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Sep 2021

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A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells
一种基于流式细胞术的分析 G0和G1期哺乳动物细胞中 DNA 末端切除的方法   

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Abstract

DNA double strand breaks (DSBs) constantly arise in cells during normal cellular processes or upon exposure to genotoxic agents, and are repaired mostly by homologous recombination (HR) and non-homologous end joining (NHEJ). One key determinant of DNA DSB repair pathway choice is the processing of broken DNA ends to generate single strand DNA (ssDNA) overhangs, a process termed DNA resection. The generation of ssDNA overhangs commits DSB repair through HR and inhibits NHEJ. Therefore, DNA resection must be carefully regulated to avoid mis-repaired or persistent DSBs. Accordingly, many approaches have been developed to monitor ssDNA generation in cells to investigate genes and pathways that regulate DNA resection. Here we describe a flow cytometric approach measuring the levels of replication protein A (RPA) complex, a high affinity ssDNA binding complex composed of three subunits (RPA70, RPA32, and RPA14 in mammals), on chromatin after DNA DSB induction to assay DNA resection. This flow cytometric assay requires only conventional flow cytometers and can easily be scaled up to analyze a large number of samples or even for genetic screens of pooled mutants on a genome-wide scale. We adopt this assay in G0- and G1- phase synchronized cells where DNA resection needs to be kept in check to allow normal NHEJ.

Keywords: DNA DSB ( DNA DSB), Resection (切除), ssDNA (ssDNA), NHEJ ( NHEJ), HR (HR), RPA (RPA), Flow cytometry (流式细胞术)

Background

Genome stability relies in large part on a timely response to and repair of DNA damage that arises during normal physiological processes such as DNA replication and exposure to genotoxic agents such as ionizing irradiation. Disruptions in these activities will lead to death of the affected cells in response to unrepairable damage or genomic rearrangement that promote oncogenic transformation (Tubbs and Nussenzweig, 2017). One of the major DNA lesions that cells encounter is DNA double strand breaks (DSBs). While all DSBs are detrimental if not repaired properly, many of them are important intermediates of critical cellular activities. For example, DSBs can be created by topoisomerase II to relieve torsional strain and supercoiled or catenated DNA during DNA replication or transcription (Nitiss, 2009). During meiosis, DSBs are induced by the nuclease SPO11 and other accessory proteins to initiate meiotic recombination (Lam and Keeney, 2014). In developing lymphocytes, DNA DSBs are generated by the RAG endonuclease, composed of RAG1 and RAG2, during V(D)J recombination to assemble functional antigen receptor genes (Schatz and Swanson, 2011).


The two major repair pathways for DNA DSBs are homologous recombination (HR) and non-homologous end joining (NHEJ). HR is utilized during S and G2 phases of the cell cycle when a sister chromatid is available as a template for accurate repair (Prakash et al., 2015). NHEJ is the predominant DNA DSB repair pathway in G0, also termed quiescence, and G1 phases of the cell cycle due to the lack of sister chromatids, although it is also functional in other cell cycle phases (Chang et al., 2017). The critical choice of HR or NHEJ for DNA DSB repair depends on whether the broken DNA ends are resected to generate extensive single strand DNA (ssDNA) overhangs, which inhibit NHEJ and are quickly bound by the trimeric ssDNA binding complex replication protein A (RPA) to initiate HR (Symington and Gautier, 2011; Ceccaldi et al., 2016; Scully et al., 2019). Given the importance of appropriately employing HR or NHEJ in different cell cycle phases and at distinct genomic territories, DNA end processing is sophisticatedly regulated by nucleases and accessory proteins that promote resection, and DNA protection proteins that counter nucleolytic activities at DSBs (Symington, 2016; Setiaputra and Durocher, 2019; Mirman and de Lange, 2020; Cejka and Symington, 2021). Proper DNA end protection is especially important for cells in G1 and G0 phases of the cell cycle as extensively resected DNA DSBs cannot be repaired by NHEJ. The most studied DNA end protection protein is 53BP1, and since the initial discovery of its DNA protection function, many downstream effectors have been identified, including RIF1, the Shieldin complex, and the CST complex (Setiaputra and Durocher, 2019; Mirman and de Lange, 2020).


To decipher the complex regulation of DNA end resection and protection, numerous approaches have been established to qualitatively and/or quantitatively monitor the levels of ssDNA in cells in response to endogenous or exogenous DNA damage. For example, a quantitative PCR approach on restriction enzyme-digested genomic DNA purified from cells with endonuclease-induced DSBs allows for low resolution detection of resected ssDNA near DSBs that are resistant to cleavage by restriction enzymes (Zhou et al., 2014). HCoDES and End-seq utilize different DNA structure capturing designs coupling with next generation sequencing to determine resected DNA end structure at nucleotide resolution (Dorsett et al., 2014; Canela et al., 2016). BrdU, when incorporated in the genome during DNA replication, can be used to reveal the presence of BrdU-labeled ssDNA in cells by anti-BrdU antibody staining and immunofluorescence imaging or flow cytometry in the absence of DNase I treatment (Mukherjee et al., 2015; Tkac et al., 2016).


Given that RPA rapidly binds to ssDNA with high affinity in vivo and in vitro, immunofluorescence imaging- or flow cytometry-based approaches have also been widely used to visualize the chromatin-bound RPA, as a surrogate of ssDNA, by anti-RPA antibody staining following detergent extraction to remove soluble RPA in cells (Forment et al., 2012; Mukherjee et al., 2015). Here we describe a high-throughput flow cytometry-based method, originally developed by Josep V. Forment, Rachael V. Walker, and Stephen P. Jackson, to monitor DNA end resection (Forment et al., 2012). The flow cytometric assay described here and in (Forment et al., 2012) can easily provide quantitative measurement of the levels of chromatin-bound RPA in large number of cells (tens to hundreds of thousands of cells) on conventional flow cytometers that are readily available to most research communities. We include steps that allow optimizing this assay in multiple cell types. While this method can be used for cells in any phase of the cell cycle, we also include protocols for synchronizing cells in G0 and for identifying G1 cells in a proliferating culture, where DNA end integrity is critical for DNA DSB repair by NHEJ (Chen et al., 2021).

