DNA damage and chromosomal instability.

Format:

Book

Contributor:

EBSCOhost

Series:

METHODS IN CELL BIOLOGY.182

Language:

English

Subjects (All):

DNA damage.

Chromosomal Instability.

DNA Damage.

Medical Subjects:

Chromosomal Instability.

DNA Damage.

Physical Description:

1 online resource

Place of Publication:

London : Academic Press, 2024.

Contents:

Intro

Methods in Cell Biology

Copyright

Contents

Contributors

DNA damage and chromosomal instability

Acknowledgments

Conflict of interest

References

Chapter 1: Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation

1. Introduction

2. Y centromere inactivation using a CENP-A gene replacement strategy

2.1. Expression of TIR1 by retroviral transduction

2.2. Fusing an auxin-inducible degron tag to CENP-A

2.3. Expression of a CENP-A C-terminal mutant rescue

3. Induction of Y chromosome-specific micronuclei

3.1. Cell culture and interphase FISH

3.2. Interphase FISH

4. Y chromosome fragmentation following micronucleus induction

4.1. Cell culture

4.2. Metaphase spread preparation

4.3. Metaphase FISH

4.4. Quantification of mitotic chromosome fragmentation

5. Y chromosome rearrangements following micronucleus induction

5.1. Insertion of a neoR selection marker into the Y chromosome

5.2. Cell culture

5.3. Chromosome rearrangement analysis by metaphase FISH

6. Summary

Chapter 2: Generation of aneuploid cells and assessment of their ability to survive in presence of chemotherapeutic agents

2. Materials

2.1. Disposables

2.2. Reagents

2.3. Software

2.4. Antibodies for immunostaining

3. Methods

3.1. Cell culture

3.2. Generation of chromosome mis-alignment

3.3. Immunofluorescence staining

3.4. Chromosome segregation fidelity analysis

3.5. Crystal violet assay

3.6. Crystal violet intensity quantification

4. Notes

Chapter 3: Genetic reporters to detect and quantify homologous recombination in yeast

2.1. Media

2.2. Yeast strains

2.3. ade2 inverted-repeat strains.

2.4. ade2 direct-repeat strains

2.5. Primers

2.6. Reagents

3.1. Quantifying spontaneous and Tus/Ter induced recombination with the ade2 inverted-repeat reporter

3.2. Quantifying DSB-induced recombination with the ade2 direct-repeat reporter

3.3. Physical monitoring of Ade+ recombination events in the ade2 direct-repeat assay

4. Limitations of the assays

Chapter 4: Mapping histone variant genomic distribution: Exploiting SNAP-tag labeling to follow the dynamics of incorporation

2.1. Disposable

2.2. Specialized equipment

2.3. Cell lines (see Notes 2, 3)

2.4. Reagents and solutions

2.5. Other materials

3.1. Cell culture and treatment

3.2. Soluble chromatin preparation

3.3. Magnetic beads preparation (see note 19 and 20)

3.4. Capture of nucleosomes

3.5. DNA purification

3.6. Quality controls, DNA quantification and MNase profile

3.7. High-throughput sequencing

Chapter 5: Nascent DNA sequencing and its diverse applications in genome integrity research

2. Methods

2.1. Synthesis associated with repair sequencing (SAR-seq)

2.1.1. Considerations for experimental design

2.1.2. Cell culture and fixation

2.2. Replication origin sequencing (EdU-Seq)

2.2.1. Cell culture and treatments

2.3. Mitotic DNA synthesis sequencing (MiDAS-seq)

2.3.1. Cell culture and treatments

2.4. Biotin-labeling of EdU using click chemistry

2.5. Library preparation for sequencing

2.5.1. DNA sonication

2.5.2. Biotinylated DNA enrichment

2.5.3. End-repair, A-tailing, and adapter ligation

2.5.4. Library PCR amplification

2.5.5. Strand-specific SAR-seq variation

3. Notes

References.

Chapter 6: Enrichment of DNA replication intermediates by EdU pull down

2. EdU labelling and genomic DNA preparation

3. Click reaction and purification conditions

4. Enrichment of replication intermediates

5. Materials and reagents

6. Concluding remarks

Chapter 7: DNA curtains for studying phase separation mechanisms of DNA-organizing proteins

2. Current methods to study phase separated droplets

3. DNA curtain assay to unmask molecular mechanisms of phase separation

3.1. Materials

3.2. Protocol

3.2.1. Single-tethered DNA curtain assay

3.2.2. Real-time DNA compaction assay

3.2.3. Characterization of protein-DNA interaction

3.2.4. Determination of DNA compaction origin with the help of tracer proteins

5. Conclusion

Chapter 8: Visualizing the dynamics of DNA replication and repair at the single-molecule level

