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DNA Damage Induction by Laser Microirradiation
激光微辐射诱导DNA损伤   

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Abstract

Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live cell microscopy. DNA lesions are introduced at pre-determined sub-nuclear sites and repair can be analyzed in real time in living cells when using fluorescently tagged repair proteins (Mortusewicz et al., 2008). Alternatively, laser microirradiation can be combined with immunofluorescence analysis to study recruitment of endogenous proteins to laser-induced DNA damage tracks that can be visualized by positive controls like, e.g., γH2AX that mark sites of DNA breaks.

Keywords: Microirradiation(微辐照), Live cell imaging(活细胞成像), DNA damage(DNA损伤), DNA repair(DNA修复), DNA lesions(DNA损害), DNA damage response(DNA损伤反应), Immunofluorescence(免疫荧光), Microscopy(显微镜检查)

Background

The genomic integrity of mammalian cells is constantly challenged by DNA damage introduced through external and internal sources. Amongst the most common DNA lesions are oxidized bases, double strand breaks, single strand breaks, inter- and intra-strand crosslinks and UV adducts. Various DNA damage signalling and repair pathways have evolved to deal with these lesions. For DNA repair to be fast, precise and efficient, numerous proteins involved in sensing, signalling and repairing specific DNA lesions have to be coordinated in space and time. Furthermore, DNA is organized into higher order chromatin structures and thus for DNA lesions to be accessible to DNA repair enzymes, chromatin has to be remodeled. Laser microirradiation in combination with advanced live cell microscopy allows studying these highly dynamic processes in the context of living cells (Mortusewicz et al., 2008). The protocol described here uses a 405 nm laser that should be readily available at most confocal or spinning disk microscopes to induce DNA damage in living cells and should therefore be cost effective and feasible in most standard cell biology laboratories. Using different sensitization methods (e.g., Heochst versus BrdU sensitization) and laser energies, the ratio between double strand breaks, single strand breaks, oxidative lesions and UV damage can be modified to the experimental needs.

Materials and Reagents

  1. Live cell microscopy compatible Petri dish or chambers (e.g., Ibidi, catalog number: 35 mm µ-Grid )
  2. Adherent cell lines, e.g., U2OS
  3. If no CO2 control is available at your microscope, use CO2 independent medium without phenol red (e.g., Leibovitz's L-15 medium or CO2 independent medium, Thermo Fisher Scientific, GibcoTM, catalog number: 18045088 ) supplemented with 10% FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10082139 ) and antibiotics (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 ). Alternatively, HEPES (Sigma-Aldrich, catalog number: H4034 ) can be added to your medium of chose.
  4. 5-bromo-2’-deoxycytidine (e.g., Santa Cruz Biotechology, catalog number: 1022-79-3 )
  5. Hoechst 33342 (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: H1399 )
  6. 4% formaldehyde in PBS (Santa Cruz Biotechology, catalog number: 30525-89-4 )
  7. Triton X-100 (Sigma-Aldrich, catalog number: 234729 )
    Note: This product has been discontinued.
  8. Mouse-anti-γH2AX (EMD Milipore, catalog number: 05-636 )
  9. Donkey anti-Mouse IgG (H+L) secondary antibody, Alexa Fluor® 488 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21202 )
  10. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503-500g )
  11. Tween 20 (Sigma-Aldrich, catalog number: P1379-500ML )
  12. Optional: plasmids encoding for fluorescently tagged proteins of interest, e.g., GFP-Timeless (Figure 1). Fluorescently tagged proteins known to be involved in different DNA repair pathways, like PARP-1, XRCC1 or 53BP1, can be used as controls to set up optimal conditions.
  13. Optional: transfection reagent of choice, e.g., self-made PEI solution or commercial distributor