Materials and Reagents

  1. 6-well plates (Corning, catalog number: 3506)

  2. 24-well plate (Corning, catalog number: 3524)

  3. Round bottom polystyrene tubes for FACS (Thermo Fisher Scientific, catalog number: 149595)

  4. Abelson leukemia viral kinase transformed pre-B cells (abl pre-B cells, custom made in lab)

  5. MCF10A (ATCC, catalog number: CRL-10317)

  6. Triton X-100 (Sigma, catalog number: T-8787)

  7. Rat monoclonal anti-RPA32 (4E4) antibody (Rat monoclonal) (Cell Signaling Technology, catalog number: 2208S)

  8. Mouse monoclonal anti-phospho-H2AX (S139) antibody (Millipore Sigma, catalog number: 05-636)

  9. Alexa Fluor 488, goat anti-rat IgG (BioLegend, catalog number: 405418)

  10. Alexa Fluor 647, mouse anti-rat IgG (BioLegend, catalog number: 405322)

  11. Click-iTTM EdU Alexa FluorTM 647 Flow Cytometry Assay Kit (Life Technologies, catalog number: C10419)

  12. 7-AAD (BD Biosciences, catalog number: 559925)

  13. Mouse FITC-anti-BrdU antibody (BD Biosciences, catalog number: 556028)

  14. BD PharmigenTM BrdU Flow Kit (BD Biosciences, catalog number: 559619)

  15. DMEM, high glucose, no glutamine (Thermo Fisher Scientific, catalog number: 11960077)

  16. Fetal bovine serum/FBS (GeminiBio, catalog number: 100-106)

  17. MEM non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140050)

  18. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081)

  19. Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360070)

  20. Penicillin Streptomycin (Thermo Fisher Scientific, catalog number: 15140122)

  21. 2-mercaptoethanol (MP Biomedicals, catalog number: 194705)

  22. DMEM/F-12 (Thermo Fisher Scientific, catalog number: 11330032)

  23. Horse serum (Thermo Fisher Scientific, catalog number: 16050122)

  24. EGF (Pepprotech, catalog number: AF-100-15)

  25. Hydrocortisone (Sigma, catalog number: H-0888)

  26. Cholera toxin (Sigma, catalog number: C-8052)

  27. Insulin (Sigma, catalog number: I-1882)

  28. Trypsin (Gibco, catalog number: 15090046)

  29. Imatinib (SelleckChem, catalog number: S2475)

  30. FACS tubes/Corning Falcon round bottom polystyrene tubes (Thermo Fisher Scientific, catalog number: 14-959-5)

  31. DMEM media (for abl pre-B cells) (see Recipes)

  32. MCF10A media (see Recipes)

  33. FACS Wash (see Recipes)

Equipment

  1. Allegra X-14R Centrifuge (Beckman Coulter)

  2. XRAD 320 irradiator (Precision X-ray Inc.)

  3. BD LSRFortessa X-20 cell analyzer (BD Biosciences)

Software

  1. FlowJo (BD Life Sciences)

Procedure

  1. Synchronization of cells in G0 phase

    1. Abl pre-B cells

      1. Dilute imatinib solution in DMEM media to 3 μM.

      2. Resuspend abl pre-B cells in DMEM + 3 μM imatinib at 2 × 106 cells/mL.

        Notes:

        1) All cells are cultured at 37°C with 5% CO2.

        2) All cells are spun in an Allegra X-14 R centrifuge (Beckman Coulter) at 1,200 rpm for 5 min in procedures spinning is required for collecting cells.

      3. Culture abl pre-B cells in DMEM + 3 μM imatinib for at least 48 h.

    2. MCF10A cells

      1. Plate 2.5 × 105 MCF10A cells in 5 mL of MCF10A media per well of 6-well tissue culture plates.

      2. Culture cells for 1 day.

      3. Aspirate media from each well and wash cells with pre-warmed 1× PBS or EGF-free MCF10A media.

      4. Add 5 mL of EGF-free MCF10A media to each well and culture cells for 2 days.

    3. Verification of G0 synchronization efficiency using BD PharmigenTM BrdU Flow Kit with modification (for both abl pre-B cells and MCF10A cells)

      1. Add BrdU to cells (1mL of imatinib-treated abl pre-B cells or 1 well of EGF-deprived MCF10A cells in a 6-well plate) to a final concentration of 10 μM.

        Note: Make sure to use proliferating cells as a positive control to ensure that the labeling and detection procedures are done appropriately.

      2. Incubate cells with BrdU for 30 min.

      3. Collect cells in FACS tubes.

        1. Abl pre-B cells:

          1. Resuspend cells briefly by pipetting and transfer cells directly to FACS tubes.

          2. Spin down cells, decant supernatant by inverting tubes quickly to pour out media and tapping the inverted tubes on a paper towel, and wash cells by resuspending them in 1mL of 1X FACS wash, followed by spinning down cells again.

            Note: Do not use vacuum to avoid loss of samples.

