Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 errors^1,2. 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 tumorigenesis^3,4. To maintain genome integrity, multiple repair systems have been developed during evolution. According to the chemical and physical properties of specific types of DNA damage, multiple repair mechanisms can be activated. Mismatches, abasic sites, single-strand breaks, and 8-oxoguanine (8-oxoG) can be removed either by mismatch repair or base-excision repair pathway^5,6. Lesions caused by UV-induced photoproducts and bulky adducts can be repaired either by nucleotide-excision repair (NER) or DNA double-strand break repair (DSBR) process^7,8. NER consists of two main sub-pathways: transcription-coupled NER (TC-NER) and global genomic NER (GG-NER). Regarding the cell cycle phase, following DNA double-strand break induction, two sub-pathways can be activated: non-homologous end joining (NHEJ) and homologous recombination (HR)^1,9. NHEJ, which is the dominant pathway in resting cells, can be activated in all cell cycle phases, representing a faster but error-prone pathway¹⁰. On the other hand, HR is an error-free pathway, in which the DSBs are repaired based on sequence-homology search of the sister chromatids, therefore it is mainly present in S and G2 cell cycle phases¹¹. Furthermore, microhomology-mediated end joining (MMEJ) is another DSB repair mechanism, distinct from the aforementioned ones, based on a KU70/80- and RAD51-independent way of re-ligation of previously resected microhomologous sequences flanking the broken DNA ends. Therefore, MMEJ is considered to be error-prone and highly mutagenic¹². During DNA repair, DSBs can induce the DNA damage response (DDR), which results in the activation of checkpoint kinases that halt the cell cycle during repair^13,14,15. The DDR is activated as a response to the recruitment and extensive spreading of initiator key players of the repair process around the lesions, contributing to the formation of a repair focus. In this early signaling cascade, the ATM (Ataxia Telangiectasia Mutated) kinase plays a pivotal role by catalyzing the phosphorylation of the histone variant H2AX at Ser139 (referred to as γH2AX) around the lesion¹⁶. This early event is responsible for the recruitment of additional repair factors and the initiation of downstream repair processes. Although the exact function of the recruited proteins at the repair focus has not yet been fully characterized, the formation and the dynamics of repair foci have been investigated by several laboratories. These markers are extensively used to follow the repair kinetics, but their precise role during the repair process remains elusive. Due to the great importance yet poor understanding of DNA repair-related cellular processes, several methods have been developed so far to induce and visualize the DDR.

Various methods and systems have been established to induce the desired type of DNA damage. For instance, some agents [such as neocarzinostatin (NCS), phleomycin, bleomycin, γ-irradiation, UV] can induce large numbers of random DNA breaks at non-predictive genomic positions, while others (endonucleases, such as AsiSI, I-PpoI or I-SceI, as well as laser striping) can induce DNA breaks at known genomic loci^{17,18,19,20,21}. Here, we focus on the endonuclease-based techniques currently used to study the DDR in mammalian and yeast cells. Aside from highlighting the principles of these techniques, we emphasize both their advantages and disadvantages.