1. Background and motivation

1.1. Biomedical relevance of studying the basic mechanisms of DNA replication and repair

1.2. Overview of KEHRMIT

1.3. Other imaging approaches used in conjunction w KEHRMIT

1.4. Examples of discoveries enabled by KEHRMIT

2.1. Extract preparation

2.2. Preparing biotinylated DNA substrates

2.3. Double-biotinylation of Lambda DNA

2.4. Preparing PEG-functionalized coverslips

2.5. Generating custom polyclonal antibodies

2.6. Purifying recombinant proteins

2.7. Fluorescent labeling of recombinant proteins

2.8. Enzymatic labeling of peptide tags

2.9. Self-modifying enzymes

2.10. Validating recombinant proteins and affinity purified antibodies via immunodepletion-rescue assay

2.11. Extract preparation

2.12. Microscope configuration and imaging settings

2.13. Detailed protocol for a KEHRMIT experiment.

2.13.1. Binding antibodies to ProteinA Sepharose beads

2.13.2. Immunodepleting GINS from egg extracts

2.13.3. Preparing licensing, replication initiation, and replication elongation mixes

2.13.4. Constructing the microfluidic reaction chamber

2.13.5. Configuring the microscope for a KEHRMIT experiment

2.13.6. Double-tethering DNA substrates in the flow cell

2.13.7. Performing the KEHRMIT experiment

2.14. Performing data analysis

3. Conclusions

Author contributions

Declaration of interests

Chapter 9: Cloning and expansion of repetitive DNA sequences

2.1. Method overview

2.2. Cloning design strategy

2.3. Plasmid repeat stability considerations

3. Protocol

3.1. Design oligonucleotides for initial cloning

3.2. Anneal oligonucleotides and clone into vector

3.3. Expand repetitive sequences

3.4. Check for stability of repeats

3.5. PlasmidSelect for higher quality substrate preparation (optional)

4. Concluding remarks

5. Notes

Chapter 10: Using an ImageJ-based script to detect replication stress and associated cell cycle exit from G2 phase by flu ...

2. Cell culture

3. Treatments

3.1. Replication stress

3.2. Labeling DNA synthesis in S-phase using edu (5-ethynyl-2-deoxyuridine)

4. Immunofluorescence

5. Image acquisition

6. Image analysis (see note 10)

6.1. Segmentation

6.2. Background correction

7. Quantification

8. Data visualization

9. Notes

10. Materials

Chapter 11: An automated image analysis pipeline to quantify the coordination and overlap of transcription and replicatio ...

1.1. Materials

1.2. Cell culture and drug treatments

1.3. TR colocalization immunofluorescence.

1.4. TRC-proximity ligation assay (TRC-PLA)

1.5. Image acquisition

1.6. Image analysis

1.7. Installation requirements

1.8. Image types

1.9. User-defined parameters

1.10. Background subtraction

1.11. Nuclei segmentation

1.12. ROI filtering

1.13. Measurements and quantifications

1.14. Outputs

2. Notes

Chapter 12: Monitoring Chk1 kinase activity dynamics in live single cell imaging assays

2. Live cell imaging tools to study cell cycle and DNA damage signaling

2.1. Monitor cell cycle progression

2.2. Monitor DNA damage signaling

3. Image acquisition protocols

3.1. Set-up acquisition and imaging

3.1.1. Materials

3.1.2. Coating and seeding of IBIDI slide

3.1.3. Imaging

5. Image analysis

5.1. Install the required software, plugins and scripts

5.1.1. Fiji scripts to track, segment, measure and annotate nuclei over time

5.1.2. Python scientific packages from Anaconda

5.2. Sort files, segmentation, tracking and pixel value measurements in Fiji

5.2.1. Sort TIFF files in the expected folder structure

5.2.2. Segmentation and tracking

5.2.3. Measuring pixel intensities

5.3. Data analysis and graph generation: Experiment #1

5.3.1. Preprocess the data

5.3.2. Generate Experiment #1 graph with matplotlib

5.4. Data analysis and graph generation: Experiment #2

5.4.1. Preprocess data tables

5.4.2. Manually review and annotate G1/S and S/G2 transition for each cell in Fiji

5.4.3. Generate graph for Experiment 2

Chapter 13: Visualizing DNA damage and repair using single molecule super resolution microscopy

2.1. Cell synchronization and drug treatment

2.2. Fixation

2.3. Labeling

2.4. Super resolution imaging and analysis

Notes:

Electronic reproduction. Ipswich, MA Available via World Wide Web.

ISBN:

9780443188992

0443188998

Publisher Number:

99995988252

Access Restriction:

Restricted for use by site license.