        2. MCF10A cells:

          1. Remove media and wash cells with 1mL of 1× PBS.

          2. Add 300 μL of 0.25% Trypsin to a well and return cells to the incubator for 5–10 min.

          3. Inspect cells under the microscope to ensure that most cells have detached off the plate surface.

          4. Add 1 mL of MCF10A media to resuspend cells and transfer cells to FACS tubes.

          5. Spin down cells, decant supernatant, and wash cells by resuspending cells in 1 mL of 1× FACS wash, followed by spinning down cells again.

    4. Fix cells in 150 μL of BD Cytofix/Cytoperm Buffer at room temperature for 20 min.

    5. Wash cells with 1 mL of 1× BD Perm/Wash Buffer.

    6. Permeabilize cells by one of the following methods

      1. Freeze-and-thaw method

        1. Freeze cells in 0.5 mL of 10% DMSO in FBS at -80°C.

        2. Thaw cells at room temperature and wash freeze-thaw cells with 1 mL of 1× BD Perm/Wash Buffer.

      2. Permeabilization with detergent

        1. Resuspend cells in 150 μL of BD Permeabilization Plus Buffer and incubate on ice for 10 min.

        2. Wash cells with 1 mL of 1× BD Perm/Wash Buffer.

    7. Fix cells in 150 μL of BD Cytofix/Cytoperm Buffer at room temperature for 5 min.

    8. Add 100 μL of 300 μg/mL DNase I (in 1× PBS) to each tube of cells and incubate at 37°C for 1 h.

    9. Wash cells with 1 mL of 1× BD Perm/Wash Buffer.

    10. Stain cells with 100 μL of 1:50 diluted FITC-anti-BrdU antibody (in 1× BD Perm/Wash Buffer) at room temperature for 1 h.

    11. Wash cells with 1 mL of 1× BD Perm/Wash Buffer.

    12. Add 15 μL of 7-AAD to each tube of cells and incubate at room temperature for 3 min.

    13. Add 300 μL of 1× PBS to each tube.

      Note: Do not wash off 7-AAD. Add 1× PBS directly to each tube and process flow cytometry analysis directly.

    14. Record DNA content (7-AAD) and BrdU incorporation (FITC) on BD FACS Fortessa X-20 or an equivalent flow cytometer.

    15. Analyze data using FlowJo (Figure 1).



    Figure 1. Flow cytometric analysis of cycling and G0-arrested abl pre-B cells and MCF10A cells following BrdU labeling.

    Dot plots depicting cell cycle profiles of cycling and cells arrested in G0 phases by (A) imatinib treatment (abl pre-B cells) or (B) EGF withdrawal (MCF10A) cells after BrdU labeling and flow cytometry analysis for BrdU incorporation (FITC) and DNA content (7-AAD). S (BrdU-positive), G1 (BrdU-negative, 2N DNA), and G2 (BrdU-negative, 4N DNA) cells in cycling cultures and G0 cells (BrdU-negative, 2N DNA) in imatinib-treated abl pre-B cell and EGF-deprived MCF10A cultures are shown in red polygon gates.


  2. Monitoring DNA end resection by flow cytometry-based chromatin-bound RPA assay after irradiation in G0-arrested and G1 phase cells in proliferating cultures

    1. Cell preparation

      1. G0 abl pre-B cells: Treat abl pre-B cells with imatinib as indicated in Step A3. Aliquot 1 mL of imatinib-treated cells in wells of a 24-well plate for irradiation.

      2. G0 MCF10A cells: Synchronize MCF10A in EGF-free MCF10A media in a 12-well plate as indicated in Step A3 for irradiation.

      3. Proliferating abl pre-B cells: Resuspend cells in pre-warmed DMEM media at 2 × 106 cells/mL and aliquot 1 mL of cells in each well of a 24-well plate for irradiation.

      4. Proliferating MCF10A cells: Plate 5 × 105 MCF10A cells 5 mL of MCF10A media in a well of a 6-well plate for 24 h before irradiation.

    2. For analysis of G1 phase abl pre-B or MCF10A cells from proliferating cultures, incubate cells with 10 μM EdU for 1 h before irradiation.

    3. Irradiate cells in XRAD 320 irradiator.

      Note: The dosage of irradiation and the time of sample collection after IR were determined empirically, based on the distinct RPA staining intensities between 53BP1-proficient (normal DNA end protection, basal levels of resection) and 53BP1-deficient (impaired DNA end protection).

      1. Imatinib-treated (G0) abl pre-B cells: 15Gy IR; cell collected 3–18 h after IR

      2. EGF-deprived (G0) MCF10A cells: 30 Gy IR; cell collected 4 h after IR

      3. Proliferating abl pre-B cells: 5 Gy IR; cell collected 3 h after IR

      4. Proliferating MCF10A cells: 25 Gy IR; cell collected 6 h after IR

    4. Collect cells in FACS tubes, spin down, and wash with 1 mL of FACS wash.

    5. Pre-extract cells with 150 mL of cold Triton X-100 in 1× PBS on ice for 10 min

      1. Imatinib-treated (G0) abl pre-B cells: 0.05% Triton X-100

      2. EGF-deprived (G0) MCF10A cells: 0.5% Triton X-100

      3. Proliferating abl pre-B cells: 0.2% Triton X-100

      4. Proliferating MCF10A cells: 0.5% Triton X-100

    6. Wash cells with 2 mL of FACS wash.

    7. Fix cells in 150 μL of BD Cytofix/Cytoperm Buffer at room temperature for 20 min.

    8. Wash cells with 1 mL of FACS wash.

    9. Stain cells 100 μL of 1:500 diluted anti-RPA32 and 1:1,000 diluted anti-phospho-H2AX (S139) antibodies (in 1× BD Perm/Wash Buffer) at room temperature for 2 h.

