Mechanisms of DNA recombination and genome rearrangements : intersection between homologous recombination, DNA replication and DNA repair / edited by Maria Spies, Anna Alkova.

Format:

Book

Contributor:

Spies, Maria, editor.

Alkova, Anna, editor.

Series:

Methods in enzymology ; Volume 601.

Methods in Enzymology, 0276-6879 ; Volume 601

Language:

English

Subjects (All):

Genetic recombination.

Recombinant DNA.

Physical Description:

1 online resource (436 pages) : illustrations (some color).

Place of Publication:

Cambridge, Massachusetts : Academic Press, 2018.

Summary:

Mechanisms of DNA Recombination and Genome Rearrangements: Intersection between Homologous Recombination, DNA Replication and DNA Repair, Volume 601, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field.Homologous genetic recombination remains the most enigmatic process in DNA metabolism. The molecular machines of recombination preserve the integrity of the genetic material in all organisms and generate genetic diversity in evolution. The same molecular machines that support genetic integrity by orchestrating accurate repair of the most deleterious DNA lesions, however, also promote survival of cancerous cells and emergence of radiation and chemotherapy resistance. This two-volume set offers a comprehensive set of cutting edge methods to study various aspects of homologous recombination and cellular processes that utilize the enzymatic machinery of recombination. The chapters are written by the leading researches and cover a broad range of topics from the basic molecular mechanisms of recombinational proteins and enzymes to emerging cellular techniques and drug discovery efforts.- contributions by the leading experts in the field of DNA repair, recombination, replication and genome stability- documents cutting edge methods

Contents:

Front Cover

Mechanisms of DNA Recombination and Genome Rearrangements: Intersection between Homologous Recombination, DNA Replication a ...

Copyright

Contents

Contributors

Preface

Chapter One: Using the Flp Recombinase to Induce Site-Specific Protein-DNA Nicks

1. Introduction

2. Generation of a Basic FLP-Nick Yeast Strain

2.1. Integration of the flpH305L-Containing Plasmid and Cure of the 2 μ Plasmid

2.1.1. Equipment

2.1.2. Buffers and Reagents

2.1.3. Procedure

2.1.4. Notes

2.2. Generation of FRT-Containing Plasmids and Insertion of the FRT Site Into the Genome

2.2.1. Equipment

2.2.2. Buffers and Reagents

2.2.3. Procedure

2.2.4. Notes

3. Growth of FLP-Nick Yeast Strains and Induction of flpH305L

3.1. Equipment

3.2. Buffers and Reagents

3.3. Procedure

3.4. Notes

4. Alkaline Denaturing Gel Analysis to Measure Nicking Activity

4.1. Equipment

4.2. Buffers and Reagents

4.3. Procedure

4.3.1. Growth of Yeast Cells

4.3.2. Preparation and Enzymatic Digestion of Genomic DNA

4.3.3. Preparation and Running Alkaline Gel

4.4. Notes

5. DSB Assay Using Agarose Plugs to Detect DSB Formation and Repair

5.1. Equipment

5.2. Buffers and Reagents

5.3. Procedure

5.3.1. Growth of Yeast Cells

5.3.2. Treatment of Aliquots/Plugs

5.3.3. Digestion of Plugs

5.3.4. Gel Casting and Gel Electrophoresis

6. ChIP Without Crosslinking to Detect FlpH305L Binding and Clevage at the FRT Site

6.1. Equipment

6.2. Buffers and Reagents

6.3. Procedure

6.3.1. Growth of Yeast Cells

6.3.2. Preparation of Buffers for ChIP

6.3.3. Coupling of Antibody to Beads

6.3.4. Making the Extract

6.3.5. Quantitative PCR

6.4. Notes

References

Chapter Two: A Proximity Ligation-Based Method for Quantitative Measurement of D-Loop Extension in S. cerevisiae.

