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May 2019

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SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
SMART(切除轨迹单分子分析)技术用于评估DNA末端切除对DNA损伤的反应   

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Abstract

DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks (SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3′ single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3′ single-strand DNA tails, generated from DSB processing.

Keywords: DNA repair (DNA修复), Homologous Recombination (同源重组), DNA end-resection (DNA末端切除), BrdU (BrdU标记), Immunofluorescence (免疫荧光)

Background

Genomic instability is one of the enabling characteristics leading to tumour development (Hanahan and Weinberg, 2011). Many sources of DNA damage, either endogenous or exogenous, induce various DNA lesions including double strand breaks (DBSs), which activate the DNA damage response (DDR)–the cell process aimed at preserving DNA integrity. Two main repair pathways are involved in the DSBs repair: non-homologous end joining (NHEJ) and homologous recombination (HR) (Mao et al., 2008). The HR process prevents the loss of genetic information upon DNA damage through a faithful repair using a complementary DNA sequence. By inhibiting the error prone NHEJ and triggering homology-directed DSB repair, DNA end resection has a crucial function in directing the repair pathway choice towards a faithful repair. So, to promote a correct HR, the DSBs are processed through the DNA end-resection machinery which is necessary to generate the long 3′ single-strand DNA (ssDNA) tails essential for the homologous strand invasion. DNA end-resection is a finely regulated process; the first step consists of the recruitment of the MRN complex (MRE11-RAD50-NBSI) and CtIP (RBBP8) onto the DNA lesions to produce short stretches of ssDNA (Stracker and Petrini, 2011) followed by further end-resection mediated by exonuclease I (EXOI) or DNA replication helicase/nuclease 2 (DNA2) in complex with the Bloom syndrome helicases (BLM) (Nimonkar et al., 2011). Subsequently, the replication protein A (RPA) complex binds the ssDNA generated by DNA end-resection, preventing the formation of DNA hairpins (Chen et al., 2013) and to facilitate the loading of RAD51 required for the strand exchange process (Krogh and Symington, 2004).

The evaluation of DNA end-resection is crucial to dissect the molecular mechanisms underlying this finely tuned process and to identify the key players involved. Various methods can be used to assess the DNA end-resection process indirectly, analyzing phosphorylated S4/8 RPA32 as mentioned above, or RAD51 foci, or detection of BrdU in ssDNA following long incubation time (Tkáč et al., 2016). A method to determine the length of resection and the extent of ssDNA at a specific DSB site was developed in 2014 by Tania Paull, although this is based on the expression of an endonuclease (such as AsiSI) to induce DSBs at specific sites within the genome, which is then followed by PCR analysis (Zhou et al., 2014).

The SMART assay was first developed by Huertas and co-authors as a reliable method to measure the length of resected DNA following exposure to any damaging agent in any cellular system, at the level of single molecules (Cruz-García et al., 2014). The assay was based on the previously developed DNA combing assay, which enables the physical stretching of DNA fibres onto a solid support allowing visualization through immunofluorescence (Alfano et al., 2016 and 2017). We recently identified the RNA binding protein HNRNPD as a new player in the HR process and used the SMART assay, along with other techniques, to evaluate DNA end-resection (Alfano et al., 2019). We pulse-labeled HeLa cells with the halogenated IdU (5-Iodo-2′-deoxyuridine)–although any other pyrimidine analogue can be used, such as BrdU (Bromodeoxyuridine)or CldU (5-Chloro-2'-deoxyuridine)–which is incorporated during S phase into the newly synthesized DNA, for approximately 24 h followed by treatment with the DNA damaging agent, camptothecin. At the end of drug incubation time, cells were lysed and the DNA fibres were extracted in non-DNA denaturing conditions (avoiding hydrochloric acid treatment); this is an important criterion for the assay, because the anti-BrdU antibody is able to recognize the DNA-incorporated IdU only when DNA is in the single strand conformation, as is the end-resected DNA. Through the SMART assay, it is possible to visualize the long 3′ single-strand DNA tails and measure the DNA tracts evaluating the efficiency of the endogenous resection machinery without genetic manipulations. Finally, this technique can be used in any model cellular system from bacteria to humans.

The DNA fibres, stretched on a microscope slide, and challenged with the anti-BrdU Ab and the secondary fluorescinated Ab were then visualized through immunofluorescence microscopy. Analysis through the SMART assay allowed us to quantify the amount of resected DNA: by using an image analysis software (Photoshop CS5), we were able to measure the length of the DNA fibre tracts analysing the efficiency of the DNA end-resection machinery.

