DNA replication-repair interface / edited by Brandt F. Eichman.

Format:

Book

Contributor:

Eichman, Brandt F., editor.

Series:

Methods in enzymology ; Volume 661.

Methods in enzymology ; Volume 661

Language:

English

Subjects (All):

DNA.

Physical Description:

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

Edition:

1st ed.

Place of Publication:

Cambridge, MA : Academic Press, [2021]

Summary:

Replication-Coupled Repair, Volume 661 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of timely topics, including the Repair of replication-born DNA breaks by sister chromatid recombination, High resolution and high throughput DNA cyclization measurements.

Contents:

Intro

The DNA Replication-Repair Interface

Copyright

Contents

Contributors

Preface

Chapter One: Eukaryotic DNA replication with purified budding yeast proteins

1. Introduction

2. Expression of proteins

2.1. Codon optimizations and strains

2.2. Protocols for protein expression

2.2.1. Expression in S. cerevisiae

2.2.1.1. Chemicals

2.2.1.2. Protocol

2.2.2. Expression in E. coli

2.2.2.1. Chemicals

2.2.2.2. Protocol

3. Purification of proteins

3.1. General strategy and buffer compositions

3.1.1. Common equipment and tubes needed for most purification methods

3.1.2. Chromatography columns

3.2. Affinity chromatography

3.2.1. Flag

3.2.1.1. Chemicals

3.2.2. Immobilized metal ion affinity chromatography (IMAC)

3.2.2.1. Chemicals

3.2.3. Calmodulin

3.2.3.1. Chemicals

3.2.4. GST-tag

3.2.4.1. Chemicals

3.2.5. IgG (only for Sld3/Sld7)

3.2.5.1. Chemicals

3.2.6. Elution by TEV protease

3.2.6.1. Chemicals

3.2.7. TEV protease removal by IMAC

3.2.7.1. Chemicals

3.3. Ion exchange chromatography

3.3.1. MonoQ and SP FF

3.3.2. Heparin

3.3.3. Hydroxyapatite

3.3.3.1. Chemicals

3.4. Size exclusion chromatography

3.4.1. Hiload Superdex 16/600 200pg and Superdex 200 increase 10/300 GL

3.5. Dialysis and concentration

3.5.1. Buffer exchange and concentration by dialysis

3.5.1.1. Equipment

3.5.2. Concentration by centrifugal filter column

3.5.2.1. Equipment

3.5.3. Protein concentration determination by SDS-PAGE

3.5.3.1. Equipment

3.5.3.2. Chemicals

3.5.4. Buffers used in the purification of proteins

3.5.4.1. Chemicals

3.6. Protocols for purification of each protein in the replication system

3.6.1. Orc

3.6.2. Cdt1/Mcm2-7

3.6.3. Cdc6

3.6.4. DDK

3.6.5. Sld3/Sld7

3.6.6. Sld2

3.6.7. Cdc45

3.6.8. GINS.

3.6.9. Dpb11

3.6.10. S-CDK

3.6.11. Pol ε

3.6.12. Pol α

3.6.13. RPA

3.6.14. Mcm10

3.6.15. Ctf4

3.6.16. TopoI

3.6.17. TopoII

3.6.18. Mrc1

3.6.19. Csm3/Tof1

3.6.20. RFC

3.6.21. PCNA

3.6.22. Pol δ

3.6.23. Ligase 1

3.6.24. Fen1

4. Optimizations of the in vitro replication assay

4.1. Setup of replication assay

4.2. Tuning of the assay

4.3. DNA replication assay

4.3.1.1. Equipment

4.3.1.2. Chemicals

4.3.1.3. Protocol

4.4. Troubleshooting

4.4.1. Salt

4.4.2. Challenging proteins

5. Summary

Acknowledgments

References

Chapter Two: Monitoring the replication of structured DNA through heritable epigenetic change

2. DT40 lines and their culture

2.1. DT40 culture reagents

2.2. DT40 culture conditions

2.3. Genetic controls

3. Protocol: Bu-1 loss variant fluctuation assay

3.1. Equipment

3.2. Reagents

3.3. Experimental design

3.4. Performing and analysing a Bu-1a fluctuation analysis experiment

3.5. A note on statistical tests and data interpretation

3.6. Calculation of per-division rate of BU-1 loss variant generation

4. Optimisation and troubleshooting

5. Applications and additional considerations

5.1. Advantages and disadvantages of the method

5.2. Applications

5.3. Discrete expression states of BU-1

5.4. Transvection between the BU-1A and BU-1B alleles

6. Summary

Chapter Three: Visualizing replication fork encounters with DNA interstrand crosslinks

2. Experimental procedures

2.1. Synthesis of digoxigenin-trimethyl psoralen (Dig-TMP)

2.1.1. Chemicals

2.1.2. Equipment

2.1.3. Synthesis of digoxigenin trimethylpsoralen (Dig-TMP)

3. DNA fiber assay

3.1. Cells

3.3. Equipment

3.4. Procedure.

3.4.1. Treatment of cells and DNA spreading

3.5. Immunostaining

3.5.1. Imaging

4. Sequential proximity ligation assay

4.1. Cells

4.2. Reagents

4.3. Equipment

4.4. Software

4.5. Procedures

4.5.1. Marking Mattek plates

4.5.2. Preparation of CellTak coated Mattek plates

4.5.3. Sequential PLA of DONSON: pMCM2 and FANCM: pMCM2 in cells treated with TMP/UVA

Contributions

Chapter Four: Single-molecule imaging of replication fork conflicts at genomic DNA G4 structures in human cells

