- Email: [email protected]
Comet and micronucleus assays for analyzing DNA damage and genome integrity
CHAPTER EIGHTEEN
Comet and micronucleus assays for analyzing DNA damage and genome integrity Hui Jianga, Swarupa Pandaa, Nelson O. Gekaraa,b,*
a The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea˚ Centre for Microbial Research (UCMR), Umea˚ University, Umea˚, Sweden b Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden *Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. The comet assay 2.1 Materials 2.2 Methods 2.3 Analysis of the result 3. The micronucleus assay 3.1 Materials 3.2 Methods 3.3 Analysis of the result 4. Summary Acknowledgments References
300 301 301 302 303 304 304 305 305 306 306 307
Abstract Detection of DNA damage in cells is fundamental for the study of DNA repair and genome-instability associated processes including carcinogenesis. Many studies often rely on cytotoxicity assays to estimate genotoxicity. However, measurements of cytotoxicity, a delayed outcome requiring high threshold genotoxicity to induce, does not provide information about the subtle, early genotoxic effects relevant for mechanistic understanding of DNA repair processes. Here describe how to combine two simple procedures for monitoring the presence of DNA damage in individual eukaryotic cells using: (1) the Comet assay for measuring initial DNA breaks and (2) the Micronucleus assay for detecting delayed outcome DNA breaks in dividing cells. We discuss the principles, experimental design considerations and troubleshooting tips for optimizing these methods. They require standard molecular biology instruments and a fluorescent microscope.
Methods in Enzymology, Volume 625 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.05.015
#
2019 Elsevier Inc. All rights reserved.
299
300
Hui Jiang et al.
1. Introduction DNA damage from endogenous and exogenous genotoxic stress is persistent threat to the integrity of all organisms (Ciccia & Elledge, 2010). Cells resolve different types of DNA lesions via distinct pathways including mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), single-strand break repair (SSBR) and double strand breaks repair (DSBR) (Caldecott, 2008; David, O’Shea, & Kundu, 2007; Harfe & Jinks-Robertson, 2000; Sancar, Lindsey-Boltz, Unsal-Kacmaz, & Linn, 2004; Scharer, 2013; Wyman & Kanaar, 2006). Of the different types of DNA lesions, Double strand DNA breaks (DSBs) are the most deleterious and results in genome fragmentation and cell death. If repaired improperly DSBs can also cause chromosome translocation or deletions which may result in malignant transformation and cancer (Basu, 2018; Roos & Kaina, 2006, 2013; Wang, 2001). The most direct approach to detecting DSB in cells is by assaying for the presence of genomic DNA fragments in the cell. The most commonly used technique is the Comet assay, a simple and sensitive method that allows the visualization and quantitative evaluation chromosome fragments at a single cell level (Fairbairn, Olive, & O’Neill, 1995). The comet assay, also known as the single cell gel electrophoresis assay, was firstly developed by Ostling and Johanson (1984). Briefly, damaged DNA fragments migrate faster thereby separating from undamaged intact genomic DNA when electrophoresed in the gel. The more DNA breaks in the cell, the more chromosome fragments generated hence the longer the tails formed by electrophoresis. Therefore, the intensity and length of the comet tail relative to the head reflects the number DSB in the cell. One of the late cellular outcomes of DSB is the generation of micronuclei (MN). How are micronuclei formed? In higher eukaryotes, cell division involves “open” mitosis, involving the complete dissolution of the nuclear membrane and subsequent reassembly as the cell partition the replicated DNA into daughter cells. During this process, whole or broken chromosome fragments can miss-segregate from the main chromatin mass and at the end of mitosis these, miss-segregated chromosome fragments can recruit nuclear membrane components to form micronuclei compartmentally distinct from the primary nucleus, see the schematic representation of micronucleus formation in Fig. 1 (Fenech et al., 2011; Gekara, 2017). Micronuclei also known as Howell–Jolly bodies and first identified in red cell precursors
Comet and micronucleus assays
301
Fig. 1 Schematic representation of micronucleus formation after DNA damage.
by William Howell and Justin Jolly 100 years ago (Howell, 1890; Jolly, 1907) are now well acknowledged as key hallmarks of genome instability. More recently micronuclei have been proposed as platforms for innate immune recognition of genomic DNA by the cytosolic DNA sensor Cyclic GMP-AMP synthase (cGAS) following DNA damage (Gekara, 2017; Harding et al., 2017; Jiang et al., 2018; Mackenzie et al., 2017). Essentially, micronuclei are DNA fragments and can be easily stained by nucleic acid dye. Because micronuclei are structurally smaller, they can readily be distinguished from the main nucleus by standard fluorescence microscopy. Here, we provide detailed protocols for the comet and micronuclei assays and illustrate their application in the analysis of genome integrity.