Materials and Reagents

  1. 6 cm dish (Corning Inc., catalog number: 430166 )
  2. Silane Prep-slide (Sigma-Aldrich, catalog number: S4651 )
  3. HeLa cells (ATCC, CCL2 , catalog number: CCL2 )
  4. Anti-BrdU clone BU1/75 (ICR1) (AbdSerotec, catalog number: OBT0030CX )
  5. Alexa Fluor 488-conjugated chicken anti-rat (Thermo Fisher Scientific, catalog number: A-21470 )
  6. Anti phospho-RPA32 S4/8 antibody (Bethyl Laboratories, catalog number: A300-245 )
  7. Roswell Park Memorial Institute (RPMI) 1640 Medium (Thermo Fisher Scientific, catalog number: 11875093 )
  8. 10% fetal bovine serum (FBS), EU Approved (South American) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  9. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
  10. Trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056 )
    Note: HeLa cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin. 
  11. 5-Iodo-2′-deoxyuridine (IdU) (Sigma-Aldrich, catalog number: I7125 , storage at 4 °C)
  12. (S)-(+)-Camptothecin (CPT) (Sigma-Aldrich, catalog number: C9911 , storage at -20 °C)
  13. Sodium dodecyl sulphate (SDS) (Sigma-Aldrich, catalog number: 71725 , storage at RT)
  14. TRIS (Sigma-Aldrich, catalog number: 17926 , storage at RT)
  15. EDTA 0.5 M pH 8.0 (Thermo Fisher Scientific, catalog number: AM9262 , storage at 4°C)
  16. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 , storage at RT)
  17. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 , storage at RT)
  18. Potassium Cloride (KCl) (Sigma-Aldrich, catalog number: P9541 , storage at RT)
  19. Hydrochloric acid 39.5% (HCl) (Carlo Erba Reagents, catalog number: 405761 , storage at RT)
  20. Methanol (Carlo Erba Reagents, catalog number: 528101 , storage at RT)
  21. Acetic Acid Glacial (Carlo Erba Reagents, catalog number: 401424 , storage at RT)
  22. Ethanol Absolute (Carlo Erba Reagents, catalog number: 414607 , storage at RT)
  23. Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A9418 , storage at 4 °C)
  24. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 , storage at RT)
  25. ProLong Gold Antifade Reagent (Thermo Fisher Scientific, catalog number: P36935 , storage at RT)
  26. Phosphate buffered saline (PBS) (see Recipes)
  27. Spreading buffer (see Recipes)

Equipment

  1. Zeiss Axiovert LSM100M confocal microscope (Carl Zeiss, Germany)
  2. CO2 incubator (Panasonic, catalog number: MCO-18AC )
  3. Centrifuge

Software

  1. Adobe Photoshop CS5
  2. GraphPad Prism 8 software

Procedure

  1. Representation of SMART assay
    Figure 1 shows a representative scheme of the SMART assay.


    Figure 1. Schematics of SMART assay. First of all, label HeLa cells with IdU for 24 h followed by CPT treatment; at the end of incubation trypsinize cells and dilute them at a 2.5 x 105 cells/ml concentration. Mix labelled and unlabelled cells 1:8 and spot 2.5 μl of this mix onto the upper part of the slide; after incubation with 7.5 μl of spreading buffer, tilt the slide form the end to allow the stretching of the DNA fibres.

    Figure 2 shows representative images acquired through confocal microscopy.


    Figure 2. Representative images of the SMART technique. Panel A shows two images from HeLawt or siEXOI cells treated with 1 μM of campthotecin for one hour; following the SMART assay images were analyzed as reported in the protocol through the Fiji software. As shown in panel B the silencing of EXOI, one of the principal exonucleases involved in the generation of single strand DNA upon DNA damage, reduces the length of the processed DNA up to two times. The Figure 3B reports the mean ± standard deviation of the fibre length measured in μm, for each condition.