Equipment

  1. Inverted confocal or spinning disk microscope equipped with a 405 nm laser and an environmental chamber or insert to control temperature and CO2/humidity for long-term live cell experiments, e.g., Zeiss LSM710 or LSM780 confocal laser scanning microscope equipped with a UV-transmitting Plan-Apochromat 63x/1.40 Oil DIC M27 or Plan-Apochromat 40x/1.30 Oil DIC M27 objective, respectively
  2. Cell incubator

Software

  1. Microscope software, e.g., ZEN from Zeiss (Zeiss)
  2. Microsoft Excel (Microsoft)
  3. Image J (https://imagej.nih.gov/ij/)

Procedure

  1. Seed 4-9 x 104 U2OS cells/ml in a 35 mm µ-Grid dish (Ibidi) 2-3 days before starting the microirradiation experiment. Use dishes with Grid to relocate cells after microirradiation and immunofluorescence staining.
  2. Optional: transfect cells with plasmid encoding a fluorescently tagged protein of interest 24-48 h before microirradiation experiment. In our hands jetPEI gives good transfection efficiencies in U2OS cells.
  3. Sensitization of cells for DNA damage induction by laser micorirradiation
    1. BrdU sensitization: incubate cells in medium containing 10 µM BrdU for at least 24 h prior to microirradiation experiment. BrdU is an analog of thymidine, which gets incorporated into DNA during DNA replication and upon irradiation with the 405 nm laser will lead to induction of DNA damage through, e.g., radical formation.
    2. Hoechst sensitization: incubate cells in medium containing 10 µg/ml Hoechst for 10 min prior to microirradiation experiment. Hoechst is a cell permeable DNA dye that binds to the minor groove of DNA. As Hoechst is excited by UV light, localized microirradiation with a 405 nm laser will generate DNA damage. Use either Hoechst or BrdU for sensitization.
  4. Add prewarmed, CO2 adjusted live cell medium supplemented with 10% FBS and antibiotics without phenol-red to cells to minimize autofluorescence.
  5. DNA damage induction followed by live cell imaging (Figure 1)
    1. Spot irradiation: For DNA damage induction in live cells expressing a fluorescently tagged protein of interest irradiate a diffraction-limited spot within the nucleus with a 405 nm diode laser (Xie et al., 2015) (405 nm laser set to 70%, 1 iteration, zoom 5, pixel dwell time 177.32 µs).
    2. Line irradiation: For DNA damage induction in live cells expressing a fluorescently tagged protein of interest irradiate a stripe of 5 pixel width with a 405 nm diode laser (Xie et al., 2015) (405 nm laser set to 24%, 1 iteration, zoom 5, pixel dwell time 12.61 µs).
    3. Live cell imaging: Before and after microirradiation record confocal image series of one mid z-section at 2 sec time interval (typically 6 pre-irradiation and 60 post-irradiation frames, frame size of 512 x 512 pixels, pixel size of 90 nm), using the laser line according to the excitation spectrum of the chosen reporter protein (e.g., 488 nm Ar laser for GFP/tagged Timeless)
  6. DNA damage induction followed by immunofluorescence staining
    1. Laser microirradiation: Use the FRAP module of the ZEN software (line scan, 405 nm laser set to 92%, 50 iterations) or the tile scan mode (Xie et al., 2015; Baldeyron et al., 2011) (3 x 3 tiles, image size 128 x 128, scan speed 177.32 µs, every 7th line scanned, 405 nm laser set to 30%).
    2. Fixation and immunofluorescence:
      1. Fix cells in 4% formaldehyde for 10 min after different repair times.
      2. 1 x wash in PBS
      3. Permeabilize with 0.5% Triton X-100 in PBS for 5 min
      4. 1 x wash in PBS
      5. Incubate in blocking solution for 40 min
      6. 1 x wash in PBS
      7. Add primary antibody, e.g., Mouse-anti-γH2AX diluted in blocking buffer and incubate overnight at 4 °C.
      8. 3 x wash in PBST for 5 min
      9. Add secondary antibody, e.g., Donkey anti-Mouse IgG (H+L) secondary antibody, Alexa Fluor® 488 diluted in 4% BSA/PBS and incubate for 1 h at RT
      10. 3 x wash in PBST for 5 min
      11. Add fresh PBS and store at 4 °C
    Note: The intensity of the 405 nm laser may vary between different microscopes. Therefore optimal laser settings have to be determined first, e.g., varying the laser intensity followed by staining with DNA damage markers like γH2AX, PAR or thymine dimers. Settings that induce clear stripes of, e.g., γH2AX should be used, whereas pan-nuclear staining indicates that the used laser intensity is too high. Sensitization methods can also be varied depending on what kind of DNA repair pathway should be analysed, e.g., single strand break repair versus double strand break repair.

Data analysis

For evaluation of the recruitment kinetics, fluorescence intensities at the irradiated region are corrected for background and for total nuclear loss of fluorescence over the time course and normalized to the pre-irradiation value. For the quantitative evaluation of microirradiation experiments, data of at least 10-20 nuclei from three independent experiments should be averaged and the mean curve and the standard error of the mean calculated and displayed using Microsoft Excel software.

  1. Image registration and measurement of fluorescence intensities
    To compensate for potential cell movements in x and y direction during image acquisition, the TurboReg plug-in from ImageJ can be used.
    1. Open file in ImageJ
      1. Display ROIs
      2. Split channels
    2. Copy the first image of the image series, this will be used as a template for the image registration
    3. Turbo reg
      1. Source – original image series
      2. Target – new image
      3. Choose Rigid body and set quality to fast
      4. Batch = movement compensation
      5. Save new file as tiff
    4. For analyses define three different regions of interest (ROIs) in ImageJ
      1. ROIi = irradiated region (spot or line ROI)
      2. ROIn = outline of the cell nucleus
      3. ROIb = background region outside of the cell
    5. Add ROIs to ROI manager (Edit→Selection→add to manager), choose multi measure and save ROIs and measurements (measurement types can be chosen in: Edit→options→input/output. Untick Row, if needed)
    6. Copy measurements into Microsoft Excel
      1. Subtract background intensity (ROIb) from fluorescence intensities of nucleus (ROIn) and irradiated region (ROIi): ROIi-b = ROIi - ROIb and ROIn-b = ROIn - ROIb
      2. To compensate for bleaching during acquisition and set the pre irradiation value to 1 use double normalization: (ROIn-b t0/ROIn-b t)/(ROIi-b t0/ROIi-b t)
        Where, ROIi-b t and ROIn-b t is the fluorescence intensity value at time point t and ROIi-b t0 and ROIn-b t0 is the fluorescence intensity at time point zero, respectively (pre-irradiation value)


        Figure 1. Determination of protein recruitment to laser-induced DNA damage sites in fixed and live cells. A. Schematic illustration of micorirradiation procedure using either the tile scanning mode to induce DNA damage in a large number of cells followed by fixation and immunofluorescence analysis or line and spot irradiation of single cells expressing fluorescently tagged proteins of interest followed by live cell imaging. B. Representative confocal images after micoirradiation using the tile scanning mode of the Zeiss LSM780. U2OS cells were microirradiated, fixed after indicated time points and stained with antibodies against γH2AX and RPA, which can be used as markers for double strand breaks and ssDNA formations to identify DNA damage sites. C. U2OS cells transfected with expression plasmid encoding GFP/tagged Timeless were microirradiated and recruitment of GFP-Timeless followed in real/time with a time interval of 2 sec.

Notes

To ensure reproducibility between experiments, a laser power meter can be used to measure the 405 nm laser intensity. Alternatively regular immunofluorescence staining with a reference antibody against, e.g., γH2AX as a positive control can be employed.

