJ in G1 Phase of the Cell Cycle -- 3.2.1. Equipment -- 3.2.2. Buffers and Reagents -- 3.2.3. Procedure -- 3.2.4. Notes -- 3.3. Monitoring DNA End Resection or Ongoing HR in Mid-S Phase of the Cell Cycle -- 3.3.1. Equipment -- 3.3.2. Reagents and Buffers -- 3.3.3. Procedure -- 3.4. Quantification of Foci -- 3.4.1. Equipment -- 3.4.2. Procedure -- 4. Determination of DNA Repair Capacity in Different Phases of the Cell Cycle -- 4.1. Cell Synchronization Utilizing Double-Thymidine Block -- 4.1.1. Buffers and Reagents -- 4.1.2. Procedure -- 4.1.3. Notes -- 4.2. Cell Cycle Analysis by Propidium Iodide Staining Followed by Flow Cytometry -- 4.2.1. Equipment -- 4.2.2. Buffers and Reagents -- 4.2.3. Procedure -- 4.2.4. Notes. , 4.3. Survival Assay With Various Fractions of Synchronous Cells Obtained From Double-Thymidine Block Method -- 4.3.1. Buffers and Reagents -- 4.3.2. Procedure -- 4.3.3. Notes -- Acknowledgments -- References -- Chapter Six: Base Excision Repair Variants in Cancer -- 1. Introduction -- 2. Identification and Prioritization of Base Excision Repair Variants Using In Silico Methods -- 2.1. In Silico Data Analysis -- 2.2. Variant Features -- 2.3. Preliminary Evaluation of Variants -- 3. Cellular Characterization of BER Variants -- 3.1. Subcloning in the pRVY-tet Vector -- 3.1.1. Equipment -- 3.1.2. Buffers and Reagents -- 3.1.3. Procedure -- 3.1.4. Notes -- 3.2. Generation of Stable MCF10A Pools and Clones Expressing the BER Variant of Interest -- 3.2.1. Equipment -- 3.2.2. Buffers and Reagents -- 3.2.3. Procedure -- 3.2.4. Notes -- 3.3. Cellular Transformation in Human Cells -- 3.3.1. Equipment -- 3.3.2. Buffers and Reagents -- 3.3.3. Procedure -- 3.3.4. Notes -- 3.4. Genomic Instability and Mutagenesis -- 3.4.1. Equipment -- 3.4.2. Buffers and Reagents -- 3.4.3. Procedure -- 3.4.4. Notes -- 3.5. Preliminary Mechanistic Analysis -- 3.5.1. Equipment -- 3.5.2. Buffers and Reagents -- 3.5.3. Procedure -- 3.5.4. Notes -- References -- Chapter Seven: Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli -- 1. Introduction -- 2. Description of the Reporter Systems -- 2.1. TEMrev-GFP -- 2.2. sfGFP-TEM -- 2.3. TEMrev-GFPrev -- 3. Experimental Validation -- 3.1. Mutagenesis -- 3.2. Readout on Solid Media -- 3.3. Readout in Liquid Media -- 3.4. Continuous Mutagenesis Detection -- 4. Discussion -- 5. Materials -- 5.1. Transformation and Mutagenesis -- 5.2. Washing Plates -- 5.3. Readout (Plates) -- 5.4. Readout (Liquid Culture Assay) -- 5.5. Plasmid Recovery -- 5.6. Sequencing Plasmids of Interest -- 6. Methods -- 6.1. Transformation and Mutagenesis. , 6.1.1. Preparation of Competent Cells for Chemical Transformation -- 6.1.2. Chemical Transformation -- 6.1.3. Mutagenesis -- 6.2. Washing Plates -- 6.3. Readout (Plates) -- 6.4. Readout (Liquid Culture Assay) -- 6.5. Plasmid Recovery -- 6.6. Sequencing Plasmids of Interest -- Acknowledgments -- References -- Chapter Eight: Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine -- 1. Introduction -- 2. Method A: Sequencing OG by Conversion to a Deletion Signature -- 2.1. Theory -- 2.2. Equipment -- 2.3. Materials -- 2.4. Notes -- 2.5. Procedure -- 3. Method B: Sequencing OG by Conversion to an Unnatural DNA Base Pair -- 3.1. Theory -- 3.2. Equipment -- 3.3. Materials -- 3.4. Notes -- 3.5. Procedure -- 4. Method C: Sequencing OG by OG-Seq -- 4.1. Theory -- 4.2. Equipment -- 4.3. Materials -- 4.4. Notes -- 4.5. Procedure -- 5. Comparison and Contrast of the three OG-Sequencing Methods -- 5.1. Sequencing Platforms -- 5.2. DNA Contexts for Sequencing OG -- 5.3. Resolution for Sequencing OG -- 5.4. Ability to Sequence More Than One OG Per Strand -- 5.5. Custom Chemical Synthesis Requirements -- Acknowledgments -- References -- Chapter Nine: Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability -- 1. Introduction -- 2. Preparation of Interphase Egg Extract -- 2.1. Equipment -- 2.2. Buffers and Reagents -- 2.3. Procedures -- 2.3.1. Female Frogs Injection -- 2.3.2. Egg Extract Preparation -- 2.4. Notes -- 3. Xenopus laevis Demembranated Sperm Nuclei Preparation -- 3.1. Equipment -- 3.2. Buffers and Reagents -- 3.3. Procedures -- 3.3.1. Male Frogs Injection -- 3.3.2. Preparation of Demembranated Sperm DNA -- 3.4. Notes -- 4. Chromatin Binding of DNA Replication Factors -- 4.1. Equipment -- 4.2. Buffers and Reagents -- 4.3. Procedure -- 5. Activation of ATM-, ATR-, and DNA-PK-Dependent DNA Damage Response. , 5.1. Introduction.

