The original GFP-Pem1 was a gift from Dr. Lei Li. This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.

In this protocol we describe the method for analysis of DNA DSB repair with chromosomally integrated reporter constructs1,2 where DSBs are induced in vivo by transient expression a rare cutting endonuclease I-SceI3. The integrated assay provides the advantage of analyzing DSB repair within the chromosomal context. However, this protocol requires prolonged cell passaging, which may be problematic when working with primary cells.
An alternative approach is an extrachromosomal assay where reporter constructs are digested in vitro with I-SceI or HindIII endonucleases and then transfected into cells as linear DNA. The exctrachromosomal assay protocol4 is similar to the integrated assay described below with the following modifications. For the extrachromosomal assay omit the integration of reporter constructs and instead co-transfect the cells with 0.5 μg of NHEJ reporter plasmid linearized in vitro with I-SceI or HindIII and 0.1 μg of pDsRed2-N1 as transfection control. For the HR assay co-transfect 2 μg HR reporter plasmid linearized with I-SceI or HindIII and 0.1 μg pDsRed2-N1. Analyze the cells by FACS three days after transfection. The extrachromosomal assay allows for the analysis of DSB repair in primary isolates, avoiding extensive cell passaging, and facilitating the analysis of multiple cell lines.
1. Reporter Constructs for NHEJ and HR
The NHEJ reporter cassette5 contains a GFP gene with an engineered 3 kb intron from the Pem1 gene (GFP-Pem1). The Pem1 intron contains an adenoviral exon flanked by recognition sequences for HindIII and I-SceI endonucleases (Figure 1a) for induction of DSBs. The I-SceI sites are in inverted orientation. I-SceI has a nonpalindromic recognition sequence, hence two inverted sites generate incompatible DNA ends (Figure 1c). Incompatible DNA ends best mimic the naturally occurring DSBs. The intact NHEJ cassette is GFP negative as the adenoviral exon disrupts the GFP ORF. Upon induction of DSBs by I-SceI, the adenoviral exon is removed and NHEJ restores function of the GFP gene (Figure 2). The unique feature of this NHEJ reporter is that it can detect a wide spectrum of NHEJ events since the intron can tolerate deletions and insertions.
The HR reporter cassette1 (Figure 1b) is also based on the GFP-Pem1. In the HR cassette, the first exon of the GFP-Pem1 contains a 22 bp deletion combined with the insertion of three restriction sites, I-SceI/HindIII/I-SceI. The deletion ensures that GFP cannot be reconstituted by an NHEJ event. The two I-SceI sites are in inverted orientation, so that I-SceI digestion leaves incompatible ends (Figure 1c). The first copy of GFP-Pem1 is followed by a promoter-less/ATG-less first exon and intron of GFP-Pem1. The intact construct is GFP-negative. Upon induction of a DSB by I-SceI digestion the functional GFP gene is reconstituted by intramolecular or intermolecular gene conversion between the two mutated copies of the first GFP-Pem1 exon. Since the second copy of the GFP gene is lacking the first ATG codon and the second exon, crossing over or single-strand annealing will not restore the GFP activity. This design allows for the exclusive detection of gene conversion, which is the predominant HR pathway in mammalian cells (Figure 3).
2. Integration of the Reporter Constructs into the Genome
<ol>
 <li>Make a high quality preparation of NHEJ or HR reporter plasmid. For best results use Qiagen Endo Free Kit. Measure plasmid concentration and purity by absorbance at 260/280 nm.</li>
 <li>Linearize 10 μg of the reporter plasmid by digesting with 50 U of NheI in 50 μl (total volume of the reaction) for 6 hours in 37°C incubator (do not use the dry block, to prevent evaporation). Purify DNA from the digestion solution with Qiagen QiaexII Kit, elute DNA into 20 μl of 10 mM Tris-HCl pH 8.0. Measure DNA concentration and purity by absorbance at 260/280 nm. Usually, you will lose 20-30% of DNA during purification. Confirm the yield and digestion by running a small aliquot (2 μl) on a gel, along with an undigested reporter construct. The purified linearized reporter construct can be stored at 20°C.</li>
 <li>In preparation for transfection, bring the cells to the best growing condition. If starting from a frozen vial, split the cells twice before transfection. If starting from a plate of confluent cells, split them once.</li>
 <li>Grow the cells to 70-80% confluence (usually two days, if plated 5x105 cells/100 mm plate, for normal human fibroblasts). It is critical for the best transfection efficiency to bring the cells into logarithmic growth. Actively growing culture contains cells in M stage (seen as rounded cells attached to the surface). </li>
 <li>Transfect the cells using Amaxa Nucleofector following manufacturer's recommendations. For fibroblasts use NHDF solution and program U20. For transfection, harvest the cells from two 100 mm plates (~2x106 cells). It is critical not to over trypsinize the cells. Stop trypsinization as soon as 80% of cells detached from the plate. Resuspend the cells in NHDF solution. Cells are sensitive to NHDF solution, therefore, minimize the incubation time and mix the cells gently. Transfect the cells with 0.5 μg of linearized reporter construct. These transfection conditions, when used with normal human fibroblasts result in the integration of a single copy of a reporter construct for the majority of integrants. </li>
 <li>Select the cells with chromosomally integrated reporter constructs by adding 1 mg/mL geneticin (G418) 24h after transfection. Continue selection for 7-10 days.</li>
 <li>Pick the G418 resistant colonies (you can pick individual colonies or pool the resistant clones). Expand the culture to approximately five 100 mm plates. Freeze three plates for future use and expand remaining two plates to the desired cell number (usually ten 100 mm plates are sufficient for five transfections). </li>
 <li> When the cells reach 70-80% confluence (two days after plating 5x105 cells per 100 mm plate for fibroblasts), the cells are ready to be transfected with I-SceI expressing plasmid.</li>
</ol>
3. Induction of DSB by Transient Expression of I-SceI Endonuclease
<ol>
 <li>Transfect ~2x106 cells (two plates) of logarithmically growing culture with a mixture of 5 μg I-SceI expressing plasmid and 0.1 μg pDsRed2-N1 (Clontech) as a transfection efficiency control using Amaxa Nucleofector (use NHDF solution and U20 program for fibroblasts). </li>
 <li>At the same time prepare three calibration controls for FACS by trasfecting ~2x106 cells with (i) 5 μg EGFP-expressing plasmid; (ii) 5 μg pDsRed2-N1; (iii) 5 μg of a control plasmid that does not express a fluorescent protein (we are using a plasmid encoding HPRT minigene).</li>
