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Summary

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.

Abstract

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 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 “foci”, 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.

Introduction

Human cells are constantly exposed to a variety of DNA damaging agents of various origins. Exogenous sources mostly consist of exposure to radiations, chemicals (including chemotherapeutic agents and some antibiotics), and viruses, while the main endogenous sources include errors in DNA replication and oxidative stress. The direct effects of genotoxic exposure can range from a modified base to a potentially lethal DNA double-strand break (DSB), depending on the stress and the exposure dose. Ultimately, unrepaired or mis-repaired DNA damage can lead to the accumulation of mutations, genomic rearrangements, genome instability and eventually lead to carcinogenesis¹. Mammalian cells have evolved complex pathways to recognize specific types of DNA damage^2,3 and repair them in a timely fashion, synchronized with the cell cycle progression.

Ionizing radiation (IR) damages the DNA double helix and creates double-strand breaks (DSBs), one of the most deleterious forms of DNA damage. The MRN (MRE11, RAD50, NBS1) complex functions as a sensor of DNA ends and activates the protein kinase ataxia telangiectasia mutated (ATM)^4,5. Following the initial activation of ATM by DNA ends, ATM triggers a cascade of DDR events at the site of the break, initiating with a key event, the phosphorylation of the histone variant H2AX⁶. H2AX phosphorylation on residue S139 activates it into γH2AX, spanning regions up to megabases around the DNA lesion^6,7,8,9. This event increases DNA accessibility, leading to the recruitment and accumulation of other DNA repair proteins⁷. Because γH2AX is abundantly and specifically induced surrounding DSBs, it can be readily visualized using specific antibodies, and is commonly used as a surrogate marker for DSBs in the DNA repair field. Once the break is signaled, cells activate their DNA repair pathways and process the DNA damage. The protein MDC1 (mediator of DNA damage checkpoint protein 1) directly binds γH2AX¹⁰, interacts with ATM¹¹ and also with NBS1^12,13. It contributes to increasing the concentration of MRN complex at the DSB and initiating a positive ATM feedback loop. γH2AX is rapidly removed once the break is repaired, consequently, allowing the monitoring of DSB clearance. Followed by microscopy, the decrease in γH2AX over time provides an indirect measurement of residual breaks and DNA repair efficiency.

Eukaryotic cells can repair DSBs by several pathways, the two main ones being non-homologous end-joining (NHEJ) and homologous recombination (HR) (Figure 1). NHEJ essentially ligates DNA double-strand ends without the use of extended homology and operates throughout the cell cycle^14,15. HR becomes predominant during S and G2 phases, and is otherwise repressed, since it requires a sister chromatid as a homologous template for repair^14,16. Pathway choice between NHEJ and HR not only depends on the physical proximity of the sister chromatid, but also on the extend of DNA end resection¹⁷, which inhibits NHEJ.

Homology-dependent DSB repair initiates by nucleolytic degradation of the 5’ strand from the break ends to generate 3’ single-strand DNA (ssDNA) tails, a process referred to as 5’-3’ resection. The MRN complex initiates DNA end resection and further resection is processed in combination with BLM/EXO1 (Bloom syndrome protein/exonuclease 1) or BLM/DNA2 (DNA replication ATP-dependent helicase/nuclease)^{18,19,20,21,22}. DNA end resection is enhanced by CtIP (CtBP-interacting protein) through its direct interaction with MRN complex²³ and recruitment of BRCA1 (breast cancer type 1 susceptibility protein)^24,25. Replication protein A (RPA) promptly binds to the ssDNA exposed and is then displaced by the recombinase protein RAD51 to form a nucleoprotein filament that catalyzes homologous search and strand invasion^26,27,28.

The initiation of resection is a critical step for repair pathway choice. Once resection has initiated, the DNA ends become poor substrates for binding by Ku70/Ku80 heterodimer (component of NHEJ pathway) and cells are committed to HR^17,29,30. The Ku70/Ku80 heterodimer binds to DSB ends, recruiting DNA-PKcs and p53 Binding Protein 1 (53BP1)^29,30. 53BP1 acts as a barrier to resection in G1, thus blocking HR while promoting NHEJ^31,32, but it is removed in a BRCA1-dependent manner in S phase, consequently allowing resection to occur^33,34. Therefore, 53BP1 and BRCA1 play opposing roles in DSB repair, with 53BP1 being a NHEJ facilitator whilst BRCA1 acts enabling breaks to repair through HR.

In the laboratory, DSB formation can be induced by ionizing radiation (IR). While this example utilizes a high dose of 4 Gy, 1 Gy and 2 Gy also create a significant amount of DSBs, suitable for the analysis of foci formation by abundant proteins. It is important to note that the type and dose of radiation used can lead to different lesions in the DNA and in the cell: while IR induces DSBs, it can also cause single strand breaks or base modification (see^35,36 for a reference on irradiation linear energy transfer (LET) and type of DNA damage). To determine the kinetics of ionizing radiation-induced foci (IRIF) formation and their clearance, which indicate repair of the damage and reversal of the activated DDR^8,9,37,38, foci formation can be monitored at different time points after ionizing radiation. Timing of activation and clearance of all major DNA damage proteins is known³⁹, and many are investigated as surrogate markers of key events. For example, pRPA, which possesses high affinity for ssDNA is used as a surrogate of the break resection, MRN proteins (MRE11, RAD50, NBS1) and exonucleases can be used to assess resection efficiency too. While RAD51, BRCA1, BRCA2 (breast cancer type 2 susceptibility protein), and PALB2 (partner and localizer of BRCA2) can be monitored to evaluate HR efficiency, the presence of the Ku proteins or 53BP1, are used as markers of NHEJ (Figure 1).

As proteins of the DNA repair machinery recruit each other to the break and assemble in super-complexes, DNA-protein and protein-protein interactions can be inferred by following their individual localization over time and analyzing co-localization of proteins, as visualized by overlapping signals in cell^40,41,42. In cell lines, the introduction of point mutations or deletion in specific DNA repair genes either through genome editing, or by overexpression of plasmid-based mutants, allows investigation of specific residues and their possible role in recognition of DNA damage (e.g., co-localization with γH2AX) or complex assembly (co-localization with another, or several, proteins), as well as their impact on DNA repair. Here, we use indirect immunofluorescence as a mean to investigate the formation and resolution of DSBs by following γH2AX foci over time. We also present one example of foci formation and co-localization analysis by a major player in DSB repair: p53 Binding Protein 1 (53BP1)³². As mentioned earlier, 53BP1 is considered central to DNA repair pathway choice. Following 53BP1 accumulation and its co-localization with γH2AX provides precious information on cell cycle phase, DNA damage accumulation, and pathway used to repair DSBs. The purpose of indirect immunolocalization is to assess the efficiency of DNA damage repair in cell lines, following IR like in this study, or after exposure to various stresses in cell, from DNA crosslinking to blockage of the replication fork (a list of DNA damaging agents is provided in Table 1).

Figure 1: DNA double strand breaks (DSB) repair pathways.
DSB repair involves two major pathways: Homologous Recombination (HR, left) and Non-Homologous End-Joining (NHEJ, right). Following the break, proteins get activated to mark the break (γH2AX), participate in end resection (MRN), coat the resected ssDNA (pRPA), promote recombination (BRCA1, PALB2, BRCA2, RAD51) or limit resection and promote NHEJ (53BP1). Other proteins participate in damage repair, but proteins listed are routinely followed by indirect immunofluorescence. Please click here to view a larger version of this figure.

DNA damaging agent	Mechanism of action	Recommended dose
γ-rays/X-rays	Radiation Formation of double-stranded breaks with some uncontrolled cellular effects	1-4 Gy
36Ar ions	Radiation Formation of double-stranded breaks	270 keV/μm
α-particles	Radiation Formation of double-stranded breaks	116 keV/μm
Bleomycin	Inhibitor of DNA synthesis	0.4-2 μg/mL
Camptothecin	Inhibitor of topoisomerase I	10-200 nM
Cisplatin	Alkylating agent (inducing intrastrand crosslinks)	0.25-2 μM
Doxorubicin	Intercalating agent Inhibitor of topoisomerase II	10-200 nM
Etoposide	Inhibitor of topoisomerase II	10 μM
Hydroxyurea	Inhibitor of DNA synthesis (by ribonucleotide reductase)	10-200 μM
Methyl methanesulfonate	Alkylating agent	0.25-2 mM
Mitomycin C	Alkylating agent	0.25-2 μM
Ultraviolet (UV) light	Formation of thymidine dimers (generating distortion of DNA chain)	50-100 mJ/cm2

Table 1: Genotoxic agents. Examples of DNA damaging agents, their mechanism of action and the damage induced based on suggested working concentration.