1. Introduction

1.1. DNA Synthesis in Homologous Recombination

1.2. Current Method for Detection of Initiation of Recombination-Associated DNA Synthesis and Its Limitations

2. Methods

2.1. Materials

2.1.2. Reagents

2.1.3. S. cerevisiae Strains

2.2. The D-Loop Extension Assay

2.2.1. Rationale

2.2.2. Procedure

2.2.2.1. Pregrowth, Site-Specific DSB Induction, and Sample Collection

2.2.2.2. Yeast Cells Spheroplasting and Restriction Sites Restoration

2.2.2.3. Restriction Digestion and Ligation

2.2.2.4. DNA Purification

2.2.2.5. Quantitative PCR

2.2.3. Analysis, Normalization, and Controls

3. Results and Discussion

3.1. DLE Kinetics in Wild-Type Cells and Genetics Requirements

3.2. Restriction Sites Restoration Allows to Distinguish DNA Synthesis Initiation and BIR Product Formation

3.3. Possible Refinements of the DLE Assay

4. Conclusion

Acknowledgments

Funding

Chapter Three: Inducing and Detecting Mitotic DNA Synthesis at Difficult-to-Replicate Loci

2. Materials

2.1. Cell Lines

2.2. Reagents

2.3. Equipment/Tools

3. Method

3.1. To Analyze MiDAS in Prometaphase

3.2. To Analyze EdU on Metaphase Chromosome Spreads

4. Notes

5. Conclusions

Chapter Four: An Approach to Detect and Study DNA Double-Strand Break Repair by Transcript RNA Using a Spliced-Antisense ...

2. Repair Systems to Detect RNA-Templated Repair

2.1. The trans System

2.2. The cis System

2.3. Note

3. Assays to Detect RNA-Templated DSB Repair in cis and trans Systems

3.1. Patching Assay

3.1.1. Materials

3.1.2. Procedure

3.1.2.1. Note

3.2. Fluctuation Assay

3.2.1. Materials

3.2.2. Procedure

3.2.2.1. Notes

References.

Chapter Five: Assaying Repair at DNA Nicks

2. Chromosomal Reporters for Analysis of Repair by Flow Cytometry

2.1. Chromosomal Reporters to Quantify Repair by Exogenous Donors

2.2. Traffic Light Reporter Construct

2.2.1. Key Genetic Elements of the Reporter

2.2.2. Scoring HDR

2.2.3. Scoring mutEJ

2.3. Donors for HDR

3. Cell Lines and Populations Harboring Chromosomal Reporters

3.1. Generating Cell Lines and Populations Harboring Chromosomal Reporters

3.1.1. Lentivirus Production and Transfection

3.1.2. Generating Cell Lines or Populations Harboring the Virus

3.2. Storage and Quality Control of Early Passage Cells

4. Assaying Repair Frequencies

4.1. Cell Culture, siRNA Depletion, and Ectopic Protein Expression

4.1.1. Cell Culture and Fixation

4.1.2. Assays Involving siRNA Depletion

4.1.3. Assays Involving Ectopic Protein Expression

4.2. Monitoring siRNA Depletion or Ectopic Protein Expression

4.3. Identification of Transfectants and Determination of Transfection Frequency

4.4. Timing of Cell Harvest and Analysis

4.5. Flow Cytometry

4.5.1. Cell Analysis and Signal Recording

4.5.2. Data Analysis Using FlowJo (Tree Star, Ashland, OR)

4.6. PCR Assays

Chapter Six: Recovery of Alternative End-Joining Repair Products From Drosophila Embryos

1.1. A Comparison of Methods for Recovering Alt-EJ Repair Products

2. Preparing and Injecting Plasmids Into Embryos

2.1. Overview of the Process

2.2. Reagent Preparation

2.2.1. Plasmid Preparation

2.2.2. Injection Mix Preparation

2.3. Embryo Collection

2.3.1. Procedure

2.4. Dechorionation of Embryos

2.4.1. Materials Needed

2.4.2. Procedure

2.5. Positioning Embryos for Injection

2.6. Microinjection of Embryos

2.6.1. Materials Needed.

2.6.2. Preparing Needles

2.6.3. Injecting the Embryos

3. Plasmid Recovery and Repair Product Sequencing

3.1. Recovery of Injected Embryos

3.2. Plasmid DNA Recovery by Alkaline Lysis

3.2.1. Reagents

3.3. Bacterial Transformation and Sanger Sequencing

3.4. Next-Generation Amplicon Sequencing

3.4.1. Materials Needed

3.4.2. Amplicon Sequencing Approach

4. Data Analysis

4.1. Analyzing Reads From Amplicon Sequencing

5. Summary and Conclusion

Chapter Seven: High-Throughput Analysis of DNA Break-Induced Chromosome Rearrangements by Amplicon Sequencing