Language: English

UID:

Format: 1 Online-Ressource (xviii, 460 Seiten) , Illustrationen, Diagramme

Edition: First edition

ISBN: 9780128118467 , 9780128118474 , 0128118474

Series Statement: Methods in enzymology volume 591

Note: Includes indexes

Additional Edition: ISBN 9780128118467

Additional Edition: Erscheint auch als Druck-Ausgabe DNA repair enzymes Cambridge, MA : Academic Press, an imprint of Elsevier, 2017 ISBN 9780128118467

Language: English

Subjects: Biology

Keywords: DNS-Reparatur ; Enzym ; Aufsatzsammlung

URL: Volltext

URL: Volltext

URL: Volltext

URL: https://www.elsevier.com/books/dna-repair-enzymes-cell-molecular-and-chemical-biology/eichman/978-0-12-811846-7

UID:

Format: 1 online resource (461 pages).

Edition: First edition.

ISBN: 0-12-811847-4 , 0-12-811846-6

Series Statement: Methods in Enzymology ; Volume 591

Note: Front Cover -- DNA Repair Enzymes: Cell, Molecular, and Chemical Biology -- Copyright -- Contents -- Contributors -- Preface -- Chapter One: Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells -- 1. Introduction -- 2. Suspension Cell Growth and Handling for iPOND and aniPOND -- 2.1. Optimizing Growth Conditions to Obtain iPOND/aniPOND Cell Numbers -- 2.2. Handling of Suspension Cells During Pulse and Chase -- 3. Optimizations to the aniPOND Protocol to Increase Functionality -- 3.1. Optimized Sonication Regimen -- 3.2. Preventing Chromatin Precipitation in Sonicated Chromatin -- 3.3. Eliminating Sources of Background -- 3.4. Optimization Limitations -- 4. Comparison of iPOND and aniPOND in Suspension Cells -- 5. Protocol for Optimized aniPOND in Suspension Cells -- 5.1. Suspension Cell Growth to aniPOND Experimental Numbers -- 5.1.1. Equipment -- 5.1.2. Buffers and Reagents -- 5.1.3. Procedure -- 5.1.4. Notes -- 5.2. EdU Pulse, Thymidine Chase, and Click Reaction -- 5.2.1. Equipment -- 5.2.2. Buffers and Reagents -- 5.2.3. Procedure -- 5.2.4. Notes -- 5.3. Solubilization and Pulldown of Biotin-Labeled Chromatin -- 5.3.1. Equipment -- 5.3.2. Buffers and Reagents -- 5.3.3. Procedure -- 5.3.4. Notes -- 5.4. Protein Elution and Quantitative Western Analysis -- 5.4.1. Equipment -- 5.4.2. Buffers and Reagents -- 5.4.3. Procedure -- 5.4.4. Notes -- 5.5. Troubleshooting -- Acknowledgments -- References -- Chapter Two: Proteomic Analyses of the Eukaryotic Replication Machinery -- 1. Introduction -- 2. Replisome Purification Approaches -- 2.1. Isolation of Proteins on Nascent DNA -- 2.1.1. iPOND-Label-Free MS -- 2.1.2. iPOND-iTRAQ-MS -- 2.1.3. iPOND-SILAC-MS -- 2.1.4. Variations on iPOND -- 2.2. Nascent Chromatin Capture -- 2.2.1. Comparison of iPOND and NCC -- 3. Additional Applications. , 3.1. Purifying Replication Stress Response Proteins -- 3.2. Analysis of Chromatin Deposition and Maturation -- 3.3. Analysis of Viral Replication and Other Opportunities -- 4. Summary and Conclusions -- Acknowledgments -- References -- Chapter Three: DNA Fiber Analysis: Mind the Gap! -- 1. Introduction -- 2. Preparation of DNA Fibers -- 2.1. Pulse-Labeling Replication Forks In Vivo -- 2.2. Preparation of DNA Fibers Using DNA Combing and maRTA -- 2.2.1. Preparation of Fibers Using DNA Combing Machine -- 2.2.2. Preparation of Fibers Using Capillary Force (or maRTA) -- 2.3. Preparation of DNA Fibers Using DNA Spreading -- 2.4. Data Acquisition and Analysis -- 3. Biological