    10. Wash cells with 1 mL of 1× BD Perm/Wash Buffer.

    11. Stain cells 100 μL of 1:500 diluted Alexa Fluro 488 goat anti-rat IgG and Alexa Fluro 647 goat anti-mouse IgG (in 1× BD Perm/Wash Buffer) at room temperature for 1 hour, protected from light.

    12. Wash cells with 1 mL of 1× BD Perm/Wash Buffer.

    13. For analysis of G1-phase cells in EdU-pulsed proliferating cultures:

      Note: Click-iT Plus EdU imaging kit is used in place of BrdU labeling kit because DNase I treatment in the procedures of BrdU labeling kit results in poor RPA staining compared to untreated cells.

      1. Prepare Click-iT Plus reaction cocktail according to the manufacturer’s instructions.

        Note: Use Click-iT Plus reaction cocktail within 15 min of preparation.

      2. Resuspend cells in each tube in 250 μL of Click-iT Plus reaction cocktail and incubate at room temperature for 30 min, protected from light.

      3. Wash cells with 2 mL of 1× BD Perm/Wash Buffer.

    14. Add 15 μL of 7-AAD and incubate at room temperature for 3 min.

    15. Add 300 μL of 1× PBS to each tube.

    16. Record DNA content (7-AAD), RPA (Alexa Fluro 488), and phospho-H2AX (S139) or EdU (Alexa Fluro 647) on BD FACS Fortessa X-20 or equivalent flow cytometer.

      Note: The choice of fluorophores should be based on the availability of reagents and the flow cytometers. As we use Alexa Fluro 647 for visualizing phospho-H2AX (S139) and EdU in our RPA assay, we typically do not perform phospho-H2AX (S139) staining in the analysis of proliferating cells.

    17. Analyze data using FlowJo (Figure 2).



    Figure 2. Flow cytometric analysis of chromatin-bound RPA levels after irradiation.

    (A, B) Histograms showing chromatin-bound RPA levels (Alexa Fluor 488) in G0-arrested WT or 53BP1-deficient abl pre-B (A) or MCF10A (B) cells after IR. (C, D) The dot plots on the left show EdU incorporation (Alexa Fluor 647) and DNA content (7-AAD) in cycling abl pre-B (C) and MCF10A (D) cells, with G1 cells shown in the red polygon gates. The histograms on the right show chromatin-bound RPA levels (Alexa Fluor 488) in G1-phase WT or 53BP1-deficient abl pre-B (C) or MCF10A (D) cells, with red polygon gated cells in the dot plots, after IR.

Data analysis

  1. When working with imatinib-treated abl pre-B cells and EGF-deprived MCF10A cells, generate a histogram with 7-AAD as the X-axis to identify cells with 2N DNA contents (using cycling cells as the control to determine cells with 2N and 4N DNA content) to specifically analyze levels of chromatin-bound RPA in G0 cells (with 2N DNA).

  2. To identify G1-phase cells in a proliferating population for analysis of chromatin-bound RPA, generate a dot plot with Alexa Fluro 647 (EdU) as the Y-axis and 7-AAD as the X-axis to identify EdU-negative cells with 2N DNA content.

Recipes

  1. DMEM media (for abl pre-B cells)

    Reagent Final concentration Amount
    DMEM, high glucose, no glutamine - 860 mL
    Heat-inactivated FBS 10% 100 mL
    L-glutamine (200 mM) 2 mM 10 mL
    Sodium pyruvate (100 mM) 1 mM 10 mL
    MEM non-essential amino acids (100×) 10 mL
    Penicillin/streptomycin (10,000 U/mL) 100 U/mL 10 mL
    2-mercaptoethanol (14.3 M) 57 mM 4 mL
    Total n/a 1,000 mL

  2. MCF10A media

    Reagent Final concentration Amount
    DMEM/F-12 - 470 mL
    Horse serum 5% 25 mL
    EGF (100 μg/mL) 20 ng/mL 100 μL
    Hydrocortisone (1 mg/mL) 0.5 μg/mL 250 μL
    Cholera toxin (1 mg/mL) 100 ng/mL 50 μL
    Insulin (10 mg/mL) 10 μg/mL 500 μL
    Penicillin/streptomycin (10,000 U/mL) 100 U/mL 5 mL
    Total - 500 mL

  3. FACS Wash

    Reagent Final concentration Amount
    Heat-inactivated FBS 2% 10 mL
    10× PBS 50 mL
    MilliQ H2O - 440 mL
    Total - 500 mL

Acknowledgments

B.P.S. is supported by National Institutes of Health grants R01 AI047829 and R01 AI074953. J.K.T. is supported by National Institutes of Health grants R01 CA095641 and R35 GM139816. B.P.S. and J.K.T. are also supported by the Starr Cancer Consortium. This protocol is largely derived from the original work of Josep V. Forment, Rachael V. Walker, and Stephen P. Jackson (Forment et al., 2012) and from Bo-Ruei Chen et al. (Chen et al., 2021).

Competing interests

The authors declare no competing interests.