2. The comet assay Among several versions of the comet assays, the alkaline comet assay is widely used since it is able to detect single-strand breaks (SSBs), doublestrand breaks (DSBs), and alkaline-labile sites (ALS). In this protocol, we described procedures for measuring DNA damage using an alkaline comet assay.
2.1 Materials • •
2-Hydroxyethyl agarose (Low gelling temperature-agarose, Sigma; cat. no. A4018) Phosphate-buffered saline (Ca2+ and Mg2+ free)
302
• • • • • • • •
Hui Jiang et al.
0.5 M EDTA (pH 8.0) 5 M NaOH. Propidium iodide (Sigma; cat. no. P4170) or DAPI may also be used, Sodium laurylsarcosinate (Sigma; cat. no. 61747) OxiSelect™ 3-well Comet Assay Slides (Cell biolabs; cat. no. STA-353) Horizontal gel electrophoresis chamber and power Fluorescence microscopy Image J software
2.2 Methods 2.2.1 Preparation of solutions for comet assay • Alkaline lysis solution: 1.2 M NaCl, 100 mM EDTA, 0.1% sodium laurylsarcosinate, 0.26 M NaOH (pH >13); store at 4 °C. This solution should be prepared freshly for each experiment. • Alkaline electrophoresis and rinse solution: 0.03 M NaOH, 2 mM EDTA (pH >13). 2.2.2 Preparation of comet agarose Dissolve 1 g 2-Hydroxyethyl agarose in 100 mL distilled water by incubating at 100 °C in water bath for 5 min to make 1% comet agarose (Microwaving is not recommended). Maintain agarose at 40 °C in water bath. 2.2.3 Preparation of cell samples Shortly before starting the comet assay, harvest cells (e.g., following exposure to genotoxic agents) and keep on ice to stop DNA repair. • Using ice-cold PBS, collect and wash the cell samples by centrifugation (1500 rpm, 5 min) at 4 °C. • Resuspend cells (1 105 cells/mL) in ice cold 1 PBS (Ca2+ and Mg2+ free, to inhibit endonuclease activities). • Include the appropriate controls, i.e., negative control (undamaged cells) and positive control (e.g., cells treated with genotoxins or γ-irradiation). • Mix 1 105 cells/mL in cold PBS with 1% low-melting agarose at 40 °C at a ratio of 1:3 (v/v). • Pipette the cell/agarose mixture onto a well of CometSlide (100 μL/ well) and use the pipette tip to spread agarose/cells over sample area to ensure complete coverage of the sample area. • Before lysing the cells, make sure the cell/agarose mixture has gelled (usually gelling for 20 min in high humidity environments).
Comet and micronucleus assays
303
2.2.4 Lysis of cells • Before lysing cells, the lysis solution should be freshly prepared and chilled at 4 °C for at least 30 min. • Submerge slide in prechilled lysis solution and leave at 4 °C for overnight in the dark. This step should be done carefully by slowly submerging the slides into solutions and maintaining the slides in horizontal position. 2.2.5 Gel electrophoresis • After cell lysis, gently remove slides from the lysis solution at 4 °C. Then immerse the slides in Alkaline electrophoresis and rinse solution for 10 min in room temperature. • Place the slides on the horizontal electrophoresis chamber already filled with Alkaline electrophoresis and rinse solution. Make sure the buffer covers the slides. • Set the power supply to 20 V (0.6 V/cm) and current at about 40 mA. Then allow electrophoresis to run for 20 min. 2.2.6 Staining • Remove slides from electrophoresis chamber and carefully rinse with distilled water twice. • Use filter paper to absorb water from the slides. Add 100 μL of a 10 μg/mL stock solution of propidium iodide directly onto one well of the slide and incubate 30 min. • Rinse slides with distilled water twice. • Keep the slides moist for further analysis (if in dry condition, the gel will shrink).