Questions and Labeling Schemes -- 3.1. Replication Fork Velocity and Symmetry -- 3.2. New Origin Firing and Interorigin Distance -- 3.3. Replication Fork Restart and Progression Upon Treatment With Genotoxic Agents -- 3.3.1. Global Replication-Stalling Drugs -- 3.3.2. DNA-Damaging Agents -- 3.4. Nucleolytic Degradation of Nascent DNA (or Resection) -- 4. Application to Study Postreplication Repair/Gap-Filling Mechanism -- 4.1. Detection of ssDNA Gaps on Ongoing Forks -- 4.1.1. Material, Solutions, and Reagents -- 4.1.2. Procedure -- 4.1.3. Data Analysis -- 4.2. Detection of Postreplication Repair Tracts (or Gap Filling) -- 4.2.1. Reagents -- 4.2.2. Protocol -- 4.2.3. Data Analysis -- 5. Concluding Remarks and Future Directions -- Acknowledgments -- References -- Chapter Four: Comet-FISH for Ultrasensitive Strand-Specific Detection of DNA Damage in Single Cells -- 1. Introduction -- 2. Design and Synthesis of Strand-Specific FISH Probes -- 2.1. Gene or Sequence Selection -- 2.2. Synthesis of Probes by PCR -- 2.3. Purification of Single-Stranded FISH Probes -- 2.4. Labeling of Single-Stranded Probes With Fluorophores -- 3. Comet Assay -- 4. Hybridization. , 5. Counterstaining and Imaging -- 6. Analysis of Strand-Specific Repair -- 7. Analysis of GGR -- 8. Conclusions -- Acknowledgments -- References -- Chapter Five: Examining DNA Double-Strand Break Repair in a Cell Cycle-Dependent Manner -- 1. Introduction -- 2. Dynamics of Repair Proteins to Laser-Generated DSBs -- 2.1. Transient Expression of YFP-Tagged Ku80 and DsRed-Tagged PCNA -- 2.1.1. Equipment -- 2.1.2. Buffers and Reagents -- 2.1.3. Procedure -- 2.1.4. Notes -- 2.2. Microscope and Laser-Irradiation Setup -- 2.2.1. Equipment -- 2.2.2. Procedure -- 2.3. DSB Repair Kinetics With Laser Microirradiation -- 2.3.1. Procedure -- 2.4. Calculation of Relative Fluorescent Intensity for Protein Recruitment Kinetics -- 2.4.1. Procedure -- 2.5. Enhancement of Laser Microirradiation With DNA Photosensitizer -- 3. Cell Cycle-Specific Immunofluorescence Assays to Examine NHEJ, DNA End Resection, and Ongoing HR -- 3.1. Pulse-Labeling Cells With EdU to Allow Differentiation of Cell Cycle Stages -- 3.1.1. Buffers and Reagents -- 3.1.2. Procedure -- 3.2. Monitoring NHEJ in G1 Phase of the Cell Cycle -- 3.2.1. Equipment -- 3.2.2. Buffers and Reagents -- 3.2.3. Procedure -- 3.2.4. Notes -- 3.3. Monitoring DNA End Resection or Ongoing HR in Mid-S Phase of the Cell Cycle -- 3.3.1. Equipment -- 3.3.2. Reagents and Buffers -- 3.3.3. Procedure -- 3.4. Quantification of Foci -- 3.4.1. Equipment -- 3.4.2. Procedure -- 4. Determination of DNA Repair Capacity in Different Phases of the Cell Cycle -- 4.1. Cell Synchronization Utilizing Double-Thymidine Block -- 4.1.1. Buffers and Reagents -- 4.1.2. Procedure -- 4.1.3. Notes -- 4.2. Cell Cycle Analysis by Propidium Iodide Staining Followed by Flow Cytometry -- 4.2.1. Equipment -- 4.2.2. Buffers and Reagents -- 4.2.3. Procedure -- 4.2.4. Notes. , 4.3. Survival Assay With Various Fractions of Synchronous Cells Obtained From Double-Thymidine Block Method -- 4.3.1. Buffers and Reagents -- 4.3.2. Procedure -- 4.3.3. Notes -- Acknowledgments -- References -- Chapter Six: Base Excision Repair Variants in Cancer -- 1. Introduction -- 2. Identification and Prioritization of Base Excision Repair Variants Using In Silico