References

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  2. Ceccaldi, R., Rondinelli, B. and D'Andrea, A. D. (2016). Repair Pathway Choices and Consequences at the Double-Strand Break. Trends Cell Biol 26(1): 52-64.
  3. Cejka, P. and Symington, L. S. (2021). DNA End Resection: Mechanism and Control. Annu Rev Genet 55: 285-307.
  4. Chang, H. H. Y., Pannunzio, N. R., Adachi, N. and Lieber, M. R. (2017). Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol 18(8): 495-506.
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简介

【摘要】
DNA 双链断裂 (DSB) 在正常细胞过程中或暴露于基因毒剂时会在细胞中不断出现,并且主要通过同源重组 (HR) 和非同源末端连接 (NHEJ) 进行修复。 DNA DSB 修复途径选择的一个关键决定因素是处理断裂的 DNA 末端以产生单链 DNA (ssDNA) 突出端,这一过程称为 DNA 切除。 ssDNA 悬垂的产生通过 HR 进行 DSB 修复并抑制 NHEJ。因此,必须仔细规范 DNA 切除,以避免错误修复或持续存在的 DSB。因此,已经开发了许多方法来监测细胞中 ssDNA 的产生,以研究调节 DNA 切除的基因和途径。在这里,我们描述了一种流式细胞术方法,测量复制蛋白 A (RPA) 复合物的水平,这是一种由三个亚基(哺乳动物中的 RPA70、RPA32 和 RPA14)组成的高亲和力 ssDNA 结合复合物,在 DNA DSB 诱导以测定 DNA 切除后染色质上的水平.这种流式细胞仪检测只需要传统的流式细胞仪,并且可以很容易地放大以分析大量样本,甚至可以在全基因组范围内对汇集的突变体进行遗传筛选。我们在需要检查 DNA 切除以允许正常 NHEJ 的 G0 期和 G1 期同步细胞中采用这种检测方法。


背景

基因组稳定性在很大程度上依赖于对正常生理过程(如 DNA 复制和暴露于基因毒剂(如电离辐射)期间出现的 DNA 损伤)的及时反应和修复。这些活动的中断将导致受影响的细胞因无法修复的损伤或促进致癌转化的基因组重排而死亡(Tubbs 和 Nussenzweig,2017 年) 。细胞遇到的主要 DNA 损伤之一是 DNA 双链断裂 (DSB)。虽然所有 DSB 如果不正确修复都是有害的,但其中许多是关键细胞活动的重要中间体。例如,可以通过拓扑异构酶 II 创建 DSB,以缓解 DNA 复制或转录过程中的扭转应变和超螺旋或链状 DNA (Nitiss, 2009) 。在减数分裂期间,核酸酶 SPO11 和其他辅助蛋白诱导 DSB 启动减数分裂重组(Lam 和 Keeney,2014) 。在淋巴细胞发育过程中,由 RAG1 和 RAG2 组成的 RAG 核酸内切酶在 V(D)J 重组过程中产生 DNA DSB,以组装功能性抗原受体基因(Schatz 和 Swanson,2011) 。
DNA DSB 的两个主要修复途径是同源重组 (HR) 和非同源末端连接 (NHEJ)。 HR 用于细胞周期的 S 和 G 2阶段,此时姐妹染色单体可用作精确修复的模板(Prakash等人,2015) 。 NHEJ 是 G 0中的主要 DNA DSB 修复途径,也称为细胞周期的静止期和 G 1期,因为缺乏姐妹染色单体,尽管它在其他细胞周期阶段也有功能(Chang et al. , 2017) . DNA DSB 修复中 HR 或 NHEJ 的关键选择取决于是否切除断裂的 DNA 末端以产生广泛的单链 DNA (ssDNA) 突出端,其抑制 NHEJ 并迅速与三聚体 ssDNA 结合复合物复制蛋白 A (RPA) 结合启动 HR (Symington 和 Gautier,2011;Ceccaldi等,2016;Scully等,2019) 。鉴于在不同细胞周期阶段和不同基因组区域适当使用 HR 或 NHEJ 的重要性,DNA 末端加工受到促进切除的核酸酶和辅助蛋白以及对抗 DSB 的核溶解活动的 DNA 保护蛋白的复杂调节(Symington,2016; Setiaputra 和 Durocher,2019 年;Mirman 和 de Lange,2020 年;Cejka 和 Symington,2021 年) 。适当的 DNA 末端保护对于细胞周期 G 1和 G 0期的细胞尤为重要,因为 NHEJ 无法修复广泛切除的 DNA DSB。研究最多的 DNA 末端保护蛋白是 53BP1,自从最初发现其 DNA 保护功能以来,已经鉴定出许多下游效应子,包括 RIF1、 Shieldin复合物和 CST 复合物(Setiaputra 和 Durocher,2019;Mirman 和 de Lange , 2020;) .
为了破译 DNA 末端切除和保护的复杂调节,已经建立了许多方法来定性和/或定量监测细胞中 ssDNA 的水平,以响应内源性或外源性 DNA 损伤。例如,对从具有内切核酸酶诱导的 DSB 的细胞中纯化的限制性酶消化的基因组 DNA 进行定量 PCR 方法,可以低分辨率检测 DSB 附近对限制性酶切割具有抗性的切除 ssDNA (Zhou et al. , 2014) 。 HCoDES和 End-seq 利用不同的 DNA 结构捕获设计与下一代测序相结合,以核苷酸分辨率确定切除的 DNA 末端结构(Dorsett等人,2014;Canela等人,2016) 。 BrdU在 DNA 复制过程中掺入基因组时,可用于通过抗BrdU抗体染色和免疫荧光成像或流式细胞术在没有 DNase I 处理的情况下揭示细胞中BrdU标记的 ssDNA的存在(Mukherjee等人, 2015 年;Tkac等人,2016 年;) 。
鉴于 RPA在体内和体外以高亲和力快速结合 ssDNA,基于免疫荧光成像或流式细胞术的方法也被广泛用于通过抗 RPA 抗体染色来可视化染色质结合的 RPA,作为 ssDNA 的替代物在去污剂提取后去除细胞中的可溶性 RPA (Forment等人,2012 年;Mukherjee等人,2015 年) 。在这里,我们描述了一种基于高通量流式细胞术的方法,最初由Josep V. Forment 、Rachael V. Walker 和 Stephen P. Jackson 开发,用于监测 DNA 末端切除(Forment等人,2012) 。此处和(Forment et al. , 2012)中描述的流式细胞仪测定 ADDIN EN.CITE 可以很容易地在常规流式细胞仪上提供对大量细胞(数万到数十万个细胞)中染色质结合 RPA 水平的定量测量,这些流式细胞仪很容易可用于大多数研究团体。我们包括允许在多种细胞类型中优化此测定的步骤。虽然这种方法可用于细胞周期任何阶段的细胞,但我们还包括用于同步 G 0细胞和识别增殖培养物中的 G 1细胞的协议,其中 DNA 末端完整性对于 NHEJ 修复 DNA DSB 至关重要。陈等人,2021) 。

