JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Published: August 21st, 2021

DOI:

10.3791/62175

1Institute of Pathology, Faculty of Medicine, University of Szeged
* These authors contributed equally

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

Our genome is constantly being challenged by various DNA damaging agents. These assaults can derive from environmental sources, such as UV light or irradiation, as well as from endogenous sources, such as metabolic by-products caused by oxidative stress or replication errors1,2. These lesions can affect the integrity of either one or both DNA strands, and if the generated errors become persistent, it frequently leads to translocations and genome instability, which may result in tumorigenesis3,4. To maintain genome integrity, multiple repair systems hav....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Immunodetection of specific proteins

  1. Preparation of cell culture and experimental setup
    1. Maintain U2OS cells in monolayers in DMEM culture medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 1% antibiotic-antimycotic solution.
      NOTE: For endonuclease-based DNA damage induction, use charcoal-treated or steroid-free medium to avoid system leakiness.
    2. Grow cells in a humidified 5% CO2 environment at 37 °C until 80% confluency, renewin.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Studying site-directed DSB-induced repair processes in cells can be achieved via either stable or transient transfection. However, it should be noted that stable transfection ensures a homogenous cell population, which gives a unified and thus more reliable cellular response. In the case of transient transfection, only a small proportion of the cell population takes up and maintains the plasmid, which introduces diversity into the experiment. Establishing ER-I-PpoI or ER-AsiSI endonuclease-based cell systems require.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Although DNA repair is a relatively recent research field, our knowledge is rapidly expanding with the help of various biochemical and microscopic methods. Preserving genetic information is crucial for cells since mutations occurring in genes involved in repair processes are among the leading causes of tumorigenesis and therefore elucidating the key steps of DNA repair pathways is essential.

Biochemical techniques (i.e., western blot, immunoprecipitation, mass-spectrometry, etc.) require large.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This research was funded by the National Research, Development and Innovation Office grant GINOP-2.3.2-15-2016-00020, GINOP-2.3.2-15-2016-00036, GINOP-2.2.1-15-2017-00052, EFOP 3.6.3-VEKOP-16-2017-00009, NKFI-FK 132080, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences BO/27/20, ÚNKP-20-5-SZTE-265, EMBO short-term fellowship 8513, and the Tempus Foundation.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
4-OHT Sigma Aldrich H7904
Agarose Lonza 50004
Antibiotic-Antimycotic Solution (100×), Stabilized Sigma Aldrich A5955
Anti-gamma H2A.X (phospho S139) antibody Abcam ab26350
Bovine Serum Fraction V albumin Biosera PM-T1725
TrackIt™ Cyan/Yellow Loading Buffer Thermo Fisher Scientific 10482035
DMEM with 1.0 g/L Glucose, without L-Glutamine Lonza 12-707F
Doxycycline Sigma Aldrich D9891
Dynabeads™ M-280 Sheep Anti-Mouse IgG Invitrogen 11202D
Dynabeads™ M-280 Sheep Anti-Rabbit IgG Invitrogen 11204D
EDTA Sigma Aldrich E6758
EGTA Sigma Aldrich E3889
Ethanol Molar Chemicals 02910-101-340
Fetal Bovine Serum (South America Origin), EU-approved Gibco ECS0180L
Formaldehyde 37% solution free from acid Sigma Aldrich 1.03999
GlutaMAX™ Supplement Thermo Fisher Scientific 35050038
Glycine Sigma Aldrich 50046
IPure kit v2 Diagenode C03010015
Isoamyl alcohol Sigma Aldrich W205702
LiCl Sigma Aldrich L9650
NaCl Sigma Aldrich S5886
Na-DOC Sigma Aldrich D6750
NaHCO3 Sigma Aldrich S5761
Neocarzinostatin from Streptomyces carzinostaticus Sigma Aldrich N9162
NP-40 Sigma Aldrich I8896
PBS Powder without Ca2+, Mg2+ Sigma Aldrich L182-50-BC
Phenol Sigma Aldrich P4557
PIPES Sigma Aldrich P1851
Polysorbate 20 (Tween 20) Molar Chemicals 09400-203-190
KCl Sigma Aldrich P5405
ProLong™ Gold Antifade Mountant with DAPI Thermo Fisher Scientific P36935
Protease Inhibitor Cocktail Set I Roche 11873580001
Proteinase K Sigma Aldrich P2308
P-S2056 DNAPKcs antibody Abcam ab18192
RNase A Roche 10109169001
CH3COONa Sigma Aldrich S2889
SDS Sigma Aldrich L3771
Tris Acetate-EDTA buffer Sigma Aldrich T6025
Tris-HCl Sigma Aldrich 91228
TRITON X-100 Molar Chemicals 09370-006-340
Trypsin from porcine pancreas Sigma Aldrich T4799
Trypsin-EDTA (0.5%), no phenol red Gibco 15400054