2. Creating Complex Libraries of DSB Repair Junctions

3. Aligning Sequence Reads

3.1. Merging Paired Reads

3.2. Trimming and Padding fastq Files

3.3. Choosing an Aligner

3.3.1. Mapping With Geneious 10

3.3.2. Mapping With CLC Genomics Workbench 7.5

3.3.3. Mapping With Bowtie2

3.3.4. Mapping With BWA

4. Breakpoint Analysis

4.1. Running Hi-FiBR

4.2. How Hi-FiBR Works

5. Results of Analysis and Discussion

5.1. Effects of Using Different Aligners for Hi-FiBR Analysis

5.2. Using Different Sequencing Platforms With Hi-FiBR

6. Summary and Conclusion

Chapter Eight: Assaying Mutations Associated With Gene Conversion Repair of a Double-Strand Break

2. Scoring Interchromosomal Template Switching (ICTS) Events

2.1. Equipment

2.2. Solutions and Reagents

2.3. Protocol for Assaying ICTS Mutation Frequency

3. Scoring Quasipalindrome (QP)-Mediated Mutations During DSB Repair

4. Scoring Frameshift Mutations During DSB Repair

4.2. Solutions and Reagents

5. Protocol for Assaying QP and Frameshift Mutation Frequency

Chapter Nine: Investigation of Break-Induced Replication in Yeast

2. Calculating BIR Efficiency

2.1. Equipment and Materials

2.3. Procedure

2.4. Notes

3. Analysis of BIR Kinetics

3.1. Determining BIR Kinetics in a Time-Course Experiment

3.1.1. Equipment

3.1.2. Solutions and Reagents

3.1.3. Procedure

3.1.4. Notes

3.2. Preparing Agarose Plugs With Embedded DNA for CHEF

3.2.1. Equipment

3.2.2. Solutions and Reagents

3.2.3. Procedure

3.2.4. Notes

3.3. Analysis of BIR Products Using CHEF Gel Electrophoresis

3.3.1. Equipment

3.3.2. Solutions and Reagents

3.3.3. Procedure

3.3.4. Notes

3.4. Capillary Transfer and Hybridization

3.4.1. Equipment

3.4.2. Solutions and Reagents

3.4.3. Procedure

3.4.4. Notes

4. Analysis of Mutagenesis Associated With BIR

4.1. Analysis of Frameshift Mutagenesis

4.1.1. Equipment

4.1.2. Solutions and Reagents

4.1.3. Procedure

4.1.4. Notes

4.2. Analysis of Base-Substitution Mutagenesis Associated With BIR

4.2.1. Equipment

4.2.2. Solutions and Reagents

4.2.3. Procedure

4.2.4. Notes

4.3. Analysis of Lys+ and Ura+ Mutations by Sanger Sequencing

4.3.1. Equipment

4.3.2. Solutions and Reagents

4.3.3. Procedure

4.3.4. Notes

4.4. Sequencing of URA3 and LYS2 Sequences Located on Either Donor or Recipient Chromosomes

4.4.1. Equipment

4.4.2. Solutions and Reagents

4.4.3. Procedure

5. Analysis of BIR Structural Intermediates by Two-Dimensional Gel Electrophoresis

5.1. Preparation of Yeast Samples

5.1.1. Equipment

5.1.2. Solutions and Reagents

5.1.3. Procedure

5.1.4. Notes

5.2. DNA Cross-linking

5.2.1. Equipment

5.2.2. Solutions and Reagents

5.2.3. Procedure

5.2.4. Notes

5.3. Extraction of DNA

5.3.1. Equipment.

5.3.2. Solutions and Reagents.

Notes:

Includes bibliographical references at the end of each chapters.

Description based on online resource; title from PDF title page (EBC, viewed April 14, 2018).

ISBN:

9780128139806

0128139803

9780128139790

012813979X