2.3 Analysis of the result Use a fluorescence microscope with an excitation of 530 nm to observe the DNA damage at suitable magnification (usually 50–100 comets in one field). The Tail Moment is the most common parameter to for evaluating extent of DNA damage. As shown in Fig. 2, use image analysis software such as Image J to quantitate tail moment length, percentage DNA in tail and tail moment (Tail Moment ¼ Tail DNA% Tail Moment Length). For each sample, 100–200 cells should be analyzed. In Fig. 3, we use γ-irradiation (IR) to induce DNA damage in HEK293 cells for different time to monitor the DNA repair process.
304
Hui Jiang et al.
Fig. 2 An example of damaged DNA in Comet. Representative comet tails of HEK293 cells exposed to γ-irradiation (10 Gy).
Fig. 3 Monitoring the DNA damage repair process by Comet assay. HEK293 cells were γ-irradiated in a 137Cs gamma-ray source (Gammacell 40 irradiator, MDS Nordion) with indicated dose. During irradiation all the cells are keep in ice to stop the DNA repair process. Thereafter, cells were transfer to 37 °C to allow DNA repair and then harvested at indicated time points for analysis.
3. The micronucleus assay 3.1 Materials • • • • •
Phosphate-buffered saline (Ca2+ and Mg2+ free) 4% paraformaldehyde 0.5% Triton X-100 DNA dye 40 ,6-diamidino-2-phenylindole (DAPI) Nocodazole (Sigma; cat. no. SML1665)
Comet and micronucleus assays
• • •
305
Dako fluorescence mounting medium (Agilent) Coverslips Confocal microscope
3.2 Methods The DNA binding innate immune sensor cGAS is abundant in micronuclei hence GFP-tagged cGAS can be used as a marker to monitor micronuclei generation ( Jiang et al., 2018). For illustration here below, we describe micronuclei formation in HEK293 that stably express GFPcGAS (HEK293-GFP-cGAS) or GFP reporter tag with a nuclear localization sequence NLS). • Seed 1 105 cells and cultured on glass coverslips in 6-well plate. • Synchronize the cells with 100 nM nocodazole for about 12 h to arrest the cell cycle at G2 phase. • Expose cells to DNA damage (e.g., genotoxins or γ-irradiation). • Released the synchronized G2 phase cells into mitosis (wash three times with PBS and add warm fresh medium). • After 24 h culture, fix the cells on coverslips by incubating them in 4% paraformaldehyde for 30 min at room temperature. • Permeabilize cells in 0.5% Triton X-100 for 5 min. • Incubate coverslips with DAPI (1 μg/mL) for 10 min in the dark at room temperature. • Wash the coverslips three times with PBS. • Mount coverslips using Dako fluorescence mounting medium (Agilent). • Image cells using the confocal microscope Fig. 4.
3.3 Analysis of the result Micronuclei are defined as discrete DNA aggregates separate from the primary nucleus in cells where interphase primary nuclear morphology is normal. Cells with an apoptotic or necrotic appearance should be excluded. Recent studies show that innate immune DNA sensor cGAS localizes to micronuclei ( Jiang et al., 2018; Mackenzie et al., 2017). Fig. 4 shows that after irradiation, the number of micronuclei in the cell is significantly increased and that GFP-cGAS foci co-localize with micronuclei (DAPI staining). For quantification, count all micronuclei as well as the total number of cells in one microscopic field, then calculate ratio: micronuclei (MN)/ cell. For each sample, at least 50 different microscopic fields with over 500 cells should be analyzed.
306
Hui Jiang et al.
Fig. 4 DNA damage-induced micronuclei generation. The synchronized HEK293 cells stably expressing GFP-cGAS were exposed (or not) to γ-irradiation and cultured for 24 h. Cells were fixed, permeabilized in 0.5% Triton X-100, stained with the DNA dye DAPI, then analyzed by confocal microscopy for micronuclei. The white arrow shows cGAS foci (green spot) co-localized with micronuclei (blue). After DNA damage, the cGAS-micronuclei foci is dramatically increased. Scale bar: 50 μm.
