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, renewing medium every 2-3 days.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Incubate cells for 24 h at 37 &#176;C in a humidified 5% CO2 atmosphere to allow attachment onto the coverslips.</li>
		<li>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.</li>
		<li>Incubate cells for 1-8 h at 37 &#176;C in a humidified 5% CO2 atmosphere to follow the kinetics of DNA repair.</li>
	</ol>
	</li>
	<li>Fixation of cells 
	&#8203;NOTE: 300-500 &#181;L 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.
	<ol>
		<li>Following DSB induction and incubation of cells, remove the medium from the attached cells and wash the cells once with 1x PBS.</li>
		<li>Fix cells with 4% formaldehyde-PBS solution for 20 min at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Permeabilization of cells
	<ol>
		<li>Remove the fixing solution and wash cells three times with 1x PBS for 5 min each.</li>
		<li>Remove the PBS and add 0.2% Triton X-100 dissolved in PBS. Incubate the samples for 20 min.</li>
	</ol>
	</li>
	<li>Blocking of non-specific binding sites
	<ol>
		<li>Wash the cells three times with 1x PBS.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>Add the proper amount of primary antibody (i.e., anti-&#947;H2AX, anti-DNA-PKcs) diluted in 1% BSA-PBST solution. Place each coverslip upside down onto a paraffin film over a 10 &#181;L droplet of the diluted anti-&#947;H2AX antibody. 
		NOTE: In case of co-immunostaining dilute appropriately both antibodies in the same 1% BSA-PBST solution.</li>
		<li>Incubate the samples in a humidity chamber for 1.5 h at 4 &#176;C. 
		NOTE: Incubation can also be performed at 4 &#176;C overnight.</li>
		<li>Place the coverslips back being side up into the 24-well plate and wash three times for 5 min with 1x PBS.</li>
		<li>Add the proper amount of secondary antibody diluted in 1% BSA-PBST. Place each coverslip upside down onto a paraffin film over a 10 &#181;L droplet of the diluted antibody.</li>
		<li>Incubate the samples in a humidity chamber at 4 &#176;C for 1 h.</li>
		<li>Place the coverslips back being side up into the 24-well plate and wash three times for 5 min with 1x PBS.</li>
		<li>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). 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
2. Chromatin immunoprecipitation
<ol>
	<li>Cell collection, crosslinking, cell and nuclear lysis, and DNA fragmentation
	<ol>
		<li>Culture approximately 5&#160;x 106 cells/mL in a 150 mm dish for each sample.</li>
		<li>Remove the culture medium and wash the cells twice with ice-cold 1x PBS.</li>
		<li>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.</li>
		<li>Stop the fixation with 125 mM glycine and incubate on an orbital shaker with gentle agitation for 5 min at 25 &#176;C.</li>
		<li>Place the plates on ice and wash twice with ice-cold 1x PBS.</li>
		<li>Scrape the cells in ice-cold 1x PBS and transfer them into 15 mL conical tubes.</li>
		<li>Centrifuge the cells at 2,500 x g for 5 min at 4 &#176;C.</li>
		<li>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.</li>
		<li>Centrifuge the cell suspension at 2,500 x g for 5 min at 4 &#176;C.</li>
		<li>Carefully discard the supernatant and resuspend the pellet in 500-1,500 &#181;L 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Reversal of crosslinking, determination of sonicated fragment sizes
	<ol>
		<li>Take out 100 &#181;L of the sonicated sample to verify the fragment size of the sonicated chromatin. The remaining chromatin should be stored at &#8722;80 &#176;C.</li>
		<li>Add 0.5 mg/mL RNase A to each 100 &#181;L of the sample and incubate them at 37 &#176;C for 20 min to activate the RNase.</li>
		<li>Incubate the samples at 65 &#176;C overnight.</li>
		<li>The next day, add 500 &#181;g/mL of Proteinase K and 0.5% SDS, and incubate the samples at 50 &#176;C for 3 h.</li>
		<li>Add 0.5 volume of phenol and 0.5 volume chloroform-isoamyl alcohol mix (24:1) to each sample.</li>
		<li>Vortex for 1 min.</li>
		<li>Centrifuge at 13,000 x g for 10 min.</li>
		<li>Transfer the upper aqueous phase to a new microcentrifuge tube.</li>
		<li>Add 1 volume chloroform-isoamyl alcohol mix (24:1) to each sample.</li>
		<li>Vortex for 1 min.</li>
		<li>Centrifuge at 13,000 x g for 10 min.</li>
		<li>Transfer the upper aqueous phase to a new microcentrifuge tube.</li>
		<li>Add 2.5 volumes of 96% ethanol and 0.1 volume of 3 M Na-Acetate pH 5.2.</li>
		<li>Incubate for at least 20 min at &#8722;80 &#176;C.</li>
		<li>Centrifuge the samples at 13,000 x g for 10 min at 4 &#176;C.</li>
		<li>Remove the ethanol and wash the pellet with 400 &#181;L of 70% ethanol.</li>
		<li>Centrifuge the samples at 13,000 x g for 10 min at 4 &#176;C.</li>
		<li>Remove the ethanol and air dry the pellet.</li>
		<li>Resuspend the pellet in 10 &#181;L of TE.</li>
		<li>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.</li>
		<li>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 &#176;C. 
		&#8203;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.</li>
		<li>Measure the DNA concentration of the chromatin samples at 260/280 nm using a spectrophotometer.</li>