2. Single molecule localization microscopy (SMLM)

2.1. Protein labeling with photoswitchable fluorescent probes

2.2. Stochastic photoactivation of labeled fluorophores

2.3. Localization precision of individual fluorophores

2.4. Analysis of SMLM data

3. Protocol

3.1. Reagents and equipment

3.2. Reagent setup

3.3. Cell seeding, drug treatments and EdU incorporation

3.4. Pre-extraction and fixation

3.5. EdU detection

3.6. Immunostaining

3.7. Assembly of imaging chamber

3.8. SMLM

3.9. SMLM analysis

Chapter Five: Studying the DNA damage response in embryonic systems

2. Studying the DNA damage response in the early Xenopus embryo

2.1. UV-C irradiation of Xenopus embryos

2.1.1. Equipment

2.1.2. Buffers and reagents

2.1.3. Procedure

2.2. Analysis of DNA damage in isolated nuclei

2.2.1. Equipment

2.2.2. Buffers and reagents

2.2.3. Procedure

2.3. Notes

2.4. UV-C irradiation of plasmid DNA

2.4.1. Equipment

2.4.2. Buffers and reagents

2.4.3. Procedure

2.5. Notes

2.6. Damaging plasmid DNA with MMS

2.6.1. Buffers and reagents

2.6.2. Procedure

2.7. Testing the efficiency of induced DNA damage in Xenopus egg extracts

2.7.1. Equipment

2.7.2. Buffers and reagents.

2.7.3. Procedure

2.8. DNA precipitation onto glass fiber filters

2.8.1. Equipment

2.8.2. Buffers and reagents

2.8.3. Procedure

2.9. Notes

2.10. Microinjection of damaged plasmid DNA into Xenopus eggs

2.10.1. Equipment

2.10.2. Buffers and reagents

2.10.3. Procedure(s)

2.10.3.1. Needles preparation and calibration

2.10.3.2. Microinjection of damaged plasmid DNA into 2-cell stage Xenopus embryos

2.11. Microinjection of in vitro synthesized mRNA into Xenopus embryos

2.11.1. In vitro transcription

2.11.1.1. Equipment

2.11.1.2. Buffers and reagents

2.11.1.3. Procedure

2.12. In vitro translation

2.12.1. Equipment

2.12.2. Buffers and reagents

2.12.3. Procedure

2.13. mRNA microinjection into 2-cell stage Xenopus embryos

2.13.1. Procedure

2.14. DNA isolation from embryos

2.14.1. Equipment

2.14.2. Buffers and reagents

2.14.3. Procedure

2.15. Notes

2.16. Xenopus embryos protein extracts

2.16.1. Equipment

2.16.2. Buffers and reagents

2.16.3. Procedure

3. Studying the DNA damage response in mESCs

3.1. Introduction

3.2. Damaging mESCs with UV-C

3.2.1. Equipment

3.2.2. Buffers and reagents

3.2.3. Procedure

3.2.4. Notes

3.3. Damaging mESCs with MMS

3.3.1. Equipment

3.3.2. Buffers and reagents

3.3.3. Procedure

3.4. Damaging mESCs with bleomycin

3.4.1. Equipment

3.4.2. Buffers and reagents

3.4.3. Procedure

3.5. Synchronization of mESCs with Nocodozale

3.5.1. Equipment

3.5.2. Buffers and reagents

3.5.3. Procedure

3.6. Notes

4. Conclusions

Chapter Six: Analysis of repair of replication-born double-strand breaks by sister chromatid recombination in yeast

1.1. The study of DSB repair mechanisms

1.2. Repair of broken replication forks.

2. The use of a mini-HO site to study SCR induced by replication-born DSBs generated by the HO endonuclease

3. The use of the FRT site and a mutant FLP to study replication-born DSB-induced SCR

4. Rationale of the TINV recombination systems

5. Reagents, materials and equipment

5.1. Reagents

5.2. Materials

5.3. Equipment

6. Protocols

6.1. Replication-born DSB induction

6.2. Genetic determination of recombination frequencies

6.3. Physical analysis of DSBs and SCR intermediates

6.3.1. DNA extraction

6.3.2. Gel electrophoresis

6.3.3. Transfer of DNA to a nylon membrane

6.3.4. Random primer radiolabeling

6.3.5. Southern-blot hybridization

7. Data analysis

Chapter Seven: Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rat ...

2. Experimental system

3. Collecting cells from the time course during BIR

3.2. Media for cell culture

3.3. Procedure

4. DNA preparation

4.1. Equipment

4.3. Procedure

5. Note

6. BIR synthesis detected by ddPCR

6.1. Equipment and supplies

6.2. Reagents

6.3. Procedures

7. Note

8. Results

9. Discussion

Funding

Chapter Eight: Genomic mapping of DNA-repair reaction intermediates in living cells with engineered DNA structure-trap pr ...

1.1. Overview of the RuvCDefGFP Holliday-junction trap and X-seq

1.2. Overview of the ExID single-stranded DNA 3-end trap and SsEND-seq

2. Considerations for experimental design with X-seq in E. coli

2.1. Interpretation of X-seq data

2.2. Bioinformatic analysis of X-seq data

3. Considerations for SsEND-seq in E. coli

3.1. Bioinformatic analysis of SsEND-seq data

4. Materials for X-seq in E. coli.

4.1. Bacterial growth, induction of RuvCDefGFP (RDG) and HJ-RDG crosslinking.

Notes:

Includes bibliographical references.

Description based on print version of record.

Description based on publisher supplied metadata and other sources.

ISBN:

0-323-90733-4

OCLC:

1285605640