Recipes

  1. Fixation solution
    4% formaldehyde in PBS
  2. Permeabilization solution
    0.5% Triton X-100 in PBS
  3. Blocking solution
    4% BSA in PBS
  4. Washing buffer
    0.05% Tween 20 in PBS

Acknowledgments

This work was supported by the Helleday foundation. The protocol has been adapted from the following publications: Walter et al. (2003), Mortusewicz et al. (2008), Xie et al. (2015), Mortusewicz et al. (2016), Mortusewicz et al. (2007), and Mortusewicz et al. (2006).

References

  1. Baldeyron, C., Soria, G., Roche, D., Cook, A. J. and Almouzni, G. (2011). HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair. J Cell Biol 193(1): 81-95.
  2. Mortusewicz, O., Amé, J. C., Schreiber, V. and Leonhardt, H. (2007). Feedback-regulated poly (ADP-ribosyl) ation by PARP-1 is required for rapid response to DNA damage in living cells. Nucleic acids research 35(22): 7665-7675.
  3. Mortusewicz, O., Evers, B. and Helleday, T. (2016). PC4 promotes genome stability and DNA repair through binding of ssDNA at DNA damage sites. Oncogene 35(6): 761-770.
  4. Mortusewicz, O., Leonhardt, H. and Cardoso, M. C. (2008). Spatiotemporal dynamics of regulatory protein recruitment at DNA damage sites. J Cell Biochem 104(5): 1562-1569.
  5. Mortusewicz, O., Rothbauer, U., Cardoso, M. C. and Leonhardt, H. (2006). Differential recruitment of DNA Ligase I and III to DNA repair sites. Nucleic Acids Res 34(12): 3523-3532.
  6. Walter, J., Cremer, T., Miyagawa, K. and Tashiro, S. (2003). A new system for laser-UVA-microirradiation of living cells. J Microsc 209(Pt 2): 71-75.
  7. Xie, S., Mortusewicz, O., Ma, H. T., Herr, P., Poon, R. Y., Helleday, T. and Qian, C. (2015). Timeless interacts with PARP-1 to promote homologous recombination repair. Mol Cell 60(1): 163-176.

简介

基因组不稳定性可导致细胞死亡,衰老和癌性转化。特异性修复途径已经进化以防止DNA损伤的累积。研究这些高度动态和特定的修复途径需要精确的空间和时间分辨率,这可以通过激光微激光和活细胞显微镜的组合实现。当使用荧光标记的修复蛋白时,在预定的亚核位点引入DNA损伤并且可以在活细胞中实时分析修复(Mortusewicz等人,2008)。或者,激光微辐照可与免疫荧光分析结合以研究内源蛋白质对激光诱导的DNA损伤轨迹的募集,其可通过阳性对照例如标记DNA断裂位点的γH2AX显现。
关键字:微辐射,活细胞成像,DNA损伤,DNA修复,DNA损伤,DNA损伤反应,免疫荧光,显微镜等

/strong>哺乳动物细胞的基因组完整性不断受到通过外部和内部来源引入的DNA损伤的挑战。最常见的DNA损伤是氧化碱基,双链断裂,单链断裂,链间和链内交联和UV加合物。已经发展了各种DNA损伤信号传导和修复途径以处理这些损伤。为了使DNA修复快速,精确和有效,涉及感测,信号传导和修复特定DNA损伤的许多蛋白质必须在空间和时间上协调。此外,DNA被组织成更高级的染色质结构,因此对于DNA损伤,DNA修复酶是可及的,染色质必须重塑。激光微照射与高级活细胞显微镜相结合允许在活细胞的上下文中研究这些高度动态的过程(Mortusewicz等人,2008)。这里描述的协议使用405 nm激光,应该很容易获得在大多数共聚焦或旋转磁盘显微镜诱导活细胞中的DNA损伤,因此应该是成本效益和可行在大多数标准细胞生物实验室。使用不同的敏化方法(例如,Heochst与BrdU致敏)和激光能量,双链断裂,单链断裂,氧化损伤和UV损伤之间的比率可以修改为实验需要。