  2. SMART assay protocol
    1. Culture HeLa cells in RPMI 1640 medium supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin.
    2. Treat HeLa cells in a 6 cm dish at 80% confluency with 10 μM of IdU (we made a 28 mM IdU stock solution in 0.2 N NaOH) and incubate at 37 °C in a standard 5% CO2 incubator for 24 h.
      Note: The length of incubation with BrdU or its analogs such as IdU has to be determined for each different cell type, depending on the cell cycle length. A pulse labelling time needs to allow BrdU incorporation during one complete cell cycle.
    3. Prepare a 6 cm dish of HeLa cells at 80% confluence as non-IdU-marked.
      Note: The dilution of IdU marked cell with the non-marked cells is essential to reduce the presence of marked fibres onto the glass slides favouring a correct immunofluorescence analysis.
    4. Treat HeLa cells with 1 μM of camptothecin in RPMI 1640 medium supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin and incubate at 37 °C in a 5% CO2 incubator for 2 h.
      Note: The incubation time and drug concentration for the DNA damage agent can change in different cell types. As control, perform a DNA damage response analysis with different conditions evaluating DNA damage and resection through Western blot using an anti phospho-RPA32 S4/8 antibody (Bethyl Laboratories).
    5. Detach HeLa cells from the dish with 1 ml of Trypsin-EDTA solution for 5 min at 37 °C in a standard 5% CO2 incubator.
    6. Inactivate the trypsin-EDTA solution with 3 ml of RPMI 1640 medium supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin.
    7. Centrifuge HeLa cells for 5 min at 800 x g (rcf).
    8. Wash the cell pellet with 5 ml of ice-cold PBS followed by centrifugation for 5 min at 800 x g (rcf). Repeat Step B8 twice.
    9. Resuspend HeLa labelled cells with the ice-cold PBS at 2.5 x 105 cells/ml. Prepare another mix of unlabelled cells at the same concentration (2.5 x 105 cells/ml).
    10. Dilute labelled cells by mixing them in a 1:8 proportion with unlabelled cells.
    11. Mix 2.5 μl of cells with 7.5 μl of spreading buffer directly on the upper part of the slides.
    12. Incubate at room temperature (RT) for 10 min.
    13. Tilt the slide, from the ends, to 15°.
    14. DNA spreads are air-dried, approximately 10 min.
    15. Fix DNA fibres in 3:1 methanol/acetic acid at -20 °C for 15 min.
    16. Wash slides in PBS twice.
      Note: The volume of PBS depends on the container used for the immersion of the slides
    17. Incubate DNA fibres in 70% ethanol and 30% water overnight at 4 °C.
    18. Wash slides with PBS twice.
      Note: This step is essential to remove all the ethanol residues before blocking DNA fibres. The volume of PBS depends on the container used for the immersion of the slides.
    19. Block DNA fibres with PBS + 5% BSA for 30 min at RT.
      Note: The volume of PBS depends on the container used for the immersion of the slides
    20. Incubate slides with 1:300 Anti-BrdU clone BU1/75 (ICR1) for one hour at 37 °C.
    21. Wash slides with PBS twice.
      Note: The volume of PBS depends on the container used for the immersion of the slides.
    22. Incubate slides with 1:400 Alexa Fluor 488-conjugated chicken anti-rat.
    23. Wash slides with PBS twice.
      Note: The volume of PBS depends on the container used for the immersion of the slides.
    24. Mount the slide with ProLong Gold Antifade Reagent.
    25. Analyse the slide through a confocal microscope (we used the Zeiss LSM100) with a minimum of 63x oil immersion objective.

Data analysis

Analyse approximately 150 fibres for each condition and measure the length of fibres in pixel through the Fiji image software analysis.
To obtain the fibre length in μm:

  1. First of all download the LSM toolbox form the plugin window.
    Note: This is an important step to open directly the confocal image in the .lsm format without jpg conversion.
  2. Open the .lsm file, as reported in the Figure 3 below, there is the resolution of the picture and the conversion in μm.


    Figure 3. A representative image opened with the Fiji software showing in the upper left part the pixel resolution and μm conversion; in the lower corner of the figure the μm dimension. A. Micrometer conversion into kilobase depends on the DNA type, the nucleosome and chromatin fibre assembly; for HeLa cells and the combing assay 1 μm of DNA fibre length, during the S-phase, should approximately correspond to 2.59 kb as reported originally by Jackson and Pombo (Jackson and Pombo, 1998) and others (Bianco et al., 2012; Jamroskovic et al., 2020; Petermann et al., 2008). B. A magnification image of DNA fibre showing in red (start) and blue (end) the dimension of the analysed DNA tracts. C. The yellow arrows indicate, in lower part of the figure, the direction and the length of the DNA fibres; these fibres cannot be used for the measurement given the high number of other DNA fibres, which reduce the identification of single DNA traits. The red arrow indicates a fibre at a density and fluorescence, which are optimal for the analysis of the single DNA traits. D. Example of a screenshot of the Straight Line tool from the Fiji software analysis.

  3. Select the Straight Line from the main window and measure the length of the fibres. The Straight Line tool of the Fiji software provides the length of the selected fibre identified through the x, y and angle coordinates; see an example of the Straight Line tool screen in Figure 3D.
  4. In this example, the mean fibre length ± standard deviation, calculated out of three independent experiments was plotted. Statistical significance was evaluated through the Student’s t-test using the GraphPad Prism 8 software in accordance with previous work (Cruz-García et al., 2014). The more informative way to represent data and the appropriate test for statistical analysis, however, will have to be determined according to the experimental plan upon checking data for normality through GraphPad Prism or similar programs.

