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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Biology

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

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

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

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

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 ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

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 ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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.