关键字:DNA DSB, 切除, ssDNA, NHEJ, HR, RPA, 流式细胞术

材料和试剂
1.6孔板(Corning,目录号:3506)
2.24孔板(Corning,目录号:3524)
3.用于 FACS 的圆底聚苯乙烯管(Thermo Fisher Scientific,目录号:149595)
4.Abelson 白血病病毒激酶转化的前 B 细胞( abl前 B 细胞,实验室定制)
5.MCF10A(ATCC,目录号:CRL-10317)
6.Triton X-100(Sigma,目录号:T-8787)
7.大鼠单克隆抗RPA32(4E4)抗体(Rat monoclonal)(Cell Signaling Technology,目录号:2208S)
8.小鼠单克隆抗磷酸化H2AX(S139)抗体(Millipore Sigma,目录号:05-636)
9.Alexa Fluor 488,山羊抗大鼠 IgG( BioLegend ,目录号:405418)
10.Alexa Fluor 647,小鼠抗大鼠 IgG( BioLegend ,目录号:405322)
11.点击-iT TM EdU Alexa Fluor TM 647 流式细胞术检测试剂盒(Life Technologies,目录号: C10419)
12.7-AAD(BD Biosciences,目录号: 559925)
13.小鼠 FITC 抗BrdU抗体(BD Biosciences,目录号: 556028)
14.BD Pharmigen TM BrdU Flow Kit (BD Biosciences,目录号: 559619)
15.DMEM,高葡萄糖,无谷氨酰胺(Thermo Fisher Scientific,目录号: 11960077)
16.胎牛血清/ FBS( GeminiBio ,目录号: 100-106)
17.MEM非必需氨基酸(Thermo Fisher Scientific,目录号: 11140050)
18.L-谷氨酰胺(Thermo Fisher Scientific,目录号: 25030081)
19.丙酮酸钠(Thermo Fisher Scientific,目录号: 11360070)
20.青霉素链霉素(Thermo Fisher Scientific,目录号: 15140122)
21.2-巯基乙醇(MP Biomedicals,目录号: 194705)
22.DMEM/F-12(Thermo Fisher Scientific,目录号: 11330032)
23.马血清(Thermo Fisher Scientific,目录号: 16050122)
24.EGF( Pepprotech ,目录号: AF-100-15)
25.氢化可的松(Sigma,目录号: H-0888)
26.霍乱毒素(Sigma,目录号: C-8052)
27.胰岛素(Sigma,目录号: I-1882)
28.胰蛋白酶(Gibco,目录号:15090046)
29.伊马替尼( SelleckChem ,目录号: S2475)
30.FACS管/Corning Falcon圆底聚苯乙烯管(Thermo Fisher Scientific,目录号: 14-959-5)
31.DMEM 培养基(用于abl pre-B 细胞)(参见食谱)
32.MCF10A 媒体(见食谱)
33.FACS 洗涤(见食谱)




设备


1.Allegra X-14R 离心机 (Beckman Coulter)
2.XRAD 320辐照器( Precision X-ray Inc.)
3.BD LSRFortessa X-20 细胞分析仪(BD Biosciences)




软件 


1.FlowJo(BD 生命科学)