  1. Borsos, B. N., Majoros, H., Pankotai, T. Ubiquitylation-Mediated Fine-Tuning of DNA Double-Strand Break Repair. Cancers (Basel). 12 (6), (2020).
  2. Borsos, B. N., Majoros, H., Pankotai, T. Emerging Roles of Post-Translational Modifications in Nucleotide Excision Repair. Cells. 9 (6), (2020).
  3. Stephens, P. J., et al. The landscape of cancer genes and mutational processes in breast cancer. Nature. 486 (7403), 400-404 (2012).
  4. Turnbull, C., et al. Gene-gene interactions in breast cancer susceptibility. Human Molecular Genetics. 21 (4), 958-962 (2012).
  5. Saxowsky, T. T., Meadows, K. L., Klungland, A., Doetsch, P. W. 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America. 105 (48), 18877-18882 (2008).
  6. Jiricny, J. The multifaceted mismatch-repair system. Nature Reviews Molecular Cell Biology. 7 (5), 335-346 (2006).
  7. Hanawalt, P. C., Spivak, G. Transcription-coupled DNA repair: two decades of progress and surprises. Nature Reviews Molecular Cell Biology. 9 (12), 958-970 (2008).
  8. Kanaar, R., Wyman, C., Rothstein, R. Quality control of DNA break metabolism: in the 'end', it's a good thing. EMBO Journal. 27 (4), 581-588 (2008).
  9. Lemaitre, C., et al. Nuclear position dictates DNA repair pathway choice. Genes & Development. 28 (22), 2450-2463 (2014).
  10. Lieber, M. R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual Review of Biochemistry. 79, 181-211 (2010).
  11. Lambert, S., Lopez, B. S. Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms. EMBO Journal. 19 (12), 3090-3099 (2000).
  12. Xu, S., et al. p300-mediated acetylation of histone demethylase JMJD1A prevents its degradation by ubiquitin ligase STUB1 and enhances its activity in prostate cancer. Cancer Research. , (2020).
  13. Kastan, M. B., Bartek, J. Cell-cycle checkpoints and cancer. Nature. 432 (7015), 316-323 (2004).
  14. Roy, R., Chun, J., Powell, S. N. BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nature Reviews Cancer. 12 (1), 68-78 (2011).
  15. Krenning, L., vanden Berg, J., Medema, R. H. Life or Death after a Break: What Determines the Choice. Molecular Cell. 76 (2), 346-358 (2019).
  16. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. Journal of Biological Chemistry. 273 (10), 5858-5868 (1998).
  17. Caron, P., et al. WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks. Genes & Development. 33 (11-12), 684-704 (2019).
  18. Caron, P., et al. Cohesin protects genes against gammaH2AX Induced by DNA double-strand breaks. PLoS Genetics. 8 (1), 1002460 (2012).
  19. Berkovich, E., Monnat, R. J., Kastan, M. B. Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites. Nature Protocols. 3 (5), 915-922 (2008).
  20. Paques, F., Duchateau, P. Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Current Gene Therapy. 7 (1), 49-66 (2007).
  21. Pankotai, T., Bonhomme, C., Chen, D., Soutoglou, E. DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks. Nature Structural & Molecular Biology. 19 (3), 276-282 (2012).
  22. Iacovoni, J. S., et al. High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome. EMBO Journal. 29 (8), 1446-1457 (2010).
  23. Poinsignon, C., et al. Phosphorylation of Artemis following irradiation-induced DNA damage. European Journal of Immunology. 34 (11), 3146-3155 (2004).
  24. Varga, D., Majoros, H., Ujfaludi, Z., Erdelyi, M., Pankotai, T. Quantification of DNA damage induced repair focus formation via super-resolution dSTORM localization microscopy. Nanoscale. 11 (30), 14226-14236 (2019).
  25. Kim, J. A., Kruhlak, M., Dotiwala, F., Nussenzweig, A., Haber, J. E. Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals. Journal of Cell Biology. 178 (2), 209-218 (2007).
  26. Daniels, D. L., Urh, M. Isolation of intracellular protein--DNA complexes using HaloCHIP, an antibody-free alternative to chromatin immunoprecipitation. Methods in Molecular Biology. 977, 111-124 (2013).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved