Name: visualizing and quantifying endonuclease-based site-specific dna damage
Uploaded: 2021-08-21T13:30:00
Description: Watch this Scientific Journal Video about Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage at JoVE.com

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 J&#225;nos Bolyai Research Scholarship of the Hungarian Academy of Sciences BO/27/20, &#218;NKP-20-5-SZTE-265, EMBO short-term fellowship 8513, and the Tempus Foundation.

1. Immunodetection of specific proteins
<ol>
	<li>Preparation of cell culture and experimental setup
	<ol>
		<li>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.</li>
		<li>Grow cells in a humidified 5% CO2 environment at 37 &#176;C until 80% confluency, renewin...

<ol>
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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...

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 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 pathway5,6. 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) process7,8. 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 repair13,14,15. 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 &#947;H2AX) 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, &#947;-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 loci17,18,19,20,21. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-OHT</td>
			<td>Sigma Aldrich</td>
			<td>H7904</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (100&#215;), Stabilized</td>
			<td>Sigma Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-gamma H2A.X (phospho S139) antibody</td>
			<td>Abcam</td>
			<td>ab26350</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Fraction V albumin</td>
			<td>Biosera</td>
			<td>PM-T1725</td>
			<td></td>
		</tr>
		<tr>
			<td>TrackIt&#8482; Cyan/Yellow Loading Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>10482035</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with 1.0 g/L Glucose, without L-Glutamine</td>
			<td>Lonza</td>
			<td>12-707F</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma Aldrich</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads&#8482; M-280 Sheep Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>11202D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads&#8482; M-280 Sheep Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>11204D</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Molar Chemicals</td>
			<td>02910-101-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (South America Origin), EU-approved</td>
			<td>Gibco</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37% solution free from acid</td>
			<td>Sigma Aldrich</td>
			<td>1.03999</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#8482; Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>IPure kit v2</td>
			<td>Diagenode</td>
			<td>C03010015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoamyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>W205702</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sigma Aldrich</td>
			<td>L9650</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-DOC</td>
			<td>Sigma Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Neocarzinostatin from Streptomyces carzinostaticus</td>
			<td>Sigma Aldrich</td>
			<td>N9162</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Powder without Ca2+, Mg2+</td>
			<td>Sigma Aldrich</td>
			<td>L182-50-BC</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma Aldrich</td>
			<td>P4557</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma Aldrich</td>
			<td>P1851</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Molar Chemicals</td>
			<td>09400-203-190</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong&#8482; Gold Antifade Mountant with DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Set I</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma Aldrich</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr>
			<td>P-S2056 DNAPKcs antibody</td>
			<td>Abcam</td>
			<td>ab18192</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Roche</td>
			<td>10109169001</td>
			<td></td>
		</tr>
		<tr>
			<td>CH3COONa</td>
			<td>Sigma Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Acetate-EDTA buffer</td>
			<td>Sigma Aldrich</td>
			<td>T6025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma Aldrich</td>
			<td>91228</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON X-100</td>
			<td>Molar Chemicals</td>
			<td>09370-006-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin from porcine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing and quantifying endonuclease-based site-specific dna damage

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.

Studying site-directed DSB-induced repair processes in cells can be achieved&#160;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...

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Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

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 ...