Protokół

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, renewing medium every 2-3 days.
   3. Aspirate the medium and wash the cells with 1x PBS. Detach cells with Trypsin-EDTA solution. When the cells detach, stop the trypsin activity by adding culture medium to the cells, yielding a cell suspension.
   4. Count the cells using a cell counting chamber. Plate 2 x 10⁴ cells/mL/well on a 24-well plate, with sterile 12 mm round coverslips in each well.
   5. Incubate cells for 24 h at 37 °C in a humidified 5% CO₂ atmosphere to allow attachment onto the coverslips.
   6. Treat the cells with 10 ng/mL of neocarzinostatin (NCS) by directly pipetting the damaging agent to the cultured medium. Incubate the cells with the NCS-containing medium for 15 min, then wash them with 1x PBS and add fresh, supplemented culture medium to the cells. Otherwise, use appropriate agent (i.e., 4-OHT) to induce DSBs via endonuclease-based systems without refreshing the medium²².
      NOTE: Alternatively, use irradiation to induce DNA damage, ranging from 30 min up to 8 h of recovery time by using neutron flux between 2-20 Gy²³.
   7. Incubate cells for 1-8 h at 37 °C in a humidified 5% CO₂ atmosphere to follow the kinetics of DNA repair.
2. Fixation of cells
   ​NOTE: 300-500 µL of solutions/well should be used in the following steps (steps 1.2-1.5) to cover all cells adequately. Each incubation and washing step (except the antibody incubation) should be performed on an orbital shaker with gentle agitation.
   1. Following DSB induction and incubation of cells, remove the medium from the attached cells and wash the cells once with 1x PBS.
   2. Fix cells with 4% formaldehyde-PBS solution for 20 min at 25 °C.
3. Permeabilization of cells
   1. Remove the fixing solution and wash cells three times with 1x PBS for 5 min each.
   2. Remove the PBS and add 0.2% Triton X-100 dissolved in PBS. Incubate the samples for 20 min.
4. Blocking of non-specific binding sites
   1. Wash the cells three times with 1x PBS.
   2. Block non-specific binding sites with 5% BSA (Bovine Serum Fraction V albumin) diluted in PBST (1x PBS supplemented with 0.1% Tween-20), and incubate the permeabilized samples for at least 20 min.
5. Immunofluorescence staining
   1. Add the proper amount of primary antibody (i.e., anti-γH2AX, anti-DNA-PKcs) diluted in 1% BSA-PBST solution. Place each coverslip upside down onto a paraffin film over a 10 µL droplet of the diluted anti-γH2AX antibody.
      NOTE: In case of co-immunostaining dilute appropriately both antibodies in the same 1% BSA-PBST solution.
   2. Incubate the samples in a humidity chamber for 1.5 h at 4 °C.
      NOTE: Incubation can also be performed at 4 °C overnight.
   3. Place the coverslips back being side up into the 24-well plate and wash three times for 5 min with 1x PBS.
   4. Add the proper amount of secondary antibody diluted in 1% BSA-PBST. Place each coverslip upside down onto a paraffin film over a 10 µL droplet of the diluted antibody.
   5. Incubate the samples in a humidity chamber at 4 °C for 1 h.
   6. Place the coverslips back being side up into the 24-well plate and wash three times for 5 min with 1x PBS.
   7. Before removing the last PBS washing solution, gently take out the coverslips using a tweezer and needle and then place them upside down onto glass slides with droplets of mounting medium (supplemented with DAPI).
      ​NOTE: Avoid the formation of air bubbles. When the mounting medium dries, it is recommended to seal the edges of the coverslips with nail polish to prevent shriveling of the samples.