 <li>Grow the cells for four days to provide sufficient time for expression of GFP and DsRed proteins.</li>
 <li> (Optional) Three days after transfection verify the efficiency of transfection and expression of GFP and DsRed proteins by fluorescent inverted microscope with filters for detection of GFP and DsRed.</li>
</ol>
4. Analysis of the GFP+ and DsRed+ Cells by Flow Cytometry
<ol>
 <li>Four days after transfection harvest I-SceI transfected cells and the control cells. Resuspend the cells in 500 μL of PBS and transfer cells to the FACS tubes. At this stage it is critical to harvest all the cells and have cells of round shape. To achieve this, it is recommended to use a longer trypsinization time or a stronger trypsin. Confirm that 99% of cells are detached from the plate and have round shape by observing the cells under microscope. Keep the cells on ice protected from light (cover ice bucket with foil). It is possible to store the cells on ice for a few hours. For the best results analyze the cells immediately after harvesting.</li>
 <li>Calibrate FACS with GFP, DsRed, and the negative controls. Adjust voltage and color compensation in order to include all the fluorescent cells in the analysis (Figure 4). Keep in mind that the fluorescent intensity of the experimental samples will be lower than that of the controls.</li>
 <li>Analyze I-SceI transfected cells by FACS. We count at least 20,000 cells for each treatment. To prevent potential interference between GFP and DsRed fluorescence it is recommended to keep the percent of GFP+ or DsRed+ cells below 25%. If the percentage of DsRed+ or GFP+ cells is higher reduce the amount of pDsRed2-N1 or I-SceI plasmid. In our experiments we only needed to adjust the amount of pDsRed2-N1 plasmid, but not of the I-SceI plasmid.</li>
 <li>The percent of GFP+ cells corresponds to the efficiency of DNA DSB repair and the percent of DsRed+ cells indicates the efficiency of transfection. Calculate the relative efficiency of DNA DSB repair as the ratio of GFP+ cells to DsRed+ cells.</li>
 <li> The repaired end junctions may be sequenced to test the accuracy of repair. The reporter constructs contain E.coli origin of replication and kanamycin resistance genes (Figure 1). This enables rescuing the constructs by digesting genomic DNA with EcoRI enzyme, recircularization, and transformation into competent E.coli cells. The rescued plasmids are then analyzed by restriction digestion and sequencing.</li>
</ol>
5. Representative Results
Typical FACS results of the control transfections and of NHEJ and HR experiments are shown in Figures 4 and 5. Fluorescent intensity and the number of fluorescent cells will be lower in the I-SceI transfected cells containing integrated NHEJ and HR constructs (Figure 5) compared to the controls (Figure 4) due to the difference in the copy number of GFP and DsRed constructs in these cells. Each cell in the experiment contains only one copy of the integrated reporter construct, thus successful repair events will reconstitute the GFP gene in a fraction of the cells. Transfection with 0.1 μg pDsRed2-N1 in human fibroblasts delivers approximately 1 plasmid copy per cell.
The efficiency of NHEJ and HR is calculated as a ratio of GFP+/DsRed+ cells. NHEJ is a more efficient process than HR in human cells2. In human fibroblasts the NHEJ efficiency is typically 0.6-1.3, and HR efficiency is 0.05-0.3. Since DSB efficiency is measured as a ratio GFP+/DsRed+ cells variations in the mixture of I-SceI and DsRed2-N1 plasmids may affect the result. The plasmid quality may also affect the results by changing transfection efficiency. Therefore, to obtain consistent measurements, it is important to use the same plasmid mix within one experiment. Furthermore, the DSB repair efficiency is affected by cell type, cell cycle phase, and chromosomal location of the integrated constructs.
 <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Nonhomologous end joining and homologous recombination reporter diagrams, DNA repair pathways." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
 Figure 1. Reporter constructs for the analysis of DNA DSB repair by NHEJ (A) and HR (B). (C) Incompatible DNA ends generated by digestion of two inverted I-SceI sites. SD, splice donor; SA, splice acceptor; shaded squares, polyadenylation sites; Neo/Kana, single ORF controlled by two promoters: SV40 conferring neomycin resistance in mammalian cells; and β-lactamase conferring kanamicyn resistance in E. coli; Ori, E. Coli pUC origin of replication.
<img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Non-homologous end joining (NHEJ) diagram; gene editing, GFP expression analysis." data-altupdatedat="1747911167000" data-alt="Figure 2"> Figure 2. Reporter construct for the analysis of NHEJ. In this construct the GFP gene is inactive; however upon induction of a DSB and successful NHEJ the construct becomes GFP+. Shown below is a repair product of this reporter by NHEJ.
<img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Gene editing diagram with repair pathways; equations: GC, X-over, SSA, NHEJ; GFP indicator." data-altupdatedat="1747911167000" data-alt="Figure 3"> Figure 3. Reporter construct for the analysis of HR by gene conversion. Upon induction of the DSBs by I-SceI, HR by gene conversion (GC) reconstitutes an active GFP gene. Shown below are repair products of this reporter by the major DNA repair pathways: GC, crossing-over (X-
over), single strand annealing (SSA), and NHEJ. X-over repair product is shown for intramolecular recombination, exramolecular recombination will give the same product but located on one chromosome. Genes expressing active GFP protein (GFP+) are indicated by green color.
<img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Flow cytometry results for GFP and DsRed controls. Scatter plots show expression levels." data-altupdatedat="1747911167000" data-alt="Figure 4"> Figure 4. Calibration of the parameters for FACS analysis. (A) Fibroblasts transfected with the pHPRT plasmid, used as a negative control for excluding auto fluorescent cells. (B) Fibroblasts transfected with pEGFP plasmid, used for setting the gating for GFP+ cells (green area). (C) Fibroblasts transfected with pDsRed2-N1 plasmid, used for setting the gating for DsRed+ cells (red area).
<img src="/files/ftp_upload/2002/2002fig5.jpg" alt="NHEJ and HR flow cytometry results; scatter plots showing GFP and DsRed expression in DNA repair study." data-altupdatedat="1747911167000" data-alt="Figure 5"> Figure 5. Typical results of the analysis of NHEJ (A) and HR (B) in normal human fibroblasts with chromosomally integrated reporter constructs.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