关键字:微辐照, 活细胞成像, DNA损伤, DNA修复, DNA损害, DNA损伤反应, 免疫荧光, 显微镜检查

材料和试剂

  1. 活细胞显微镜兼容的培养皿或腔室(例如,Ibidi,目录号:35mmμ-栅格)
  2. 粘附细胞系,例如,U2OS
  3. 如果在您的显微镜下没有CO 对照,则使用不含酚红的CO 2独立培养基(例如,Leibovitz的L-15培养基或补充有10%FBS(Thermo Fisher Scientific,Gibco< sup>)的DMEM培养基(CO 2 CO 2独立培养基,Thermo Fisher Scientific,Gibco <-目录号:18045088) ,目录号:10082139)和抗生素(Thermo Fisher Scientific,Gibco< sup>,目录号:15140-122)。或者,可以将HEPES(Sigma-Aldrich,目录号:H4034)加入到您选择的培养基中
  4. 5-溴-2'-脱氧胞苷(例如,Santa Cruz Biotechology,目录号:1022-79-3)
  5. Hoechst 33342(Thermo Fisher Scientific,Molecular Probes TM ,目录号:H1399)
  6. PBS中的4%甲醛(Santa Cruz Biotechology,目录号:30525-89-4)
  7. Triton X-100(Sigma-Aldrich,目录号:234729) 注意:此产品已停产。
  8. 小鼠抗γH2AX(EMD Milipore,目录号:05-636)
  9. 驴抗小鼠IgG(H + L)第二抗体Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen TM,目录号:A-21202)
  10. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503-500g)
  11. 吐温20(Sigma-Aldrich,目录号:P1379-500ML)
  12. 任选的:编码感兴趣的荧光标记的蛋白质的质粒,例如GFP-永久的(图1)。已知涉及不同DNA修复途径的荧光标记的蛋白质,如PARP-1,XRCC1或53BP1,可用作控制以建立最佳条件。
  13. 可选:选择的转染试剂,例如。,自制PEI解决方案或商业分销商

设备

  1. 装备有405nm激光器和环境室或插入物以控制温度和CO 2 2 /湿度用于长期活细胞实验的倒置共聚焦或旋转圆盘显微镜,例如 。 ,装备有UV透射Plan-Apochromat 63x/1.40 Oil DIC M27或Plan-Apochromat 40x/1.30 Oil DIC M27物镜的Zeiss LSM710或LSM780共聚焦激光扫描显微镜,分别为
  2. 细胞培养箱

软件

  1. 显微镜软件,例如来自Zeiss(Zeiss)的ZEN,
  2. Microsoft Excel(Microsoft)
  3. 图片J( https://imagej.nih.gov/ij/