Methods -- 2.1. In Silico Data Analysis -- 2.2. Variant Features -- 2.3. Preliminary Evaluation of Variants -- 3. Cellular Characterization of BER Variants -- 3.1. Subcloning in the pRVY-tet Vector -- 3.1.1. Equipment -- 3.1.2. Buffers and Reagents -- 3.1.3. Procedure -- 3.1.4. Notes -- 3.2. Generation of Stable MCF10A Pools and Clones Expressing the BER Variant of Interest -- 3.2.1. Equipment -- 3.2.2. Buffers and Reagents -- 3.2.3. Procedure -- 3.2.4. Notes -- 3.3. Cellular Transformation in Human Cells -- 3.3.1. Equipment -- 3.3.2. Buffers and Reagents -- 3.3.3. Procedure -- 3.3.4. Notes -- 3.4. Genomic Instability and Mutagenesis -- 3.4.1. Equipment -- 3.4.2. Buffers and Reagents -- 3.4.3. Procedure -- 3.4.4. Notes -- 3.5. Preliminary Mechanistic Analysis -- 3.5.1. Equipment -- 3.5.2. Buffers and Reagents -- 3.5.3. Procedure -- 3.5.4. Notes -- References -- Chapter Seven: Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli -- 1. Introduction -- 2. Description of the Reporter Systems -- 2.1. TEMrev-GFP -- 2.2. sfGFP-TEM -- 2.3. TEMrev-GFPrev -- 3. Experimental Validation -- 3.1. Mutagenesis -- 3.2. Readout on Solid Media -- 3.3. Readout in Liquid Media -- 3.4. Continuous Mutagenesis Detection -- 4. Discussion -- 5. Materials -- 5.1. Transformation and Mutagenesis -- 5.2. Washing Plates -- 5.3. Readout (Plates) -- 5.4. Readout (Liquid Culture Assay) -- 5.5. Plasmid Recovery -- 5.6. Sequencing Plasmids of Interest -- 6. Methods -- 6.1. Transformation and Mutagenesis. , 6.1.1. Preparation of Competent Cells for Chemical Transformation -- 6.1.2. Chemical Transformation -- 6.1.3. Mutagenesis -- 6.2. Washing Plates -- 6.3. Readout (Plates) -- 6.4. Readout (Liquid Culture Assay) -- 6.5. Plasmid Recovery -- 6.6. Sequencing Plasmids of Interest -- Acknowledgments -- References -- Chapter Eight: Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine -- 1. Introduction -- 2. Method A: Sequencing OG by Conversion to a Deletion Signature -- 2.1. Theory -- 2.2. Equipment -- 2.3. Materials -- 2.4. Notes -- 2.5. Procedure -- 3. Method B: Sequencing OG by Conversion to an Unnatural DNA Base Pair -- 3.1. Theory -- 3.2. Equipment -- 3.3. Materials -- 3.4. Notes -- 3.5. Procedure -- 4. Method C: Sequencing OG by OG-Seq -- 4.1. Theory -- 4.2. Equipment -- 4.3. Materials -- 4.4. Notes -- 4.5. Procedure -- 5. Comparison and Contrast of the three OG-Sequencing Methods -- 5.1. Sequencing Platforms -- 5.2. DNA Contexts for Sequencing OG -- 5.3. Resolution for Sequencing OG -- 5.4. Ability to Sequence More Than One OG Per Strand -- 5.5. Custom Chemical Synthesis Requirements -- Acknowledgments -- References -- Chapter Nine: Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability -- 1. Introduction -- 2. Preparation of Interphase Egg Extract -- 2.1. Equipment -- 2.2. Buffers and Reagents -- 2.3. Procedures -- 2.3.1. Female Frogs Injection -- 2.3.2. Egg Extract Preparation -- 2.4. Notes -- 3. Xenopus laevis Demembranated Sperm Nuclei Preparation -- 3.1. Equipment -- 3.2. Buffers and Reagents -- 3.3. Procedures -- 3.3.1. Male Frogs Injection -- 3.3.2. Preparation of Demembranated Sperm DNA -- 3.4. Notes -- 4. Chromatin Binding of DNA Replication Factors -- 4.1. Equipment -- 4.2. Buffers and Reagents -- 4.3. Procedure -- 5. Activation of ATM-, ATR-, and DNA-PK-Dependent DNA Damage Response. , 5.1. Introduction.