4. Summary In this protocol, we describe two simple methods for measuring DNA damage in eukaryotic cells. The Comet assay for initial DNA damage event and micronucleus assays for later DNA damage event. Both approaches are valuable and useful not only for experimental studies but also for clinical studies. For comet assay, optimization of the time course is the key to understanding of DNA damage repair process. For micronuclei assays, the appropriate cell seeding density and sample collection time point are very important for successful experiment (Micronuclei arise from cell proliferation, hence high cell density will inhibit cell proliferation). Taken together, these two technologies are among the most straightforward, sensitive methods for monitoring DNA damage/repair processes.
Acknowledgments This work was supported by Stockholm University, the Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea˚ University, the Swedish Research Council (grants 2015-02857 and 2016-00890 to N.O.G.) and the Swedish Cancer Foundation (grant, CAN 2017/421 to N.O.G.).
Comet and micronucleus assays
307
References Basu, A. K. (2018). DNA damage, mutagenesis and cancer. International Journal of Molecular Sciences, 19, 970. Caldecott, K. W. (2008). Single-strand break repair and genetic disease. Nature Reviews. Genetics, 9, 619–631. Ciccia, A., & Elledge, S. J. (2010). The DNA damage response: Making it safe to play with knives. Molecular Cell, 40, 179–204. David, S. S., O’Shea, V. L., & Kundu, S. (2007). Base-excision repair of oxidative DNA damage. Nature, 447, 941–950. Fairbairn, D. W., Olive, P. L., & O’Neill, K. L. (1995). The comet assay: A comprehensive review. Mutation Research, 339, 37–59. Fenech, M., Kirsch-Volders, M., Natarajan, A. T., Surralles, J., Crott, J. W., Parry, J., et al. (2011). Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis, 26, 125–132. Gekara, N. O. (2017). DNA damage-induced immune response: Micronuclei provide key platform. The Journal of Cell Biology, 216, 2999–3001. Harding, S. M., Benci, J. L., Irianto, J., Discher, D. E., Minn, A. J., & Greenberg, R. A. (2017). Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature, 548, 466–470. Harfe, B. D., & Jinks-Robertson, S. (2000). DNA mismatch repair and genetic instability. Annual Review of Genetics, 34, 359–399. Howell, W. H. (1890). The life-history of the formed elements of the blood, especially the red blood corpuscles. Journal of Morphology, 4, 57–116. Jiang, H., Panda, S., Xue, X., Liang, F., Sung, P., & Gekara, N. O. (2018). The innate immune DNA sensor cGAS is a negative regulator of DNA repair hence promotes genome instability and cell death. Biorxiv. 465401. Jolly, J. (1907). Recherches sur la formation des globules rouges des mammife`res. In 9. Archives d’anatomie microscopique (pp. 133–314). http://www.sudoc.abes.fr/xslt/ CMD?DB¼2.1&ACT¼SRCHA&PRS¼HOL&HLIB¼751052312&IKT¼8910&TRM¼ 492828151&COOKIE¼U10178,Klecteurweb,I250,B341720009+,SY,NLECTEUR+ WEBOPC,D2.1,Eb0b33e21-ac,A,H,R94.255.148.162,FY. Mackenzie, K. J., Carroll, P., Martin, C. A., Murina, O., Fluteau, A., Simpson, D. J., et al. (2017). cGAS surveillance of micronuclei links genome instability to innate immunity. Nature, 548, 461–465. Ostling, O., & Johanson, K. J. (1984). Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochemical and Biophysical Research Communications, 123, 291–298. Roos, W. P., & Kaina, B. (2006). DNA damage-induced cell death by apoptosis. Trends in Molecular Medicine, 12, 440–450. Roos, W. P., & Kaina, B. (2013). DNA damage-induced cell death: From specific DNA lesions to the DNA damage response and apoptosis. Cancer Letters, 332, 237–248. Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., & Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry, 73, 39–85. Scharer, O. D. (2013). Nucleotide excision repair in eukaryotes. Cold Spring Harbor Perspectives in Biology, 5, a012609. Wang, Z. (2001). DNA damage-induced mutagenesis : A novel target for cancer prevention. Molecular Interventions, 1, 269–281. Wyman, C., & Kanaar, R. (2006). DNA double-strand break repair: All’s well that ends well. Annual Review of Genetics, 40, 363–383.