Recipes

  1. Phosphate buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
  2. Spreading Buffer
    0.5% Sodium dodecyl sulphate (SDS)
    200 mM Tris-HCl pH 7.4
    50 mM EDTA pH 8

Acknowledgments

We are grateful to the Sbarro Health Research Organization (http://www.shro.org), for its support.
    We are grateful to Andres Cruz-García, AnaLópez-Saavedra and Pablo Huertas who first developed the SMART assay (Cruz-García et al., 2014), from which this protocol was adapted.

Competing interests

The authors declare no conflict of interest.

References

  1. Alfano, L., Costa, C., Caporaso, A., Altieri, A., Indovina, P., Macaluso, M., Giordano, A. and Pentimalli, F. (2016). NONO regulates the intra-S-phase checkpoint in response to UV radiation. Oncogene 35(5): 567-576.
  2. Alfano, L., Giordano, A. and Pentimalli, F. (2017). DNA Fiber Assay upon Treatment with Ultraviolet Radiations. Bio-protocol 7(11): e2301.
  3. Alfano, L., Caporaso, A., Altieri, A., Dell'Aquila, M., Landi, C., Bini, L., Pentimalli, F. and Giordano, A. (2019). Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation. Nucleic Acids Res 47(8): 4068-4085.
  4. Bianco, J. N., Poli, J., Saksouk, J., Bacal, J., Silva, M. J., Yoshida, K., Lin, Y. L., Tourriere, H., Lengronne, A. and Pasero, P. (2012). Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing. Methods 57(2): 149-157.
  5. Chen, H., Lisby, M. and Symington, L. S. (2013). RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol Cell 50(4): 589-600.
  6. Cruz-García, A., López-Saavedra, A. and Huertas, P. (2014). BRCA1 accelerates CtIP-mediated DNA-end resection. Cell Rep 9(2): 451-459.
  7. Hanahan, D. and Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell 144(5): 646-674.
  8. Jackson, D. A. and Pombo, A. (1998). Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J Cell Biol 140(6): 1285-1295.
  9. Jamroskovic, J., Doimo, M., Chand, K., Obi, I., Kumar, R., Brannstrom, K., Hedenstrom, M., Nath Das, R., Akhunzianov, A., Deiana, M., Kasho, K., Sulis Sato, S., Pourbozorgi, P. L., Mason, J. E., Medini, P., Ohlund, D., Wanrooij, S., Chorell, E. and Sabouri, N. (2020). Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization. J Am Chem Soc 142(6): 2876-2888.
  10. Krogh, B. O. and Symington, L. S. (2004). Recombination proteins in yeast. Annu Rev Genet 38: 233-271.
  11. Mao, Z., Bozzella, M., Seluanov, A. and Gorbunova, V. (2008). Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst) 7(10): 1765-1771.
  12. Nimonkar, A. V., Genschel, J., Kinoshita, E., Polaczek, P., Campbell, J. L., Wyman, C., Modrich, P. and Kowalczykowski, S. C. (2011). BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev 25(4): 350-362.
  13. Petermann, E., Helleday, T. and Caldecott, K. W. (2008). Claspin promotes normal replication fork rates in human cells. Mol Biol Cell 19(6): 2373-2378.
  14. Stracker, T. H. and Petrini, J. H. (2011). The MRE11 complex: starting from the ends. Nat Rev Mol Cell Biol 12(2): 90-103.
  15. Tkáč, J., Xu, G., Adhikary, H., Young, J.T.F., Gallo, D., Escribano-Díaz, C., Krietsch, J., Orthwein, A., Munro, M., Sol, W., et al. (2016). HELB Is a Feedback Inhibitor of DNA End Resection. Mol Cell 61: 405-418.
  16. Zhou, Y., Caron, P., Legube, G. and Paull, T. T. (2014). Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res 42(3): e19.

简介

[摘要] DNA双链断裂(dsb)是影响基因组完整性的最具毒性的损伤之一。dsb主要通过非同源末端连接(NHEJ)和同源重组(HR)进行修复。HR过程的一个关键步骤是通过DNA末端切除,产生一个长的3′单链DNA链,这是在DNA链入侵后,以同源区域为模板进行DNA合成所必需的。DNA末端切除抑制NHEJ并触发同源定向的DSB修复,最终保证DNA的可靠修复。已建立的评价DNA末端切除过程的方法是免疫荧光法分析磷酸化S4/8rpa32蛋白病灶(DNA末端切除的标志物)或磷酸化S4/8rpa32蛋白水平。近年来,切除轨迹单分子分析(SMART)被认为是一种可靠的方法,可以通过免疫荧光法观察S期特异性DNA损伤剂(如喜树碱)处理细胞后产生的长3′单链DNA尾。然后,通过图像分析软件(如Photoshop)测量DNA束长度,评价DNA末端切除机的处理能力。DNA纤维的制备是在非变性条件下进行的,因此免疫荧光只检测DSB处理产生的特定的3′单链DNA尾。