Language: English

UID:

Format: 1 online resource (xix, 431 pages) : , illustrations.

ISBN: 0-323-90733-4

Series Statement: Methods in enzymology ; Volume 661

Language: English

UID:

Format: 1 online resource (xix, 431 pages) : , illustrations.

ISBN: 0-323-90733-4

Series Statement: Methods in enzymology ; Volume 661

Language: English

UID:

Format: 1 Online-Ressource (xx, 492 Seiten) , Illustrationen, Diagramme

Edition: First edition

ISBN: 9780128125151 , 9780128125168

Series Statement: Methods in enzymology volume 592

Content: DNA Repair Enzymes, Part B, Volume 592,is the latest volume in the Methods in Enzymologyseries and the first part of a thematic that focuses DNA Repair Enzymes.Includes contributions from leading authorities working in enzymologyFocuses on DNA repair enzymesInforms and updates on all the latest developments in the field of enzymology

Content: Title page -- Table of Contents -- Copyright -- Contributors -- Preface -- Chapter One: MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes -- Abstract -- 1 Introduction -- 2 Entry Cloning Into MacroBac Series 11 or 438 by LIC -- 3 Building Complexes by Restriction and Ligation: MacroBac Series 11 -- 4 Building Complexes by LIC: MacroBac Series 438 -- 5 Bacmid Preparation and Virus Production -- 6 Protein Expression and Viral Stock Maintenance -- 7 Trouble Shooting -- 8 Future Considerations -- Acknowledgments -- Chapter Two: Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes -- Abstract -- 1 Introduction -- 2 Cloning -- 3 Case Studies -- 4 Nucleosome Sliding and Histone Exchange Kinetic Assays -- 5 Nucleosome Sliding by Recombinant hINO80 Chromatin Remodeling Complex -- 6 Histone Exchange by Recombinant ySWR1 Chromatin Remodeling Complex -- Chapter Three: Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR -- Abstract -- 1 Introduction -- 2 Small-Angle X-Ray Scattering -- 3 Nuclear Magnetic Resonance -- 4 Summary and Conclusion-Using SAX and NMR to Characterize Architectural Remodeling -- Chapter Four: Use of Single-Cysteine Variants for Trapping Transient States in DNA Mismatch Repair -- Abstract -- 1 Introduction -- 2 Generation of Defined Cysteine Variants -- 3 Cross-linking of Defined Cysteine Variants -- 4 Use of Single-Cysteine Variants to Study Conformational Changes in DNA Mismatch Repair -- 5 Concluding Remarks -- Acknowledgments -- Chapter Five: Expression and Structural Analyses of Human DNA Polymerase θ (POLQ) -- Abstract -- 1 Introduction -- 2 Purification and Structure Determination of Human Pol θ -- 3 Crystallization and Structure Determination of Human Pol θ -- 4 Conclusions -- Acknowledgments