程序

  1. 在开始微辐射实验之前2-3天,在35mm的μ网格盘(Ibidi)中种子4-9×10 4 U2OS细胞/ml。在微辐照和免疫荧光染色后,使用带网格的盘来重新定位细胞
  2. 可选:在微辐照实验前24-48小时用编码感兴趣的荧光标记的蛋白质的质粒转染细胞。在我们的手jetPEI在U2OS细胞中提供良好的转染效率
  3. 激光微注射对细胞DNA损伤诱导的敏感性
    1. BrdU致敏:在微辐射实验前将细胞在含有10μMBrdU的培养基中孵育至少24小时。 BrdU是胸苷的类似物,其在DNA复制期间掺入DNA,并且在用405nm激光照射时,将通过例如自由基形成诱导DNA损伤。
    2. Hoechst致敏:在微辐照实验前将细胞在含有10μg/ml Hoechst的培养基中孵育10分钟。 Hoechst是一种细胞渗透性DNA染料,其结合到DNA的小沟。当Hoechst被紫外光激发时,使用405nm激光的局部微辐射将产生DNA损伤。使用Hoechst或BrdU进行敏化
  4. 将补充有10%FBS和不含酚红的抗生素的预热的CO 2 2活性细胞培养基加入细胞中以使自发荧光最小化。
  5. DNA损伤诱导随后活细胞成像(图1)
    1. 点照射:对于表达感兴趣的荧光标记的蛋白质的活细胞中的DNA损伤诱导,用405nm二极管激光器照射细胞核内的衍射受限斑点(Xie等人,2015)(405nm激光设置为70%,1次迭代,缩放5,像素停留时间177.32μs)
    2. 线照射:对于表达感兴趣的荧光标记的蛋白质的活细胞中的DNA损伤诱导,用405nm二极管激光器照射5像素宽的条带(Xie等人,2015)(405nm激光设备到24%,1次迭代,缩放5,像素停留时间12.61μs)。
    3. 活细胞成像:在2秒时间间隔(通常6个预照射和60个后照射帧,帧大小512×512像素,像素大小90nm)的一个中间z剖面的微射线记录共焦图像系列之前和之后, ,使用激光线根据所选择的报道蛋白的激发光谱(例如,对于GFP /标记的永恒的,488nm Ar激光),
  6. DNA损伤诱导,随后免疫荧光染色
    1. 激光微辐射:使用ZEN软件的FRAP模块(线扫描,405nm激光设置为92%,50次迭代)或瓦片扫描模式(Xie等人,2015; Baldeyron& 2011)(3×3块,图像大小128×128,扫描速度177.32μs,每扫描7次扫描,405nm激光设置为30%)。
    2. 固定和免疫荧光:
      1. 在不同的修复时间后,将细胞在4%甲醛中固定10分钟
      2. 1×在PBS中洗涤
      3. 用0.5%Triton X-100在PBS中渗透5分钟
      4. 1×在PBS中洗涤
      5. 在封闭溶液中孵育40分钟
      6. 1×在PBS中洗涤
      7. 添加初级抗体,例如。用封闭缓冲液稀释的小鼠抗γH2AX,并在4℃下孵育过夜。
      8. 3×在PBST中洗涤5分钟
      9. 加入在4%BSA/PBS中稀释的二抗,例如抗驴抗小鼠IgG(H + L)二抗,Alexa Fluor 488,并孵育1小时在RT
      10. 3×在PBST中洗涤5分钟
      11. 加入新鲜的PBS并储存在4°C
    注意:405nm激光的强度可以在不同的显微镜之间变化。因此,必须首先确定最佳激光设置,例如改变激光强度,然后用诸如γH2AX,PAR或胸腺嘧啶二聚体的DNA损伤标记物染色。应该使用引起例如γH2AX的清晰条纹的设置,而泛核染色表明所使用的激光强度太高。致敏方法也可以根据应该分析什么样的DNA修复途径而改变,例如单链断裂修复与双链断裂修复。

数据分析

为了评估募集动力学,对照射区域的荧光强度针对背景和荧光在整个时间过程中的总核损失进行校正,并归一化为预照射值。对于微辐照实验的定量评价,来自三次独立实验的至少10-20个核的数据应当被平均,并且使用Microsoft Excel软件计算和显示平均值曲线和平均值的标准误差。

  1. 图像配准和荧光强度测量
    为了在图像采集期间补偿x和y方向上的潜在单元移动,可以使用来自ImageJ的TurboReg插件。
    1. 在ImageJ中打开文件
      1. 显示投资回报率
      2. 拆分渠道
    2. 复制图像系列的第一个图像,这将用作图像注册的模板
    3. Turbo reg
      1. 源 - 原始图像系列
      2. 目标 - 新图片
      3. 选择刚性身体,并设置质量快速
      4. 批次=运动补偿
      5. 将新文件另存为tiff
    4. 对于分析,定义ImageJ中的三个不同的感兴趣区域(ROI)
      1. ROI i =照射区域(点或线ROI)
      2. ROI n =细胞核的轮廓
      3. ROI b =单元格外的背景区域
    5. 向ROI经理添加ROI(编辑→选择→添加到经理),选择多个测量并保存投资回报和测量(可以在编辑→选项→输入/输出中选择测量类型)
    6. 将度量复制到Microsoft Excel
      1. 从核(ROI n)和照射区域(ROI i)的荧光强度中减去背景强度(ROI b):ROI ib = ROI i - ROI b 和ROI nb = ROI n
      2. 为了补偿在采集期间的漂白并将预照射值设置为1,使用双归一化:(ROI /ROI> nb /sub>/ROI ib t
        其中,ROI和ROI是在时间点t的荧光强度值,并且ROI ib t0 和ROI nb t0 分别是在时间点零时的荧光强度(预照射值)