程序


A.0期细胞同步


1.Abl前B细胞
a.将 DMEM 培养基中的伊马替尼溶液稀释至3 μM 。
b.在 DMEM + 3 μ M伊马替尼中以 2 × 10 6 个细胞/ mL重悬abl pre-B 细胞。
笔记:
1) 所有细胞均在 37 °C和 5% CO 2下培养。
2) 所有细胞在 Allegra X-14 R 离心机 (Beckman Coulter) 中以 1,200 rpm 的速度旋转 5 分钟,收集细胞需要旋转。
c.μ M伊马替尼中培养abl pre-B 细胞至少 48 小时。
2.MCF10A 细胞
a.板 2.5 × 10 5 MCF10A 细胞在 5 mL 的 MCF10A 培养基中,每孔 6 孔组织培养板。
b.培养细胞 1 天。
c.从每个孔中吸出培养基并用预热的 1 × PBS 或不含 EGF 的 MCF10A 培养基洗涤细胞。
d.在每口井中加入 5 mL 的无 EGF 的 MCF10A 培养基,培养细胞 2 天。
3.Pharmigen TM验证 G 0同步效率 带修饰的BrdU Flow Kit(用于abl pre-B 细胞和 MCF10A 细胞)
a.将BrdU添加到细胞(1mL 伊马替尼处理的abl pre- B细胞或 1 孔 EGF 剥夺的 MCF10A 细胞在 6 孔板中)至最终浓度为 10 μM 。
注意:确保使用增殖细胞作为阳性对照,以确保正确完成标记和检测程序。
b.BrdU孵育细胞30 分钟。
c.在 FACS 管中收集细胞。
i.Abl前 B 细胞:
1)通过移液将细胞直接重悬并直接转移到 FACS 管中。
2)旋转细胞,通过快速倒置管倒出培养基并在纸巾上敲击倒置管来倒出上清液,然后通过将细胞重新悬浮在 1mL 的 1X FACS 洗涤液中来洗涤细胞,然后再次旋转细胞。
注意:不要使用真空以避免样品损失。
ii.MCF10A 细胞:
1)去除培养基并用 1mL 的 1 × PBS 清洗细胞。
2)将 300 μL的 0.25% 胰蛋白酶添加到孔中,并将细胞返回培养箱5-10 分钟。
3)在显微镜下检查细胞,以确保大多数细胞已脱离板表面。
4)添加 1 mL 的 MCF10A 介质以重新悬浮细胞并将细胞转移到 FACS 管中。
5)× FACS 洗涤液中重新悬浮细胞,然后再次旋转细胞来旋转细胞、倾析上清液并洗涤细胞。
4.在室温下将细胞固定在 150 μL BD Cytofix / Cytoperm Buffer 中 20 分钟。
5.用 1 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
6.通过以下方法之一通透细胞
a.冻融法
i.° C下将细胞冷冻在 0.5 mL 的 10% DMSO 中的 FBS 中。
ii.在室温下解冻细胞并用 1 mL 的 1 × BD Perm/Wash Buffer 清洗冻融细胞。
b.用洗涤剂渗透
i.在 150 μL BD Permeabilization Plus Buffer 中重悬细胞,并在冰上孵育 10 分钟。
ii.用 1 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
7.在室温下将细胞固定在 150 μL BD Cytofix / Cytoperm缓冲液中 5 分钟。
8.向每管细胞中加入 100 μL的 300 μg / mL DNase I(在 1 × PBS 中),并在 37 °C下孵育 1小时。
9.用 1 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
10.用 100 μL 1:50 稀释的 FITC 抗BrdU抗体(在 1 × BD Perm/Wash Buffer 中)在室温下染色细胞 1 小时。
11.用 1 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
12.加入 15 μL的 7-AAD,并在室温下孵育 3 分钟。
13.在每管中加入 300 μL的 1 × PBS。
注意:不要洗掉 7-AAD。将 1 × PBS 直接添加到每个管中并直接进行流式细胞术分析。
14.在 BD FACS Fortessa X-20 或等效流式细胞仪上记录 DNA 含量 (7-AAD) 和BrdU掺入 (FITC)。
15.FlowJo分析数据(图 1)。




 


BrdU标记后循环和 G 0阻滞abl pre-B 细胞和 MCF10A 细胞的流式细胞术分析。 
通过(A)伊马替尼处理 ( abl pre-B 细胞) 或(B)在BrdU标记和流式细胞术分析BrdU掺入 (FITC ) 后 EGF 戒断 (MCF10A) 细胞在 G 0期停滞的细胞周期曲线) 和 DNA 含量 (7-AAD)。 S( BrdU-阳性)、G 1 ( BrdU-阴性,2N DNA)和 G 2 ( BrdU-阴性,4N DNA)细胞在循环培养中和 G 0细胞( BrdU-阴性,2N DNA)在伊马替尼处理的abl中pre-B 细胞和缺乏 EGF 的 MCF10A 培养物以红色多边形门显示。


B.在增殖培养物中 G 0期阻滞和 G 1期细胞照射后,通过基于流式细胞术的染色质结合 RPA 测定监测 DNA 末端切除


1.细胞制备
a.G 0 abl pre-B 细胞:如步骤 A3 所示,用伊马替尼处理abl pre-B 细胞。等分 1 mL 的伊马替尼处理细胞在 24 孔板的孔中进行照射。
b.G 0 MCF10A 细胞:在 12 孔板中将 MCF10A 与无 EGF 的 MCF10A 培养基同步,如步骤 A3 所示用于照射。
c.增殖abl pre-B 细胞:在预热的 DMEM 培养基中以 2 × 10 6重悬细胞 细胞/mL 和等分 1 mL 的细胞在 24 孔板的每个孔中进行照射。
d.增殖的 MCF10A 细胞:板5 × 10 5 MCF10A 细胞 5 mL MCF10A 培养基在 6 孔板的孔中放置 24 小时,然后照射。
2.为了分析来自增殖培养物的 G 1期abl pre-B 或 MCF10A 细胞,用 10 μ M孵育细胞 EdU照射前 1 小时。
3.XRAD 320辐照器中辐照细胞。
注意:根据 53BP1 熟练(正常 DNA 末端保护,切除的基础水平)和 53BP1 缺陷(DNA 末端保护受损)之间不同的 RPA 染色强度,凭经验确定照射剂量和 IR 后样品收集时间.
a.伊马替尼处理的 (G 0 ) abl pre-B 细胞:15Gy IR; IR 后3 – 18 小时收集细胞
b.EGF 剥夺 (G 0 ) MCF10A 细胞:30 Gy IR; IR 后 4 小时收集细胞
c.增殖的abl pre-B 细胞:5 Gy IR; IR 后 3 小时收集细胞
d.增殖的 MCF10A 细胞:25 Gy IR; IR 后 6 小时收集细胞
4.收集 FACS 管中的细胞,向下旋转,并用 1 mL 的 FACS 洗涤液洗涤。
5.× PBS 中在冰上预提取细胞10 分钟
a.伊马替尼处理的 (G 0 ) abl pre-B 细胞:0.05% Triton X-100
b.EGF 剥夺 (G 0 ) MCF10A 细胞:0.5% Triton X-100
c.增殖的abl pre-B 细胞:0.2% Triton X-100
d.增殖的 MCF10A 细胞:0.5% Triton X-100
6.用 2 mL 的 FACS 洗涤细胞。
7.在室温下将细胞固定在 150 μL BD Cytofix / Cytoperm Buffer 中 20 分钟。
8.用 1 mL 的 FACS 洗涤细胞。
9.100 μL 1:500 稀释的抗 RPA32 和 1:1,000 稀释的抗磷酸 H2AX (S139) 抗体(在 1 × BD Perm/Wash Buffer 中)在室温下染色 2 小时。
10.用 1 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
11.100 μL 1:500 稀释的 Alexa Fluro 488 山羊抗大鼠 IgG 和 Alexa Fluro 647 山羊抗小鼠 IgG(在 1 × BD Perm/Wash Buffer 中)在室温下染色 1 小时,避光。
12.用 1 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
13.为了分析EdU脉冲增殖培养物中的 G 1期细胞:
注意:使用 Click- iT Plus EdU成像试剂盒代替BrdU标记试剂盒,因为与未处理的细胞相比, BrdU标记试剂盒程序中的 DNase I 处理导致 RPA 染色较差。
a.根据制造商的说明制备 Click - iT Plus 反应混合物。
注意:在制备后 15 分钟内使用 Click- iT Plus 反应混合物。
b.每管中的细胞重悬于 250 μL Click- iT Plus 反应混合物中,避光在室温下孵育 30 分钟。
c.用 2 mL 的 1 × BD Perm/Wash 缓冲液清洗细胞。
14.加入 15 μL的 7-AAD 并在室温下孵育 3 分钟。
15.在每管中加入 300 μL的 1 × PBS。
16.在 BD FACS Fortessa X-20 或等效流式细胞仪上记录 DNA 含量 (7-AAD)、RPA (Alexa Fluro 488) 和 phospho-H2AX (S139) 或EdU (Alexa Fluro 647)。
注意:荧光团的选择应基于试剂和流式细胞仪的可用性。由于我们在 RPA 分析中使用 Alexa Fluro 647 可视化磷酸化 H2AX (S139) 和EdU ,因此我们通常不会在增殖细胞分析中进行磷酸化 H2AX (S139) 染色。
17.FlowJo分析数据(图 2)。