Content: Chapter Six: Structural Studies of RNases H2 as an Example of Crystal Structure Determination of Protein-Nucleic Acid Complexes -- Abstract -- 1 Introduction -- 2 Purification of RNases H2 -- 3 Biochemical Assays for RNases H2 -- 4 General Considerations in the Crystallization of Protein-Nucleic Acid Complexes -- 5 Crystallization and Structure Determination of RNases H2 -- 6 Concluding Remarks -- Acknowledgment -- Chapter Seven: DNA-PKcs, Allostery, and DNA Double-Strand Break Repair: Defining the Structure and Setting the Stage -- Abstract -- 1 Introduction -- 2 Defining the Structure of DNA-PKcs -- 3 Exploiting Multiple Se-Met Substitutions -- 4 Using Se-Met Sites to Check Sequence Registration -- 5 DNA-PKcs as a Stage for the Assembly of the Actors in NHEJ -- Acknowledgments -- Chapter Eight: Single-Particle Electron Microscopy Analysis of DNA Repair Complexes -- Abstract -- 1 Introduction -- 2 Sample Preparation -- 3 Grid Preparation -- 4 Data Collection -- 5 Data Processing -- Acknowledgments -- Chapter Nine: Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair -- Abstract -- 1 Introduction -- 2 Methods to Study DNA Repair Complexes With AFM -- 3 Image Analysis -- 4 DREEM Imaging -- 5 Complementary Techniques -- 6 Conclusions -- Acknowledgments -- Chapter Ten: Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time -- Abstract -- 1 Introduction -- 2 Preparation of Defined Lesion Substrates for AFM and DNA Tightrope Assay -- 3 Atomic Force Microscopy -- 4 Single-Molecule DNA Tightrope Assay -- 5 Conclusions -- Acknowledgments -- Chapter Eleven: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination -- Abstract -- 1 Introduction -- 2 Methods -- 3 Applications -- 4 Notes

Content: Acknowledgments -- Chapter Twelve: Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography -- Abstract -- 1 Introduction -- 2 DNA Polymerase η -- 3 Ribonuclease H1 -- 4 Endonuclease V -- 5 X-Ray Data Processing -- 6 Applications and Future Prospects -- Acknowledgments -- Chapter Thirteen: Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases -- Abstract -- 1 Introduction -- 2 Purification and Structures of Nonclassical Polymerases -- 3 Catalytic Activities of Nonclassical Polymerases -- 4 Interactions of Nonclassical Polymerases -- 5 Conclusions -- Acknowledgments -- Chapter Fourteen: Kinetic Methods for Studying DNA Glycosylases Functioning in Base Excision Repair -- Abstract -- 1 Introduction -- 2 Kinetic Methods to Study Catalysis by DNA Glycosylases -- 3 Analytical Methods for Monitoring DNA Glycosylase Activity -- 4 Determining Rate Constants From Kinetics Experiments -- Acknowledgment -- Chapter Fifteen: Transient Kinetic Methods for Mechanistic Characterization of DNA Binding and Nucleotide Flipping -- Abstract -- 1 Introduction -- 2 General Considerations for Monitoring Binding -- 3 Association Kinetics -- 4 Comparison of Binding and Flipping Mechanisms for AAG and AlkA -- 5 Use of Berkeley Madonna for Evaluating Kinetic Models -- 6 Useful Equations for Kinetic Analysis -- Acknowledgments -- Chapter Sixteen: What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses -- Abstract -- 1 Introduction -- 2 Metabolic Signaling: NAD and DDR by PARP, PARG, and AIF -- 3 DNA Double-Strand Break Responses -- 4 Taking Structural Mechanisms Into Cells by Advanced Imaging -- 5 What's Ahead -- Acknowledgments -- Author Index -- Subject Index

Additional Edition: ISBN 9780128125151

Additional Edition: Erscheint auch als Druck-Ausgabe DNA repair enzymes Cambridge, MA : Academic Press, an imprint of Elsevier, 2017 ISBN 0128125152

Additional Edition: ISBN 9780128125151

Additional Edition: Print version Eichman, Brandt DNA Repair Enzymes: Structure, Biophysics, and Mechanism Saint Louis : Elsevier Science,c2017 ISBN 9780128125151

Language: English

Subjects: Biology

Keywords: DNS-Reparatur ; Enzym ; Aufsatzsammlung

URL: Volltext

URL: Volltext

URL: Volltext

URL: https://www.elsevier.com/books/dna-repair-enzymes-structure-biophysics-and-mechanism/eichman/978-0-12-812515-1