2. Chromatin immunoprecipitation

1. Cell collection, crosslinking, cell and nuclear lysis, and DNA fragmentation
   1. Culture approximately 5 x 10⁶ cells/mL in a 150 mm dish for each sample.
   2. Remove the culture medium and wash the cells twice with ice-cold 1x PBS.
   3. Fix the cells with 1% formaldehyde-PBS solution, place the plates on an orbital shaker, and agitate gently for 20 min.
      NOTE: Formaldehyde is volatile; always prepare a fresh working solution. In some cases, the formaldehyde solution contains methanol to stabilize it, but it is better to use a methanol-free solution to avoid interference with downstream reactions.
   4. Stop the fixation with 125 mM glycine and incubate on an orbital shaker with gentle agitation for 5 min at 25 °C.
   5. Place the plates on ice and wash twice with ice-cold 1x PBS.
   6. Scrape the cells in ice-cold 1x PBS and transfer them into 15 mL conical tubes.
   7. Centrifuge the cells at 2,500 x g for 5 min at 4 °C.
   8. Carefully aspirate the supernatant and resuspend the pellet in 2 mL of cell lysis buffer [5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1x PIC (protease inhibitor cocktail)] and incubate on ice for 10 min.
   9. Centrifuge the cell suspension at 2,500 x g for 5 min at 4 °C.
   10. Carefully discard the supernatant and resuspend the pellet in 500-1,500 µL of nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 0.8% SDS, 1x PIC) and incubate on ice for 30-60 min. Transfer the lysate into a polystyrene conical tube suitable for sonication.
       NOTE: Since the nuclear lysis buffer contains SDS, it will precipitate on ice, and the solution will turn white. The solution should turn transparent following sonication.
   11. Sonicate the lysate to shear DNA to an average fragment size of 300-1,000 bp.
       NOTE: The appropriate sonication cycles and conditions should be set according to the cell type and the sonication equipment. Fragments smaller than 200 bp are not suitable for ChIP, because the nucleosome-DNA interactions can be disrupted.
2. Reversal of crosslinking, determination of sonicated fragment sizes
   1. Take out 100 µL of the sonicated sample to verify the fragment size of the sonicated chromatin. The remaining chromatin should be stored at −80 °C.
   2. Add 0.5 mg/mL RNase A to each 100 µL of the sample and incubate them at 37 °C for 20 min to activate the RNase.
   3. Incubate the samples at 65 °C overnight.
   4. The next day, add 500 µg/mL of Proteinase K and 0.5% SDS, and incubate the samples at 50 °C for 3 h.
   5. Add 0.5 volume of phenol and 0.5 volume chloroform-isoamyl alcohol mix (24:1) to each sample.
   6. Vortex for 1 min.
   7. Centrifuge at 13,000 x g for 10 min.
   8. Transfer the upper aqueous phase to a new microcentrifuge tube.
   9. Add 1 volume chloroform-isoamyl alcohol mix (24:1) to each sample.
   10. Vortex for 1 min.
   11. Centrifuge at 13,000 x g for 10 min.
   12. Transfer the upper aqueous phase to a new microcentrifuge tube.
   13. Add 2.5 volumes of 96% ethanol and 0.1 volume of 3 M Na-Acetate pH 5.2.
   14. Incubate for at least 20 min at −80 °C.
   15. Centrifuge the samples at 13,000 x g for 10 min at 4 °C.
   16. Remove the ethanol and wash the pellet with 400 µL of 70% ethanol.
   17. Centrifuge the samples at 13,000 x g for 10 min at 4 °C.
   18. Remove the ethanol and air dry the pellet.
   19. Resuspend the pellet in 10 µL of TE.
   20. Run the samples on a 0.8% agarose gel. The sonicated chromatin size should be around 500 bp.
       NOTE: Use bromophenol blue-free loading buffer because the size of this dye is approximately 500 bp, which can disturb the proper detection of chromatin fragments. Instead, it is recommended to use loading buffer complemented with xylene-cyanole, which is approximately 3,000 bp.
   21. If the chromatin size is acceptable, dilute the frozen chromatin samples from step 2.1. in 3 volumes of dilution buffer (10 mM Tris-HCl pH 8.0, 0.5 mM EGTA pH 8.0, 1% Triton X-100, 140 mM NaCl, 1x PIC) and mix the samples via rotation for 10 min at 4 °C.
       ​NOTE: This step is necessary to dilute the SDS present in the nuclear lysis buffer to avoid interference with downstream reactions, including the measurement of chromatin concentration.
   22. Measure the DNA concentration of the chromatin samples at 260/280 nm using a spectrophotometer.