[背景] 基因组不稳定性是导致肿瘤发展的有利特征之一(Hanahan和Weinberg,2011)。许多DNA损伤源,无论是内源性的还是外源性的,都会引起各种DNA损伤,包括双链断裂(DBSs),这种损伤激活了DNA损伤反应(DDR)——一种旨在保持DNA完整性的细胞过程。DSBs修复涉及两种主要的修复途径:非同源末端连接(NHEJ)和同源重组(HR)(Mao等人,2008)。HR过程通过使用互补DNA序列的忠实修复来防止DNA损伤时遗传信息的丢失。通过抑制容易出错的NHEJ并触发同源定向DSB修复,DNA末端切除在指导修复路径选择以实现可靠修复方面起着至关重要的作用。因此,为了提高正确的心率,dsb通过DNA末端切除机进行处理,这是产生同源链入侵所必需的长3′单链DNA(ssDNA)尾巴所必需的。DNA末端切除是一个精细调节的过程;第一步包括将MRN复合物(MRE11-RAD50-NBSI)和CtIP(RBBP8)募集到DNA损伤处,产生短片段的ssDNA(Stracker和Petrini,2011),然后在DNA末端切除由核酸外切酶I(EXOI)或DNA复制螺旋酶/核酸酶2(DNA2)介导布鲁姆综合征螺旋酶类(BLM)复合物(Nimonkar等人,2011年)。随后,复制蛋白A(RPA)复合物结合DNA末端切除产生的ssDNA,防止DNA发夹的形成(Chen等人,2013),并促进链交换过程所需的RAD51的装载(Krogh和Symington,2004)。

DNA末端切除术的评估对于剖析这一精细调节过程的分子机制和确定参与其中的关键参与者至关重要。可以使用各种方法间接评估DNA末端切除过程,如上文所述分析磷酸化S4/8 RPA32,或RAD51病灶,或在长时间培养后检测ssDNA中的BrdU(Tkč等人,2016年)。2014年,Tania Paull开发了一种确定切除长度和特定DSB位点ssDNA范围的方法,尽管这是基于一种内切酶(如AsiSI)的表达来诱导基因组内特定位点的DSB,然后进行PCR分析(Zhou等人,2014)。

SMART分析首先由Huertas及其合著者开发,是一种可靠的方法,可在单分子水平上测量任何细胞系统中暴露于任何损伤剂后切除的DNA的长度(Cruz-García等人,2014年)。该分析基于先前开发的DNA组合分析,该方法能够将DNA纤维物理拉伸到固体载体上,从而通过免疫荧光进行可视化(Alfano等人,2016年和2017年)。我们最近确定RNA结合蛋白HNRNPD是HR过程中的一个新参与者,并使用SMART分析和其他技术评估DNA末端切除(Alfano等人,2019年)。我们用卤化IdU(5-碘-2′-脱氧尿苷)脉冲标记HeLa细胞——尽管可以使用任何其他嘧啶类似物,如BrdU(溴脱氧尿嘧啶)或CldU(5-氯-2'-脱氧尿嘧啶),在S期并入新合成的DNA中,持续约24小时,然后用DNA损伤剂处理,喜树碱。药物孵育结束后,在非DNA变性条件下(避免盐酸处理)裂解细胞并提取DNA纤维;这是检测的一个重要标准,因为只有当DNA处于单链构象时,抗BrdU抗体才能够识别掺入DNA的IdU,最后切除的DNA也是一样。通过智能检测技术,可以在不进行遗传操作的情况下,观察长3′单链DNA尾和测量DNA束来评价内生切除机的效率。最后,这项技术可以应用于从细菌到人类的任何模型细胞系统。

用二次荧光显微镜对纤维进行荧光染色,并用荧光显微镜对纤维进行拉伸。通过智能分析的分析使我们能够量化切除的DNA的数量:通过使用图像分析软件(Photoshop CS5),我们能够测量DNA纤维束的长度,分析DNA末端切除机的效率。

关键字:DNA修复, 同源重组, DNA末端切除, BrdU标记, 免疫荧光

材料和试剂


 


1.     6厘米碟子(康宁公司,产品编号:430166)


2.     硅烷制备载玻片(Sigma-Aldrich,目录号:S4651)


3.     HeLa单元(ATCC,CCL2,目录号:CCL2)


4.     抗BrdU克隆BU1/75(ICR1)(AbdSerotec,目录号:OBT030Cx)


5.     Alexa Fluor 488共轭鸡抗鼠(Thermo Fisher Scientific,目录号:A-21470)


6.     抗磷酸化RPA32 S4/8抗体(Bethyl Laboratories,目录号:A300-245)