 


图 2. 辐照后染色质结合 RPA 水平的流式细胞仪分析。 
(A,B)直方图显示 G 0停滞的 WT 或 53BP1 缺陷abl pre-B (A)或 MCF10A (B)细胞中染色质结合的 RPA 水平(Alexa Fluor 488) 红外后。 (C, D)左侧的点图显示了循环abl pre-B (C)和 MCF10A (D)细胞中的EdU掺入 (Alexa Fluor 647) 和 DNA 含量 (7-AAD) ,其中 G 1细胞显示在红色多边形大门。右侧的直方图显示 G 1 期WT 或 53BP1 缺陷abl pre-B (C)或 MCF10A (D)细胞中染色质结合的 RPA 水平 (Alexa Fluor 488), 和 在 IR 之后,点图中的红色多边形门控单元。




数据分析


1.当使用伊马替尼处理的abl pre-B 细胞和 EGF 剥夺的 MCF10A 细胞时,生成以 7-AAD 作为 X 轴的直方图,以识别具有 2N DNA 含量的细胞(使用循环细胞作为对照来确定具有 2N 和4N DNA 含量)专门分析 G 0细胞(含 2N DNA)中染色质结合的 RPA 水平。
2.要识别增殖群体中的 G 1 期细胞以分析染色质结合的 RPA,请生成以 Alexa Fluro 647 ( EdU ) 为 Y 轴和 7-AAD 作为 X 轴的点图,以识别EdU阴性细胞2N DNA 含量。




食谱


1.DMEM 培养基(用于abl pre-B 细胞)


试剂最终浓度数量
DMEM,高糖,不含谷氨酰胺-860 毫升
热灭活胎牛血清10%100 毫升
L-谷氨酰胺 (200 毫米)2毫米10 毫升
丙酮酸钠 (100 毫米)1毫米10 毫升
MEM 非必需氨基酸 (100 × )1 ×10 毫升
青霉素/链霉素 (10,000 U/mL)100 单位/毫升10 毫升
2-巯基乙醇 (14.3 M)57毫米 4毫升
全部的不适用1,000 毫升


2.MCF10A 媒体


试剂最终浓度数量
DMEM/F-12-470 毫升
马血清5%25 毫升
EGF (100微克/毫升) 20 纳克/毫升100微升
氢化可的松 (1 毫克/毫升)0.5微克/毫升250微升
霍乱毒素 (1 mg/mL)100 纳克/毫升50微升
胰岛素 (10 毫克/毫升)10微克/毫升500微升
青霉素/链霉素 (10,000 U/mL)100 单位/毫升5 毫升
全部的-500 毫升



3.流式细胞仪清洗


试剂最终浓度数量
热灭活胎牛血清2%10 毫升
10 × PBS1 ×50 毫升
MilliQ H 2 O-440 毫升
全部的-500 毫升




致谢


BPS 得到美国国立卫生研究院 R01 AI047829 和 R01 AI074953 的资助。 JKT 得到了美国国立卫生研究院R01 CA095641 和 R35 GM139816 的资助。 BPS 和 JKT 也得到了 Starr Cancer Consortium 的支持。该协议主要源自Josep V. Forment 、Rachael V. Walker 和 Stephen P. Jackson (Forment等人,2012 年)以及 Bo - Ruei Chen等人的原创作品。 (陈等人,2021) 。




利益争夺


作者声明没有竞争利益。




参考


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Copyright Chen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, B. R., Tyler, J. K. and Sleckman, B. P. (2022). A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells. Bio-protocol 12(10): e4413. DOI: 10.21769/BioProtoc.4413.
  2. Chen, B. R., Wang, Y., Tubbs, A., Zong, D., Fowler, F. C., Zolnerowich, N., Wu, W., Bennett, A., Chen, C. C., Feng, W., et al. (2021). LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells. Elife 10: e68466.
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