Protocol

1. HeLa cell culture Pre-treat round glass coverslips with 1 M HCl at 50 °C for 16-18 hours. Rinse with ddH2O and store in 100% EtOH. Prepare cell culture medium: add 10% (v/v) FBS to DMEM medium. Grow 4.0 x 104 cells/well in sterile 12-well plate with an 18 mm round glass coverslip at 37 °C and 5% CO2 in a humidified incubator. Grow cells to 80% confluency (approximately 24 hours). 2. Cell treatment with radiation (IR) To induce the formation of double-strand breaks, expose cells to 4 Gy γ-irradiation (control: No irradiation, t=0). In the Gamma Cell -40, this corresponds to 4.54 min, at 0.815 Gy per min. Incubate cells at 37 °C and 5% CO2 in a humidified incubator for the appropriate time-length (time points chosen here t=1, 2, 4, 16 h). 3. Nuclear extraction and cell fixation Prepare stock solutions: 0.2 M PIPES (pH 6.8), 5 M NaCl, 2 M sucrose, 1 M MgCl2, 0.1 M EGTA (pH 8.0). Prepare nuclear extraction buffer (NEB): dissolve 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0) and 0.5% (v/v) Triton X-100 in ddH2O. Mix until dissolved completely. Prepare 4% (v/v) paraformaldehyde (PFA): dissolve 10 mL of 16% PFA aqueous solution in 30 mL PBS. Mix until dissolved completely. At appropriate time point (t=0, 1, 2, 4, 16 h), wash cells twice with 1 mL of PBS. Remove PBS completely. Add 200 μL of NEB to each well for cell nuclei extraction (cytoplasm is degraded, only nucleus remains) (Figure 2). Incubate for 2 minutes at room temperature and remove completely. NOTE: Do not exceed 2 minutes. Figure 2: Nuclear extraction. Representative images of cells prior to (left) and post (right) nuclear extraction. Cytoplasm should be digested but the nuclear structure should remain intact post extraction (right). (A) 20x magnification; scale bar = 20 μm and (B) 40x magnification; scale bar = 10 μm. Please click here to view a larger version of this figure. Wash cells with 1 mL of PBS. Remove PBS completely. Add PBS carefully, cells are very fragile at this step. Add 200 μL of 4% (v/v) PFA to each well for cell fixation. Incubate for 10 minutes at 4 °C. Remove PFA completely. Add 1 mL of PBS. NOTE: Cells can be stored in PBS at 4 °C. 4. Immunofluorescence staining Prepare blocking solution: dissolve 5% BSA (w/v) in PBS and add 0.3% (v/v) Triton X-100. Mix until completely dissolved. Prepare dilution buffer: dissolve 1% BSA (w/v) in PBS and add 0.3% (v/v) Triton X-100. Mix until completely dissolved. For blocking, add 200 μL of blocking solution to each well. Incubate for 2 hours at room temperature or 16-18 hours at 4 °C. NOTE: If goat antibody will be used, add 5% goat serum to blocking solution. Dilute primary antibody in dilution buffer (1:500; see Table 2 for antibodies list) and vortex until well mixed. NOTE: If goat antibody is used, add 1% goat serum to dilution buffer. In a humidity/incubation box, adhere a piece of parafilm. Add 10 μL of primary antibody (in a single drop). Align one edge of the coverslip with the drop and slowly lower onto the parafilm, for the liquid to spread throughout (avoid bubbles if possible). Incubate for 2 hours at room temperature. Wash coverslips three times in PBS for 1 minute. Dilute secondary antibody in dilution buffer (final concentration: 2 μg/mL) and vortex until well mixed. Apply 10 μL of secondary antibody as described for the primary antibodies. Incubate for 2 hours at room temperature. NOTE: Protect from light. Antibody Company Reference Source 53BP1 Cell Signaling 4937 Rabbit Anti-Mouse IgG H&L (Alexa Fluor 647) Abcam ab150103 Donkey Anti-phospho-Histone H2A.X (Ser139), clone JBW301 Millipore 05-636 Mouse Anti-Rabbit IgG H&L (Alexa Fluor 488) Abcam ab150081 Goat Table 2: Antibodies used. List of antibodies used in this study. Wash coverslips three times in PBS for 1 minute. Wash coverslips with H2O for 1 minute. Counterstain DNA with DAPI: apply 10 μL of 300 nM DAPI (as described for antibodies), incubate for 30 minutes at room temperature and then mount onto glass slide with a glycerol based mounting media. Alternatively, add one drop (10 μL) of commercial antifade mounting media containing DAPI onto a slide and apply a coverslip. Gently press the coverslip and remove excess fluid around it with a paper towel. Seal coverslips with transparent nail polish and let them dry for 20 minutes. Store slides at 4 °C. 5. Image acquisition Place a drop of immersion oil onto the 60x objective lens. Use DAPI to locate the nuclei through eye piece. For XYZ image acquisition, open acquisition software and select parameters: Scanner type: Galvano; Scanner mode: Roundtrip; Image size: 512×512; PMT mode: VBF; PMT average: frame (4 times); PMT sequential scan: line. Select the dye and the detectors: Channel (CH1), Dye (DAPI), Detector (SD1) Channel (CH2), Dye (Alexa Fluor 488), Detector (HSD3) Channel (CH3), Dye (Alexa Fluor 647), Detector (HSD4) Select ON in “Z”. Adjust the live image. Press the Live button on the Live window. Adjust the focus and set laser intensity (%), sensitivity (HV), gain and offset on “PMT” tool window. Adjust the laser intensity (%): for brightness and bleaching. The higher the laser intensity, the stronger the signal, but the specimen will photobleach. Adjust the sensitivity (HV): noise level. The higher the HV, the stronger the signal, but image will be noisy if too high. NOTE: Always keep voltage constant. Adjust the offset: background level. Select Start/End (15 slices), for Z stacks. Start the acquisition. Select the folder to save images. Press the LSM Start button to start acquiring the image. Press the Series Done button to complete the image acquisition. 6. Data analysis For image analysis, open the analysis software. Press the Batch tool window, select the images to analyze and select output folder. Press the Analysis tool window and select Projection (will display the maximum intensity projection from 15 slices). Under Input/Output setting, select the batch created. Press Process for images to be processed. Export images as .tiff files. For nuclear foci quantification, open CellProfiler. Open the γH2AX and 53BP1 foci quantification pipeline (see Supplemental information). Graph data using a table software. For co-localization analysis, open CellProfiler. Open the Colocalization pipeline (see Supplemental information). A spreadsheet file will be created and saved in the preferred location. However, graphs themselves will not be automatically saved. It is suggested to take a snapshot of the windows to keep for record of the results. Graph data using a table software.

