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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
4-OHTSigma AldrichH7904
AgaroseLonza50004
Antibiotic-Antimycotic Solution (100×), StabilizedSigma AldrichA5955
Anti-gamma H2A.X (phospho S139) antibodyAbcamab26350
Bovine Serum Fraction V albuminBioseraPM-T1725
TrackItâ„¢ Cyan/Yellow Loading BufferThermo Fisher Scientific10482035
DMEM with 1.0 g/L Glucose, without L-GlutamineLonza12-707F
DoxycyclineSigma AldrichD9891
Dynabeadsâ„¢ M-280 Sheep Anti-Mouse IgGInvitrogen11202D
Dynabeadsâ„¢ M-280 Sheep Anti-Rabbit IgGInvitrogen11204D
EDTASigma AldrichE6758
EGTASigma AldrichE3889
EthanolMolar Chemicals02910-101-340
Fetal Bovine Serum (South America Origin), EU-approvedGibcoECS0180L
Formaldehyde 37% solution free from acidSigma Aldrich1.03999
GlutaMAXâ„¢ SupplementThermo Fisher Scientific35050038
GlycineSigma Aldrich50046
IPure kit v2DiagenodeC03010015
Isoamyl alcoholSigma AldrichW205702
LiClSigma AldrichL9650
NaClSigma AldrichS5886
Na-DOCSigma AldrichD6750
NaHCO3Sigma AldrichS5761
Neocarzinostatin from Streptomyces carzinostaticusSigma AldrichN9162
NP-40Sigma AldrichI8896
PBS Powder without Ca2+, Mg2+Sigma AldrichL182-50-BC
PhenolSigma AldrichP4557
PIPESSigma AldrichP1851
Polysorbate 20 (Tween 20)Molar Chemicals09400-203-190
KClSigma AldrichP5405
ProLongâ„¢ Gold Antifade Mountant with DAPIThermo Fisher ScientificP36935
Protease Inhibitor Cocktail Set IRoche11873580001
Proteinase KSigma AldrichP2308
P-S2056 DNAPKcs antibodyAbcamab18192
RNase ARoche10109169001
CH3COONaSigma AldrichS2889
SDSSigma AldrichL3771
Tris Acetate-EDTA bufferSigma AldrichT6025
Tris-HClSigma Aldrich91228
TRITON X-100Molar Chemicals09370-006-340
Trypsin from porcine pancreasSigma AldrichT4799
Trypsin-EDTA (0.5%), no phenol redGibco15400054

References

  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 o....

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