3. Preparation of beads, pre-clearing, and immunoprecipitation
   1. Prepare beads (sheep anti-rabbit or mouse IgG) for the pre-clearing and immunoprecipitation steps. Wash beads twice for 10 min at 4 °C with RIPA buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na-DOC, 0.1% SDS, 150 mM NaCl and 1X PIC).
   2. Resuspend the beads in the same volume of RIPA buffer as in step 2.3.1.
   3. Pre-clear 25-30 µg chromatin of each sample with 4 µL of the beads via rotation for 1-2 h at 4 °C.
      NOTE: Add RIPA buffer to each chromatin sample up to 500 µL of final volume to let the samples mix properly under rotation. Do not forget to take out chromatin for NAC (No Antibody Control) and TIC (Total Input Control) in the case of each sample set. TICs only require a final volume of up to 200 µL.
   4. Precipitate the beads with a magnet and transfer the supernatant to a new microcentrifuge tube.
   5. Add the appropriate amount of antibody to each chromatin sample (except NAC and TIC) and rotate overnight at 4 °C.
   6. Next day, add 40 µL of washed beads to each sample (except TIC) and incubate them overnight, rotating at 4 °C.
4. Washing
   1. Wash once with 300 µL of Low Salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS, 1x PIC) for 10 min via rotation at 4 °C.
   2. Wash once with 300 µL of High Salt buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 2 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS, 1x PIC) for 10 min via rotation at 4 °C.
   3. Wash once with 300 µL of LiCl buffer (250 mM LiCl, 1% NP-40, 1% Na-DOC, 1 mM EDTA pH 8.0, 10 mM Tris-HCl pH 8.0, 1x PIC) for 10 min via rotation at 4 °C.
   4. Wash twice with 300 µL of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) for 10 min via rotation, for the first wash at 4 °C, and the second wash at 25 °C.
5. Elution
   1. Add 200 µL of the elution buffer (1% SDS and 100 mM NaHCO₃) to the beads and incubate at 65 °C in a thermo-shaker for 15 min with continuous shaking (approx. 400 RPM). Transfer the supernatant to a new tube and elute beads again in 200 µL of Elution buffer. Combine eluates (400 µL final volume).
   2. Add NaCl to a final concentration of 200 mM in each sample. Supplement the TIC samples with 200 µL of Elution buffer and add NaCl as well.
      ​NOTE: From this step, TIC should be handled under the same conditions as the other samples.
   3. Incubate the samples at 65 °C (without shaking) for at least 6 h.
   4. Add 1 mL of cold 100% ethanol to each sample, rotate the tubes twice to mix, and precipitate DNA overnight at −80 °C.
   5. The next day, centrifuge for 30 min at 13,000 x g at 4 °C.
   6. Discard the supernatant and wash the pellet with 70% EtOH.
   7. Centrifuge the samples at 13,000 x g for 10 min at 4 °C.
   8. Discard the supernatant and air dry the pellets.
   9. Resuspend the pellets in 100 µL of TE and add 0.5 mg/mL of RNase A to each sample. Incubate at 37 °C for 20 min to activate the RNase.
6. Reversal of crosslinking
   1. Add 500 µg/mL of Proteinase K and 0.5% SDS then incubate the samples at 50 °C for 2 h.
      NOTE: If proceeding to ChIP-seq, avoid phenol-chloroform extraction, as it inhibits the downstream NGS process. Instead, it is recommended to use a commercially available kit (see Table of Materials).
   2. Add 0.5 volume of phenol and 0.5 volume of chloroform-isoamyl alcohol mix (24:1) to each sample.
   3. Vortex for 1 min.
   4. Centrifuge at 13,000 x g for 10 min.
   5. Transfer the upper aqueous phase to a new microcentrifuge tube.
   6. Add 1 volume of chloroform-isoamyl alcohol mix (24:1) to each sample.
   7. Vortex for 1 min.
   8. Centrifuge at 13,000 x g for 10 min.
   9. Transfer the upper aqueous phase to a new microcentrifuge tube.
7. DNA extraction
   1. Add 2.5 volumes of 96% ethanol and 0.1 volume of 3 M Na-Acetate pH 5.2.
   2. Incubate for at least 20 min at −80 °C.
   3. Centrifuge the samples at 13,000 x g for 10 min at 4 °C.
   4. Remove the ethanol and wash the pellet with 400 µL of 70% ethanol.
   5. Centrifuge the samples at 13,000 x g for 10 min at 4 °C.
   6. Remove the ethanol and air dry the pellet.
   7. Resuspend the pellet in 50 µL of TE.

Wyniki

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...

Dyskusje

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...

Materiały

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