7.     罗斯韦尔公园纪念研究所(RPMI)1640 Medium(赛默飞世尔科学公司,目录号:11875093)


8.     10%胎牛血清(FBS),欧盟批准(南美)(Thermo Fisher Scientific,GibcoTM,目录号:10270-106)


9.     青霉素链霉素(Thermo Fisher Scientific,GibcoTM,目录号:15070-063)


10.  胰蛋白酶EDTA(赛默飞世尔科技公司,目录号:25200056)


注:HeLa细胞培养于添加10%FBS、1μg/ml青霉素和1µg/ml链霉素的RPMI 1640中。


11.  5-碘-2′-脱氧尿苷(IdU)(Sigma-Aldrich,目录号:I7125 4°C下储存),


12.  (S) -(+)-喜树碱(CPT) (Sigma-Aldrich,目录号:C9911,-20°C下储存)


13.  十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:71725,RT储存)


14.  特里斯(Sigma Aldrich,目录号:17926,RT储存)


15.  EDTA 0.5 M pH 8.0(赛默飞世尔科技公司,目录号:AM9262,4°C下储存)


16.  磷酸二钠(Na2HPO4)(Sigma-Aldrich,目录号:255793,室温储存)


17.  磷酸二氢钾(KH2PO4)(Sigma-Aldrich,目录号:P0662,RT储存)


18.  氯化钾(KCl)(Sigma-Aldrich,目录号:P9541,室温储存)


19.  盐酸39.5%(HCl)(Carlo Erba试剂,目录号:405761,RT储存)


20.  甲醇(Carlo Erba试剂,目录号:528101,RT储存)


21.  冰醋酸(Carlo Erba试剂,目录号:401424,室温储存)


22.  无水乙醇(Carlo Erba试剂,目录号:414607,室温储存)


23.  牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418,4°C保存)


24.  氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888,室温储存)


25.  延长金防褪色试剂(赛默飞世尔科技公司,目录号:P36935,室温储存)


26.  磷酸盐缓冲盐水(PBS)(见配方)


27.  铺展缓冲液(见配方)




 


设备


 


1.     蔡司Axiovert LSM100M共焦显微镜(德国卡尔蔡司)


2.     二氧化碳培养箱(松下,目录号:MCO-18AC)


3.     离心机


 


软件


 


1.     Adobe Photoshop CS5


2.     GraphPad Prism 8软件


 


程序


 


A、 智能分析的表示


图1显示了SMART分析的一个代表性方案。


 






图1。智能分析示意图。首先,用IdU标记HeLa细胞24小时,然后进行CPT处理;在培养结束时,对细胞进行胰蛋白酶处理,并以2.5×105个细胞/ml的浓度稀释。将已标记和未标记的细胞按1:8的比例混合,并将2.5μl的混合液点在载玻片的上部;在用7.5μl扩散缓冲液孵育后,将载玻片从末端倾斜,以允许DNA纤维的伸展。




 


图2显示了通过共焦显微镜获得的代表性图像。


 






图2。智能技术的代表性图像。面板A显示用1μM campthotecin处理1小时的HeLawt或siEXOI细胞的两张图像;随后通过Fiji软件分析协议中报告的SMART分析图像。如图B所示,在DNA损伤时参与单链DNA生成的主要外切酶之一,EXOI的沉默使处理后的DNA长度缩短了两倍。图3B报告了每种情况下测量的纤维长度的平均值±标准偏差(μm)。


 


B、 智能分析协议


1.     在RPMI1640培养基中培养HeLa细胞,培养基中添加10%FBS、1μg/ml青霉素和1µg/ml链霉素。


2.     在6 cm培养皿中用10μM的IdU在80%的浓度下处理HeLa细胞(我们在0.2 N NaOH中制备28 mM的IdU储备溶液),并在37°C的标准5%CO2培养箱中培养24小时。


注:与BrdU或其类似物(如IdU)的孵育时间必须根据细胞周期的长短来确定。脉冲标记时间需要允许BrdU在一个完整的细胞周期内结合。


3.     准备一个6厘米的HeLa细胞培养皿,在80%的汇合处作为非IdU标记。


注:用非标记细胞稀释IdU标记细胞对于减少玻片上有标记纤维的存在至关重要,有利于正确的免疫荧光分析。


4.     用1μM喜树碱在RPMI 1640培养基中处理HeLa细胞,RPMI 1640培养基中添加10%FBS、1μg/ml青霉素和1µg/ml链霉素,并在37℃下在5%CO2培养箱中培养2 h。