Comet and micronucleus assays for analyzing DNA damage and genome integrity Hui Jianga, Swarupa Pandaa, Nelson O. Gekaraa,b,*
a The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea˚ Centre for Microbial Research (UCMR), Umea˚ University, Umea˚, Sweden b Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden *Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. The comet assay 2.1 Materials 2.2 Methods 2.3 Analysis of the result 3. The micronucleus assay 3.1 Materials 3.2 Methods 3.3 Analysis of the result 4. Summary Acknowledgments References
300 301 301 302 303 304 304 305 305 306 306 307
Abstract Detection of DNA damage in cells is fundamental for the study of DNA repair and genome-instability associated processes including carcinogenesis. Many studies often rely on cytotoxicity assays to estimate genotoxicity. However, measurements of cytotoxicity, a delayed outcome requiring high threshold genotoxicity to induce, does not provide information about the subtle, early genotoxic effects relevant for mechanistic understanding of DNA repair processes. Here describe how to combine two simple procedures for monitoring the presence of DNA damage in individual eukaryotic cells using: (1) the Comet assay for measuring initial DNA breaks and (2) the Micronucleus assay for detecting delayed outcome DNA breaks in dividing cells. We discuss the principles, experimental design considerations and troubleshooting tips for optimizing these methods. They require standard molecular biology instruments and a fluorescent microscope.
Methods in Enzymology, Volume 625 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.05.015
#
2019 Elsevier Inc. All rights reserved.
299
300
Hui Jiang et al.
1. Introduction DNA damage from endogenous and exogenous genotoxic stress is persistent threat to the integrity of all organisms (Ciccia & Elledge, 2010). Cells resolve different types of DNA lesions via distinct pathways including mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), single-strand break repair (SSBR) and double strand breaks repair (DSBR) (Caldecott, 2008; David, O’Shea, & Kundu, 2007; Harfe & Jinks-Robertson, 2000; Sancar, Lindsey-Boltz, Unsal-Kacmaz, & Linn, 2004; Scharer, 2013; Wyman & Kanaar, 2006). Of the different types of DNA lesions, Double strand DNA breaks (DSBs) are the most deleterious and results in genome fragmentation and cell death. If repaired improperly DSBs can also cause chromosome translocation or deletions which may result in malignant transformation and cancer (Basu, 2018; Roos & Kaina, 2006, 2013; Wang, 2001). The most direct approach to detecting DSB in cells is by assaying for the presence of genomic DNA fragments in the cell. The most commonly used technique is the Comet assay, a simple and sensitive method that allows the visualization and quantitative evaluation chromosome fragments at a single cell level (Fairbairn, Olive, & O’Neill, 1995). The comet assay, also known as the single cell gel electrophoresis assay, was firstly developed by Ostling and Johanson (1984). Briefly, damaged DNA fragments migrate faster thereby separating from undamaged intact genomic DNA when electrophoresed in the gel. The more DNA breaks in the cell, the more chromosome fragments generated hence the longer the tails formed by electrophoresis. Therefore, the intensity and length of the comet tail relative to the head reflects the number DSB in the cell. One of the late cellular outcomes of DSB is the generation of micronuclei (MN). How are micronuclei formed? In higher eukaryotes, cell division involves “open” mitosis, involving the complete dissolution of the nuclear membrane and subsequent reassembly as the cell partition the replicated DNA into daughter cells. During this process, whole or broken chromosome fragments can miss-segregate from the main chromatin mass and at the end of mitosis these, miss-segregated chromosome fragments can recruit nuclear membrane components to form micronuclei compartmentally distinct from the primary nucleus, see the schematic representation of micronucleus formation in Fig. 1 (Fenech et al., 2011; Gekara, 2017). Micronuclei also known as Howell–Jolly bodies and first identified in red cell precursors
Comet and micronucleus assays
301
Fig. 1 Schematic representation of micronucleus formation after DNA damage.
by William Howell and Justin Jolly 100 years ago (Howell, 1890; Jolly, 1907) are now well acknowledged as key hallmarks of genome instability. More recently micronuclei have been proposed as platforms for innate immune recognition of genomic DNA by the cytosolic DNA sensor Cyclic GMP-AMP synthase (cGAS) following DNA damage (Gekara, 2017; Harding et al., 2017; Jiang et al., 2018; Mackenzie et al., 2017). Essentially, micronuclei are DNA fragments and can be easily stained by nucleic acid dye. Because micronuclei are structurally smaller, they can readily be distinguished from the main nucleus by standard fluorescence microscopy. Here, we provide detailed protocols for the comet and micronuclei assays and illustrate their application in the analysis of genome integrity.
