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 ...

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Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

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Principles of Site-Specific Recombinase (SSR) Technology

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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