	</ol>
	</li>
	<li>Preparation of beads, pre-clearing, and immunoprecipitation
	<ol>
		<li>Prepare beads (sheep anti-rabbit or mouse IgG) for the pre-clearing and immunoprecipitation steps. Wash beads twice for 10 min at 4 &#176;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).</li>
		<li>Resuspend the beads in the same volume of RIPA buffer as in step 2.3.1.</li>
		<li>Pre-clear 25-30 &#181;g chromatin of each sample with 4 &#181;L of the beads via rotation for 1-2 h at 4 &#176;C. 
		NOTE: Add RIPA buffer to each chromatin sample up to 500 &#181;L 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 &#181;L.</li>
		<li>Precipitate the beads with a magnet and transfer the supernatant to a new microcentrifuge tube.</li>
		<li>Add the appropriate amount of antibody to each chromatin sample (except NAC and TIC) and rotate overnight at 4 &#176;C.</li>
		<li>Next day, add 40 &#181;L of washed beads to each sample (except TIC) and incubate them overnight, rotating at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Washing
	<ol>
		<li>Wash once with 300 &#181;L 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 &#176;C.</li>
		<li>Wash once with 300 &#181;L 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 &#176;C.</li>
		<li>Wash once with 300 &#181;L 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 &#176;C.</li>
		<li>Wash twice with 300 &#181;L 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 &#176;C, and the second wash at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Elution
	<ol>
		<li>Add 200 &#181;L of the elution buffer (1% SDS and 100 mM NaHCO3) to the beads and incubate at 65 &#176;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 &#181;L of Elution buffer. Combine eluates (400 &#181;L final volume).</li>
		<li>Add NaCl to a final concentration of 200 mM in each sample. Supplement the TIC samples with 200 &#181;L of Elution buffer and add NaCl as well. 
		&#8203;NOTE: From this step, TIC should be handled under the same conditions as the other samples.</li>
		<li>Incubate the samples at 65 &#176;C (without shaking) for at least 6 h.</li>
		<li>Add 1 mL of cold 100% ethanol to each sample, rotate the tubes twice to mix, and precipitate DNA overnight at &#8722;80 &#176;C.</li>
		<li>The next day, centrifuge for 30 min at 13,000 x g at 4 &#176;C.</li>
		<li>Discard the supernatant and wash the pellet with 70% EtOH.</li>
		<li>Centrifuge the samples at 13,000 x g for 10 min at 4 &#176;C.</li>
		<li>Discard the supernatant and air dry the pellets.</li>
		<li>Resuspend the pellets in 100 &#181;L of TE and add 0.5 mg/mL of RNase A to each sample. Incubate at 37 &#176;C for 20 min to activate the RNase.</li>
	</ol>
	</li>
	<li>Reversal of crosslinking
	<ol>
		<li>Add 500 &#181;g/mL of Proteinase K and 0.5% SDS then incubate the samples at 50 &#176;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).</li>
		<li>Add 0.5 volume of phenol and 0.5 volume of chloroform-isoamyl alcohol mix (24:1) to each sample.</li>
		<li>Vortex for 1 min.</li>
		<li>Centrifuge at 13,000 x g for 10 min.</li>
		<li>Transfer the upper aqueous phase to a new microcentrifuge tube.</li>
		<li>Add 1 volume of chloroform-isoamyl alcohol mix (24:1) to each sample.</li>
		<li>Vortex for 1 min.</li>
		<li>Centrifuge at 13,000 x g for 10 min.</li>
		<li>Transfer the upper aqueous phase to a new microcentrifuge tube.</li>
	</ol>
	</li>
	<li>DNA extraction
	<ol>
		<li>Add 2.5 volumes of 96% ethanol and 0.1 volume of 3 M Na-Acetate pH 5.2.</li>
		<li>Incubate for at least 20 min at &#8722;80 &#176;C.</li>
		<li>Centrifuge the samples at 13,000 x g for 10 min at 4 &#176;C.</li>
		<li>Remove the ethanol and wash the pellet with 400 &#181;L of 70% ethanol.</li>
		<li>Centrifuge the samples at 13,000 x g for 10 min at 4 &#176;C.</li>
		<li>Remove the ethanol and air dry the pellet.</li>
		<li>Resuspend the pellet in 50 &#181;L of TE.</li>
	</ol>
	</li>
</ol>

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A., Kruhlak, M., Dotiwala, F., Nussenzweig, A., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+is+refractory+to+gamma-H2AX+modification+in+yeast+and+mammals.">Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals.</a> Journal of Cell Biology. 178 (2), 209-218 (2007).</li><li>Daniels, D. L., Urh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intracellular+protein--DNA+complexes+using+HaloCHIP,+an+antibody-free+alternative+to+chromatin+immunoprecipitation.">Isolation of intracellular protein--DNA complexes using HaloCHIP, an antibody-free alternative to chromatin immunoprecipitation.</a> Methods in Molecular Biology. 977, 111-124 (2013).</li></ol>

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...

Watch this Scientific Journal Video about Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage at JoVE.com

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 ...

visualizing-quantifying-endonuclease-based-site-specific-dna

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3.5K Views.  University of Szeged. 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. 

Video: Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...