2. The comet assay Among several versions of the comet assays, the alkaline comet assay is widely used since it is able to detect single-strand breaks (SSBs), doublestrand breaks (DSBs), and alkaline-labile sites (ALS). In this protocol, we described procedures for measuring DNA damage using an alkaline comet assay.
2.1 Materials • •
2-Hydroxyethyl agarose (Low gelling temperature-agarose, Sigma; cat. no. A4018) Phosphate-buffered saline (Ca2+ and Mg2+ free)
302
• • • • • • • •
Hui Jiang et al.
0.5 M EDTA (pH 8.0) 5 M NaOH. Propidium iodide (Sigma; cat. no. P4170) or DAPI may also be used, Sodium laurylsarcosinate (Sigma; cat. no. 61747) OxiSelect™ 3-well Comet Assay Slides (Cell biolabs; cat. no. STA-353) Horizontal gel electrophoresis chamber and power Fluorescence microscopy Image J software
2.2 Methods 2.2.1 Preparation of solutions for comet assay • Alkaline lysis solution: 1.2 M NaCl, 100 mM EDTA, 0.1% sodium laurylsarcosinate, 0.26 M NaOH (pH >13); store at 4 °C. This solution should be prepared freshly for each experiment. • Alkaline electrophoresis and rinse solution: 0.03 M NaOH, 2 mM EDTA (pH >13). 2.2.2 Preparation of comet agarose Dissolve 1 g 2-Hydroxyethyl agarose in 100 mL distilled water by incubating at 100 °C in water bath for 5 min to make 1% comet agarose (Microwaving is not recommended). Maintain agarose at 40 °C in water bath. 2.2.3 Preparation of cell samples Shortly before starting the comet assay, harvest cells (e.g., following exposure to genotoxic agents) and keep on ice to stop DNA repair. • Using ice-cold PBS, collect and wash the cell samples by centrifugation (1500 rpm, 5 min) at 4 °C. • Resuspend cells (1 105 cells/mL) in ice cold 1 PBS (Ca2+ and Mg2+ free, to inhibit endonuclease activities). • Include the appropriate controls, i.e., negative control (undamaged cells) and positive control (e.g., cells treated with genotoxins or γ-irradiation). • Mix 1 105 cells/mL in cold PBS with 1% low-melting agarose at 40 °C at a ratio of 1:3 (v/v). • Pipette the cell/agarose mixture onto a well of CometSlide (100 μL/ well) and use the pipette tip to spread agarose/cells over sample area to ensure complete coverage of the sample area. • Before lysing the cells, make sure the cell/agarose mixture has gelled (usually gelling for 20 min in high humidity environments).
Comet and micronucleus assays
303
2.2.4 Lysis of cells • Before lysing cells, the lysis solution should be freshly prepared and chilled at 4 °C for at least 30 min. • Submerge slide in prechilled lysis solution and leave at 4 °C for overnight in the dark. This step should be done carefully by slowly submerging the slides into solutions and maintaining the slides in horizontal position. 2.2.5 Gel electrophoresis • After cell lysis, gently remove slides from the lysis solution at 4 °C. Then immerse the slides in Alkaline electrophoresis and rinse solution for 10 min in room temperature. • Place the slides on the horizontal electrophoresis chamber already filled with Alkaline electrophoresis and rinse solution. Make sure the buffer covers the slides. • Set the power supply to 20 V (0.6 V/cm) and current at about 40 mA. Then allow electrophoresis to run for 20 min. 2.2.6 Staining • Remove slides from electrophoresis chamber and carefully rinse with distilled water twice. • Use filter paper to absorb water from the slides. Add 100 μL of a 10 μg/mL stock solution of propidium iodide directly onto one well of the slide and incubate 30 min. • Rinse slides with distilled water twice. • Keep the slides moist for further analysis (if in dry condition, the gel will shrink).