注:DNA损伤剂的孵育时间和药物浓度随细胞类型的不同而变化。作为对照,使用抗磷酸化RPA32 S4/8抗体(Bethyl Laboratories)在不同条件下进行DNA损伤反应分析,评估DNA损伤和切除。


5.     将HeLa细胞从培养皿中分离出来,用1ml胰蛋白酶EDTA溶液在37℃的标准5%二氧化碳培养箱中培养5分钟。


6.     用3 ml RPMI 1640培养基灭活胰蛋白酶EDTA溶液,添加10%FBS、1μg/ml青霉素和1µg/ml链霉素。


7.     以800 x g(rcf)离心分离HeLa细胞5分钟。


8.     用5 ml冰冷PBS洗涤细胞颗粒,然后以800 x g(rcf)离心5分钟。重复步骤B8两次。


9.     用2.5×105个细胞/毫升的冰冷PBS使HeLa标记的细胞复苏。准备另一个相同浓度(2.5×105个细胞/ml)的未标记细胞混合物。


10.  将标记细胞与未标记细胞按1:8的比例混合稀释。


11.  将2.5μl细胞与7.5μl扩散缓冲液直接混合在载玻片的上部。


12.  在室温(RT)下培养10分钟。


13.  将滑块从两端倾斜15°。


14.  DNA涂片风干,大约10分钟。


15.  将DNA纤维固定在-20°C的3:1甲醇/乙酸中15分钟。


16.  在PBS中清洗载玻片两次。


注:PBS的体积取决于用于浸泡载玻片的容器


17.  将DNA纤维在70%乙醇和30%水中于4℃下孵育过夜。


18.  用PBS清洗载玻片两次。


注意:这一步对于在阻断DNA纤维之前清除所有乙醇残留物是至关重要的。PBS的体积取决于用于浸泡载玻片的容器。


19.  用PBS+5%BSA在RT条件下30分钟阻断DNA纤维。


注:PBS的体积取决于用于浸泡载玻片的容器


20.  用1:300抗BrdU克隆BU1/75(ICR1)在37℃下培养载玻片1小时。


21.  用PBS清洗载玻片两次。


注:PBS的体积取决于用于浸泡载玻片的容器。


22.  用1:400 Alexa Fluor 488结合鸡抗鼠培养载玻片。


23.  用PBS清洗载玻片两次。


注:PBS的体积取决于用于浸泡载玻片的容器。


24.  在载玻片上涂上长效金防腐蚀剂。


25.  通过共焦显微镜(我们使用蔡司LSM100)和至少63倍油浸物镜分析载玻片。


数据分析


 


分析每种情况下大约150根光纤,并通过斐济图像软件分析以像素为单位测量光纤长度。


要获得以μm为单位的纤维长度:


1.     首先从插件窗口下载LSM工具箱。


注意:这是一个重要的步骤,直接打开的共焦图像在.lsm格式没有jpg转换。


2.     打开.lsm文件,如下图3所示,有图片的分辨率和以μm为单位的转换。


 






图3。使用斐济软件打开的代表性图像在左上角显示像素分辨率和μm转换;在图形的下角显示μm尺寸。 A、 千分尺转换成千碱基取决于DNA类型、核小体和染色质纤维装配;对于HeLa细胞和结合分析,在S期,1μm的DNA纤维长度应大约对应于2.59 kb,如Jackson和Pombo(Jackson和Pombo,1998)和其他人(Bianco等人,2012)最初报告的那样;Jamroskovic等人,2020年;Petermann等人,2008年)。B、 DNA纤维的放大图像,以红色(开始)和蓝色(结束)显示分析的DNA束的尺寸。C、 黄色箭头在图的下半部分显示了DNA纤维的方向和长度;鉴于其他DNA纤维数量较多,这些纤维不能用于测量,这减少了单个DNA特征的识别。红色箭头表示密度和荧光的纤维,这是分析单个DNA性状的最佳选择。D、 斐济软件分析中直线工具的屏幕截图示例。


 


3.     从主窗口选择一条直线并测量光纤的长度。斐济软件的直线工具提供通过x、y和角度坐标确定的选定光纤的长度;参见图3D中的直线工具屏幕示例。


4.     在本例中,绘制了从三个独立实验中计算出的平均纤维长度±标准偏差。根据之前的工作,通过使用GraphPad Prism 8软件的学生t检验来评估统计显著性(Cruz García等人,2014年)。然而,在通过GraphPad Prism或类似程序检查数据的正态性时,必须根据实验计划来确定更具信息性的数据表示方式和统计分析的适当测试。


 


食谱


 


1.     磷酸盐缓冲盐水(PBS)


137毫米氯化钠


2.7毫米氯化钾


10毫米Na2HPO4


1.8毫米KH2PO4


2.     扩展缓冲器


0.5%十二烷基硫酸钠(SDS)