        图1.确定在固定和活细胞中激光诱导的DNA损伤位点的蛋白质募集。A.使用平铺扫描模式在大量细胞中诱导DNA损伤的微辐照程序的示意图随后通过固定和免疫荧光分析或表达感兴趣的荧光标记蛋白的单细胞的线和点照射,随后进行活细胞成像。 B.使用Zeiss LSM780的瓷砖扫描模式的微光辐照后的代表性共聚焦图像。将U2OS细胞进行显微照射,在指定的时间点后固定,并用针对γH2AX和RPA的抗体染色,其可用作双链断裂和ssDNA形成的标记以鉴定DNA损伤位点。 C.用编码GFP /标记的永恒的表达质粒转染的U2OS细胞进行微照射,并且GFP-Timeless的募集在实时/时间中以2秒的时间间隔进行。

笔记

为了确保实验之间的再现性,可以使用激光功率计来测量405nm激光强度。或者,可以使用针对例如γH2AX的参照抗体作为阳性对照的常规免疫荧光染色。

食谱

  1. 固定溶液
    4%甲醛的PBS溶液
  2. 渗透溶液
    0.5%Triton X-100的PBS溶液中
  3. 封锁解决方案
    4%BSA的PBS溶液
  4. 洗涤缓冲液
    0.05%Tween 20的PBS溶液

致谢

这项工作由Helleday基金会支持。该协议已经改编自以下出版物:Walter等人。 (2003),Mortusewicz等人。 (2008),Xie等人。 (2015),Mortusewicz等人。 (2016),Mortusewicz等人。 (2007)和Mortusewicz等人。 (2006)。

参考文献

  1. Baldeyron,C.,Soria,G.,Roche,D.,Cook,AJ和Almouzni,G。(2011)。 
  2. Mortusewicz,O.,Amé,JC,Schreiber,V.and Leonhardt,H。(2007)。  PARP-1的反馈调节的聚(ADP-核糖基)是快速应答活细胞中DNA损伤所必需的。 em> 35(22):7665-7675。
  3. Mortusewicz,O.,Evers,B. and Helleday,T。(2016)。  PC4通过在DNA损伤位点结合ssDNA来促进基因组稳定性和DNA修复。 癌基因 35(6):761-770。
  4. Mortusewicz,O.,Leonhardt,H. and Cardoso,MC(2008)。  在DNA损伤位点调节蛋白质募集的时空动力学。


    J Cell Biochem 104(5):1562-1569。
  5. Mortusewicz,O.,Rothbauer,U.,Cardoso,MC和Leonhardt,H。(2006)。  DNA连接酶I和III到DNA修复位点的差异募集。核酸研究 34(12):3523-3532。
  6. Walter,J.,Cremer,T.,Miyagawa,K.and Tashiro,S。(2003)。  用于活细胞激光UVA微照射的新系统。 J Microsc 209(Pt 2):71-75。
  7. Xue,S.,Mortusewicz,O.,Ma,HT,Herr,P.,Poon,RY,Helleday,T。和Qian,C。(2015)。  Timeless与PARP-1相互作用以促进同源重组修复。 Mol细胞 60 (1):163-176。
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引用:Tampere, M. and Mortusewicz, O. (2016). DNA Damage Induction by Laser Microirradiation. Bio-protocol 6(23): e2039. DOI: 10.21769/BioProtoc.2039.
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