This methodology video introduces the crucial steps of immunostaining and chromatin immunoprecipitation protocols regularly used to study DNA damage-related cellular process and to visualize and quantify the recruitment of proteins implicated in DNA repair. This technique provides powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus, as well as their post-translational modifications necessary for the fine-tuned regulation during DNA repair. Since these techniques can be applied for studying different cellular processes, they have potential relevance if a laser microirradiation or the nucleus-based DNA damaging inducing systems are used.Recently, several powerful tools have been developed to induce site-specific DNA damage that can be used to extend our understanding of DNA repair. Applying biochemical and genetic approaches, these utensils are essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. Moreover, these techniques allow for the study of these processes at single cell resolution using both fixed and living cells.Maintain U2OS cells in monolayers in DMEM culture medium supplemented with 10%fetal calf serum, 2 millimolar glutamine, and the 1%antibiotic-antimycotic solution. Grow cells in a humidified 5%carbon dioxide environment at 37 degree until 80%confluence is achieved, renewing medium every two to three days. Aspirate the medium and wash the cells with PBS.Detach cells with Trypsin-EDTA solution. When the cells detach, stop the activity of the trypsine by adding culture media to the cells, yielding a cell suspension. Count the cells using a cell counting chamber.Plate 20, 000 cells per milliliter per well on the 24-well plate with sterile 12 millimeter round cover slips in each well. Incubate cells for 24 hours at 37 degree in a humidified 5%carbon dioxide environment to allow attachment onto the cover slips. Treat the cells with 10 nanogram per milliliter neocarzinostatin or use the appropriate method to induce double-strand breaks via endonucleuse-based systems.Incubate cells for one to eight hours at 37 degree in a humidified 5%carbon dioxide environment to follow the kinetics of the DNA repair. Fix cells with 4%formaldehyde PBS solution for 20 minutes at 25 degree. Remove the fixing solution and wash cells three times with PBS for five minutes each.Remove the PBS and add 0.2%Triton X dissolved in PBS. Incubate the samples for 20 minutes. Wash the cells three times with PBS.Block non-specific binding with 5%BSA diluted in BSA-PBST and incubate the samples for at least 20 minutes. Add the proper amount of primary antibody diluted in 1%BSA-PBST solution. Place each cover slip upside down onto a parafilm or 10 microliter droplet of the diluted antibody.Incubate the samples in a humidity chamber for one and the half hour at four degree. Place the cover slips back being side up into the 24-well plate and the wash three times with PBS. Add the proper amount of secondary antibody diluted in 1%BSA-PBST.Place each cover slip upside down onto parafilm over a 10 microliter droplet of the diluted antibody. Incubate the samples in a humidity chamber at four degree for an hour. Place the cover slips back being set up into the 24-well plate and wash three times with PBS.Before removal of the last PBS washing solution, gently take out the cover slips with the half of the tweezer and needle, and then put them upside down onto the glass slides with droplets of mounting medium. Chromatin immunoprecipitation is a widely used technique to identify binding patterns of proteins to DNA. To reveal the occupancy of a desire repair protein around the DSB, a specific antibody is used to immunoprecipitate the protein of interest from the chromatin preparation together with the DNA region to which it is bound.Furthermore, ChIP has been commonly applied to map the presence of specific post-translational modification in use by DSBs at a given genomic locus. Approximately 20 million cells per milliliter should be cultured in a 15 centimeter dish for each sample. Remove culture medium and wash the cells twice with ice cold PBS.Fix the cells with 1%formaldehyde-PBS solution. Place the plate on an orbital shaker and agitate gently for 20 minutes. Stop fixation with glycine to reach a final concentration of 125 millimolar and incubate on an orbiter shaker with gentle agitation for five minutes at 25 degree.Place the plate on ice and wash twice with ice cold PBS. Scrape the cells in ice cold PBS and transfer them into 15 milliliter conical tubes. Centrifuge the cells at 5, 000 rpm for five minutes at four degree.Carefully aspirate the supernatant and resuspend the pellet in two milliliter cell lysis buffer and incubate on ice for 10 minutes. Centrifuge at 5, 000 rpm for five minutes at four degree. Carefully discard the supernatant and resuspend the pellet in 500 to 1, 500 microliter nuclear lysis buffer and incubate on ice for 30 to 60 minutes.Transfer the lysate into a polystyrene conical tube suitable for sonication. Sonicate the lysate to shear the DNA to an average fragment size of 300 to 1000 base pairs. Prepare Dynabeads for the pre-cleaning and immunoprecipitation steps.Wash beads twice for 10 minutes at four degree with RIPA buffer. Resuspend Dynabeads in the same volume of RIPA buffer as you took out from the original stock solution in steps 3.1. Pre-clear 25 to 30 microgram chromatin of each sample with four microliter Dynabeads via rotation for one to two hours at four degree.Precipitate the Dynabeads with a magnet and transfer the supernatant to a new Eppendorf tube. Add the appropriate amount of antibody to each chromatin sample and rotate overnight at four degree. Next day, add 40 microliter of washed Dynabeads to each sample and incubate them overnight rotating at four degree.Wash once with 300 microliter low salt buffer for 10 minutes with rotation at four degree. Wash once with 300 microliter high salt buffer for 10 minutes with rotation at four degree. Wash once with 300 microliter lithium chloride buffer for 10 minutes with rotation at four degree.Wash twice with 300 microliter TE for 10 minutes with rotation for the first wash at four degree and the second wash at 25 degree. Add 200 microliter elution buffer to the beads and incubate them at 65 degree in a thermo-shaker for 15 minutes with continuous shaking. Transfer the supernatant to a new tube and elute beads again in 200 microliter elution buffer.Add 500 microgram per milliliter proteinase-K and half percent SDS and incubate the samples at 50 degree for two hours. Perform chloroform extraction and transfer the upper aqueous phase to a new Eppendorf tube. Add two and a half volumes of absolute ethanol and 0.1 volume three molar sodium acetate pH 5.2.Incubate for at least 20 minutes at minus 80 degree. Remove the ethanol and air dry the pellet. Resuspend the pellet in 50 microliter TE.To study their special distribution on DNA, chromatin immunoprecipitation should be applied.A typical experimental results obtained by ChIP-qPCR is presented in this figure, which demonstrates the temporal enrichment of gamma histone AX as a response to I-PpoI-induced induced DNA damage. On the left part of the image, the timely detected gamma histone AX signal is shown at the break side, while on the right part, gamma histone AX distribution is presented at the control gene region at which demonstrated breaks have not been induced. Although DNA repair is a relatively recent research field, our knowledge is rapidly increasing with the help of both biochemical and microscopic methods.Nonetheless, biochemical techniques, such as a Western blot, immunoprecipitation, and mass spectrometry require a large amount of cells. The study repair processes represent a snapshot of the desired cell population. The microscopic field has been revolutionized by high-resolution techniques, such as super resolution microscope, which allows the visualization of DNA damage-induced cellular processes at the nucleosomal level, as well as ensuring the accurate mapping of protein co-localization.On the other hand, chromatin immunoprecipitation is a powerful tool, especially by combining it with sequencing methods to visualize its genome-wide binding pattern of a desired DNA repair related proteins and double strand breaks in either a chromatin or heterochromatin regions.

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