Representative Results

On day 2, or 24 h post seeding cells on coverslips, cells have undergone one division and are 80% confluent. Specific knock downs or mutations in DNA repair proteins can increase doubling time, or sensitize cells to genotoxic treatment, and seeding density as well as treatment doses should be adjusted accordingly. To determine best working conditions, timing of the DNA damage response can be established by Western blotting of γH2AX over time, and sensitivity to irradiation can be identified by colony forming assay.<…

Discussion

Analysis of the timing and efficiency of DNA damage repair by microscopy has proven essential to understand how the DNA repair machinery functions, in normal cells and in human pathologies such as cancer.

The development of specific antibodies that allow detection of activated proteins in their phosphorylated version (such as γH2AX, pRPA, pRAD50 and others^{10,23,39,43<…}

Materials

16% (v/v) paraformaldehyde (PFA) aqueous solution	Electron Microscopy Sciences	15710	Microscopy quality of the PFA ensures best images. If using "home-made PFA", filter prior to use.
Bovine serum albumin (BSA)	Sigma-Aldrich	A3059	Heat-shock fraction is recommended, to avoid precipitation/background.
Coverglass #1, 18 mm round (coverslips)	Neuvitro	NC0308920	Coverslips need to be cleaned and sterilized prior using, with HCl or ethanol.
DMEM, High Glucose [(+) 4.5 g/L D-Glucose, (+) L-Glutamine, (-) Sodium Pyruvate]	Gibco	11965118	Adjust the growing media to the needs of cell line used.
DPBS, no calcium, no magnesium	Gibco	14190144	PBS for tissue culture.
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)	Research Products International	E57060	Nuclear extraction buffer.
Fetal Bovine Serum (FBS)	Life Technologies	104370028	The quality of FBS can be assessed by testing gH2AX foci formation. If traces of genotoxic endotoxin are present in the batch, gH2AX will be positive in the absence of stress.
Magnesium chloride (MgCl2)	Research Products International	M24000	Nuclear extraction buffer.
Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)	Research Products International	P40150	Nuclear extraction buffer.
SlowFade Diamond Antifade Mountant with DAPI	Invitrogen	S36973	300 nM DAPI with VECTASHIELD Antifade Mounting Medium can be used instead.
Sodium chloride (NaCl)	Research Products International	S23020	Nuclear extraction buffer.
Sucrose	Research Products International	S24060	Nuclear extraction buffer.
Superfrost Plus Microscope Slides	Fisherbrand	1255015	Polysine Slides can be used instead.
TC-Treated Multiple Well Plates, size 12 wells	Costar	3513	Seeding on coverslips is done in 12-wells plate.
Triton X-100	AmericanBio	AB02025	Nuclear extraction buffer.
TrypLE Express Enzyme (1X), No Phenol Red	Gibco	12604021	Trypsin-EDTA can be used instead.
Trypsin-EDTA (0.5%), No Phenol Red	Gibco	15400054	TrypLE can be used instead.