2.3 Analysis of the result Use a fluorescence microscope with an excitation of 530 nm to observe the DNA damage at suitable magnification (usually 50–100 comets in one field). The Tail Moment is the most common parameter to for evaluating extent of DNA damage. As shown in Fig. 2, use image analysis software such as Image J to quantitate tail moment length, percentage DNA in tail and tail moment (Tail Moment ¼ Tail DNA% Tail Moment Length). For each sample, 100–200 cells should be analyzed. In Fig. 3, we use γ-irradiation (IR) to induce DNA damage in HEK293 cells for different time to monitor the DNA repair process.
304
Hui Jiang et al.
Fig. 2 An example of damaged DNA in Comet. Representative comet tails of HEK293 cells exposed to γ-irradiation (10 Gy).
Fig. 3 Monitoring the DNA damage repair process by Comet assay. HEK293 cells were γ-irradiated in a 137Cs gamma-ray source (Gammacell 40 irradiator, MDS Nordion) with indicated dose. During irradiation all the cells are keep in ice to stop the DNA repair process. Thereafter, cells were transfer to 37 °C to allow DNA repair and then harvested at indicated time points for analysis.
3. The micronucleus assay 3.1 Materials • • • • •
Phosphate-buffered saline (Ca2+ and Mg2+ free) 4% paraformaldehyde 0.5% Triton X-100 DNA dye 40 ,6-diamidino-2-phenylindole (DAPI) Nocodazole (Sigma; cat. no. SML1665)
Comet and micronucleus assays
• • •
305
Dako fluorescence mounting medium (Agilent) Coverslips Confocal microscope
3.2 Methods The DNA binding innate immune sensor cGAS is abundant in micronuclei hence GFP-tagged cGAS can be used as a marker to monitor micronuclei generation ( Jiang et al., 2018). For illustration here below, we describe micronuclei formation in HEK293 that stably express GFPcGAS (HEK293-GFP-cGAS) or GFP reporter tag with a nuclear localization sequence NLS). • Seed 1 105 cells and cultured on glass coverslips in 6-well plate. • Synchronize the cells with 100 nM nocodazole for about 12 h to arrest the cell cycle at G2 phase. • Expose cells to DNA damage (e.g., genotoxins or γ-irradiation). • Released the synchronized G2 phase cells into mitosis (wash three times with PBS and add warm fresh medium). • After 24 h culture, fix the cells on coverslips by incubating them in 4% paraformaldehyde for 30 min at room temperature. • Permeabilize cells in 0.5% Triton X-100 for 5 min. • Incubate coverslips with DAPI (1 μg/mL) for 10 min in the dark at room temperature. • Wash the coverslips three times with PBS. • Mount coverslips using Dako fluorescence mounting medium (Agilent). • Image cells using the confocal microscope Fig. 4.
3.3 Analysis of the result Micronuclei are defined as discrete DNA aggregates separate from the primary nucleus in cells where interphase primary nuclear morphology is normal. Cells with an apoptotic or necrotic appearance should be excluded. Recent studies show that innate immune DNA sensor cGAS localizes to micronuclei ( Jiang et al., 2018; Mackenzie et al., 2017). Fig. 4 shows that after irradiation, the number of micronuclei in the cell is significantly increased and that GFP-cGAS foci co-localize with micronuclei (DAPI staining). For quantification, count all micronuclei as well as the total number of cells in one microscopic field, then calculate ratio: micronuclei (MN)/ cell. For each sample, at least 50 different microscopic fields with over 500 cells should be analyzed.
306
Hui Jiang et al.
Fig. 4 DNA damage-induced micronuclei generation. The synchronized HEK293 cells stably expressing GFP-cGAS were exposed (or not) to γ-irradiation and cultured for 24 h. Cells were fixed, permeabilized in 0.5% Triton X-100, stained with the DNA dye DAPI, then analyzed by confocal microscopy for micronuclei. The white arrow shows cGAS foci (green spot) co-localized with micronuclei (blue). After DNA damage, the cGAS-micronuclei foci is dramatically increased. Scale bar: 50 μm.