No conflicts of interest declared.

The fluorescent NHEJ and HR reporter assays provide a quantitative way for separately measuring each DSB repair pathway in vivo. The assays are very sensitive, as FACS can reliably detect 10 GFP+ cells in 20,000 cells. The assays may be adapted for measuring repair events in "real time" by detecting the appearance of GFP+ cells within minutes or hours after induction of DSBs2. Furthermore, the analysis of GFP+ cells does not rely on additional cell proliferation, allowing DSB repair to be analyzed a...

<table><tbody><tr><td>EndoFree Plasmid Maxi kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Qiaex II Gel Extraction Kit</td><td>Qiagen</td><td>20021</td><td></td></tr><tr><td>Amaxa Nucleofector</td><td>Lonza Inc.</td><td>AAD-1001</td><td></td></tr><tr><td>Geneticin (G418)</td><td>Invitrogen</td><td>11811-031</td><td></td></tr><tr><td>pDsRed2-N1</td><td>Clontech Laboratories</td><td>632406</td><td></td></tr><tr><td>Round bottom tubes</td><td>BD Biosciences</td><td>352058</td><td>FACS tubes</td></tr></tbody></table>

analysis of dna double-strand break (dsb) repair in mammalian cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

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31.6K Views.  University of Rochester. Dit artikel beschrijft op basis van GFP-fluorescentie In vivo Testen die homologe recombinatie afzonderlijk te kwantificeren en niet-homologe eind mee in zoogdiercellen.

Video: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Cancer Research

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Genetics

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins.
Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In ...

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

Detection of G1 Phase Single-Stranded DNA Foci via RPA2 Immunofluorescent Staining

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms.
This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

Author Spotlight: Visualizing Single-Stranded DNA During DNA Repair for Therapeutic Insights 

The following protocol presents the detection of single-stranded DNA foci in the G1 phase of the cell cycle utilizing cell cycle synchronization followed by RPA2 immunofluorescent staining.

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...

FACS Analysis of GFP and mCherry Reporter Cells to Evaluate NHEJ & HR Efficiency

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

Author Spotlight: Decoding DNA Repair by Extrachromosomal NHEJ Assay and HR Assays

This protocol describes an extrachromosomal nonhomologous end joining (NHEJ) assay and homologous recombination (HR) assay to quantify the efficiency of NHEJ and HR in HEK-293T cells.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

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