200mm Tris HCl pH值7.4


50毫米EDTA pH值8


 


致谢


 


我们感谢斯巴罗健康研究组织(http://www.shro.org)为它的支持。


我们感谢安德烈Cruz García、AnaLópez Saavedra和Pablo Huertas首先开发了SMART分析法(Cruz-García等人,2014年),从中修改了本协议。


 


相互竞争的利益


 


作者声明没有利益冲突。


 


工具书类


 


1.     Alfano,L.,Costa,C.,Caporaso,A.,Altieri,A.,Indovina,P.,Macaluso,M.,Giordano,A.和Pentimalli,F.(2016年)。NONO调节S期内检查点以响应紫外线辐射。癌基因35(5):567-576。


2.     Alfano,L.,Giordano,A.和Pentimalli,F.(2017年)。紫外线照射后的DNA纤维分析。生物协议7(11):e2301。


3.     Alfano,L.,Caporaso,A.,Altieri,A.,Dell'Aquila,M.,Landi,C.,Bini,L.,Pentimalli,F.和Giordano,A.(2019年)。RNA结合蛋白HNRNPD的缺失通过抑制DNA末端切除和诱导R-环积累来损害同源重组。核酸研究47(8):4068-4085。


4.     Bianco,J.N.,Poli,J.,Saksouk,J.,Bacal,J.,Silva,M.J.,Yoshida,K.,Lin,Y.L.,Touriere,H.,Lengronne,A.和Pasero,P.(2012年)。用DNA梳合法分析酵母和哺乳动物细胞的DNA复制谱。方法57(2):149-157。


5.     Chen,H.,Lisby,M.和Symington,L.S.(2013年)。RPA协调DNA末端切除,防止DNA发夹的形成。摩尔细胞50(4):589-600。


6.     Cruz García,a.,López Saavedra a.和Huertas,P.(2014年)。BRCA1加速CtIP介导的DNA末端切除。细胞报告9(2):451-459。


7.     Han R.Weinberg和Han R.D.2011年)。癌症的特征:下一代。手机144(5):646-674。


8.     杰克逊,D.A.和Pombo,A.(1998年)。复制子簇是染色体结构的稳定单位:核组织有助于人类细胞S期的有效激活和增殖。细胞生物学杂志140(6):1285-1295。


9.     Jamroskovic,J.,Doimo,M.,Chand,K.,Obi,I.,Kumar,R.,Brannstrom,K.,Hedenstrom,M.,Nath Das,R.,Akhunzianov,A.,Deiana,M.,Kasho,K.,Sulis Sato,S.,Pourbozorgi,P.,Mason,J.E.,Medini,P.,Ohlund,D.,Wanrooij,R.,Chorell,E.和N.Sabouri(2020年)。喹唑啉配体通过选择性抑制STAT3和G-四链体稳定诱导癌细胞死亡。美国化学学会杂志142(6):2876-2888。


10.  Krogh,B.O.和Symington,L.S.(2004年)。酵母中的重组蛋白。遗传学年鉴38:233-271。


11.  (2008年11月,Goruaz,Goruaz,和Bunzza,2008年)。人细胞非同源末端连接与同源重组的比较。DNA修复(Amst)7(10):1765-1771。


12.  Nimonkar,A.V.,Genschel,J.,Kinoshita,E.,Polaczek,P.,Campbell,J.L.,Wyman,C.,Modrich,P.和Kowalczykowski,S.C.(2011年)。BLM-DNA2-RPA-MRN和EXO1-BLM-RPA-MRN是两种用于人类DNA断裂修复的DNA末端切除机。基因进化25(4):350-362。


13.  Petermann,E.,Helleday,T.和Caldecott,K.W.(2008年)。卡环蛋白促进人类细胞正常的复制分叉率。分子生物学细胞19(6):2373-2378。


14.  Stracker,T.H.和Petrini,J.H.(2011年)。MRE11综合体:从末端开始。分子细胞生物学12(2):90-103。


15.  Tkách,J.,Xu,G.,Adhikary,H.,Young,J.T.F.,Gallo,D.,Escribano-Díaz,C.,Krietsch,J.,Orthwein,A.,Munro,M.,Sol,W.,等。(2016年)。HELB是DNA末端切除的反馈抑制剂。分子细胞61:405-418。


16.  Zhou,Y.,Caron,P.,Legube,G.和Paull,T.T.(2014年)。人细胞DNA双链断裂切除中间产物的定量分析。核酸Res 42(3):e19。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Altieri, A., Dell'Aquila, M., Pentimalli, F., Giordano, A. and Luigi, A. (2020). SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage. Bio-protocol 10(15): e3701. DOI: 10.21769/BioProtoc.3701.
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