4. Summary In this protocol, we describe two simple methods for measuring DNA damage in eukaryotic cells. The Comet assay for initial DNA damage event and micronucleus assays for later DNA damage event. Both approaches are valuable and useful not only for experimental studies but also for clinical studies. For comet assay, optimization of the time course is the key to understanding of DNA damage repair process. For micronuclei assays, the appropriate cell seeding density and sample collection time point are very important for successful experiment (Micronuclei arise from cell proliferation, hence high cell density will inhibit cell proliferation). Taken together, these two technologies are among the most straightforward, sensitive methods for monitoring DNA damage/repair processes.
Acknowledgments This work was supported by Stockholm University, the Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea˚ University, the Swedish Research Council (grants 2015-02857 and 2016-00890 to N.O.G.) and the Swedish Cancer Foundation (grant, CAN 2017/421 to N.O.G.).
Comet and micronucleus assays
307
References Basu, A. K. (2018). DNA damage, mutagenesis and cancer. International Journal of Molecular Sciences, 19, 970. Caldecott, K. W. (2008). Single-strand break repair and genetic disease. Nature Reviews. Genetics, 9, 619–631. Ciccia, A., & Elledge, S. J. (2010). The DNA damage response: Making it safe to play with knives. Molecular Cell, 40, 179–204. David, S. S., O’Shea, V. L., & Kundu, S. (2007). Base-excision repair of oxidative DNA damage. Nature, 447, 941–950. Fairbairn, D. W., Olive, P. L., & O’Neill, K. L. (1995). The comet assay: A comprehensive review. Mutation Research, 339, 37–59. Fenech, M., Kirsch-Volders, M., Natarajan, A. T., Surralles, J., Crott, J. W., Parry, J., et al. (2011). Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis, 26, 125–132. Gekara, N. O. (2017). DNA damage-induced immune response: Micronuclei provide key platform. The Journal of Cell Biology, 216, 2999–3001. Harding, S. M., Benci, J. L., Irianto, J., Discher, D. E., Minn, A. J., & Greenberg, R. A. (2017). Mitotic progression following DNA damage enables pattern recognition within micronuclei. Nature, 548, 466–470. Harfe, B. D., & Jinks-Robertson, S. (2000). DNA mismatch repair and genetic instability. Annual Review of Genetics, 34, 359–399. Howell, W. H. (1890). The life-history of the formed elements of the blood, especially the red blood corpuscles. Journal of Morphology, 4, 57–116. Jiang, H., Panda, S., Xue, X., Liang, F., Sung, P., & Gekara, N. O. (2018). The innate immune DNA sensor cGAS is a negative regulator of DNA repair hence promotes genome instability and cell death. Biorxiv. 465401. Jolly, J. (1907). Recherches sur la formation des globules rouges des mammife`res. In 9. Archives d’anatomie microscopique (pp. 133–314). http://www.sudoc.abes.fr/xslt/ CMD?DB¼2.1&ACT¼SRCHA&PRS¼HOL&HLIB¼751052312&IKT¼8910&TRM¼ 492828151&COOKIE¼U10178,Klecteurweb,I250,B341720009+,SY,NLECTEUR+ WEBOPC,D2.1,Eb0b33e21-ac,A,H,R94.255.148.162,FY. Mackenzie, K. J., Carroll, P., Martin, C. A., Murina, O., Fluteau, A., Simpson, D. J., et al. (2017). cGAS surveillance of micronuclei links genome instability to innate immunity. Nature, 548, 461–465. Ostling, O., & Johanson, K. J. (1984). Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochemical and Biophysical Research Communications, 123, 291–298. Roos, W. P., & Kaina, B. (2006). DNA damage-induced cell death by apoptosis. Trends in Molecular Medicine, 12, 440–450. Roos, W. P., & Kaina, B. (2013). DNA damage-induced cell death: From specific DNA lesions to the DNA damage response and apoptosis. Cancer Letters, 332, 237–248. Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., & Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry, 73, 39–85. Scharer, O. D. (2013). Nucleotide excision repair in eukaryotes. Cold Spring Harbor Perspectives in Biology, 5, a012609. Wang, Z. (2001). DNA damage-induced mutagenesis : A novel target for cancer prevention. Molecular Interventions, 1, 269–281. Wyman, C., & Kanaar, R. (2006). DNA double-strand break repair: All’s well that ends well. Annual Review of Genetics, 40, 363–383.