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Laser Microirradiation and Temporal Analysis of XRCC1 Recruitment to Single-strand DNA Breaks
微量激光辐射(诱导损伤)后XRCC1招募到单链DNA断裂处的时序分析   

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Abstract

The DNA molecule is exposed to a multitude of damaging agents that can compromise its integrity: single (SSB) and double strand breaks (DSB), intra- or inter-strand crosslinks, base loss or modification, etc. Many different DNA repair pathways coexist in the cell to ensure the stability of the DNA molecule. The nature of the DNA lesion will determine which set of proteins are needed to reconstitute the intact double stranded DNA molecule. Multiple and sequential enzymatic activities are required and the proteins responsible for those activities not only need to find the lesion to be repaired among the millions and millions of intact base pairs that form the genomic DNA but their activities have to be orchestrated to avoid the accumulation of toxic repair intermediates. For example, in the repair of Single Strand Breaks (SSB) the proteins PARP1, XRCC1, Polymerase Beta and Ligase III will be required and their activities coordinated to ensure the correct repair of the damage.
Furthermore, the DNA is not free in the nucleus but organized in the chromatin with different compaction levels. DNA repair proteins have therefore to deal with this nuclear organization to ensure an efficient DNA repair. A way to study the distribution of DNA repair proteins in the nucleus after damage induction is the use of the laser microirradiation with which a particular type of DNA damage can be induced in a localized region of the cell nucleus. The wavelength and the intensity of the laser used will determine the predominant type of damage that is induced. It is important to note that other lesions can also be generated at the microirradiated site.
Living cells transfected with the fluorescent protein XRCC1-GFP are micro-irradiated under a confocal microscope and the kinetics of recruitment of the fluorescent protein is followed during 1 min. In our protocol the 405 nm laser is used to induce SSB.

Keywords: DNA damage(DNA损伤), Single-strand DNA breaks(单链DNA的旅行车), DNA repair(DNA修复), Microirradiation(microirradiation), Live imaging(Live成像)

Materials and Reagents

  1. Glass bottom 35 mm Petri dishes (MatTek, catalog number: P35G-1.5-20-C )
  2. HeLa adherent cells in culture
  3. Plasmids coding for the fluorescent protein XRCC1-GFP
    Note: The XRCC1 sequence (NCBI P18887) was cloned in the Clontech pEGFP-N1 Vector at the restriction sites EcoRI/ApaI.
  4. Transfection agents (LipoFectamine 2000) (Life Technologies, catalog number: 11668027 )
    Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 11668027”.
  5. DMEM [DMEM(1x) + GlutaMAX™ (Thermo Fisher Scientific, Gibco™, catalog number: 31966-021 )] containing 10% of fetal bovine serum (Thermo Fisher Scientific, Gibco™, catalog number: 16000-044 )

Equipment

  1. Cell incubator (Thermo Fisher Scientific, model: NAPCO 5400 )
    Note: Currently, it is “BioSurplus, catalog number: NAPCO 5400 ”.
  2. Nikon A1 inverted confocal microscope equipped with an environmental chamber allowing the control of temperature, humidity and gas mixture
    Note: The lasers used are the following: 405 nm laser for the micro-irradiation, and a 488 nm Argon-laser to visualize the GFP fluorescence. The objective 63x was used.

Software

  1. The NIS Software
    Note: It was used for quantification of the fluorescence intensity at the microirradiation region during time, using the Time Measurement function.

Procedure

  1. HeLa cells were seeded in 35 mm glass bottom Petri dishes (250,000 cells per dish) and transfected 24 h later with the XRCC1-GFP plasmid using Lipofectamine 2000 according to manufacturer recommendations.
  2. 24 h after transfection, microirradiation was carried out with a 405 nm diode laser set to 5% power. The laser power at the exit of the fiber was of 4 mW, and around 1 mW watts at the exit of the 10x Obj.
  3. Measurements were performed by immobilizing the laser at 100% in a point bleach with the Digital Handheld Optical Power PM100D from THORLABS.
    1. Stimulation and acquisition were performed with the 60x objective at a zoom of 4 using an image size of 512 x 512.
    2. A stimulation line of 5 µm was defined and microirradiation performed for 6 sec. Six images were taken before micro-irradiation, to calculate the basal level of fluorescence. After micro- irradiation, an image was taken every 2 sec during 1 min.
    3. Between 10 and 20 cells are micro-irradiated in each experiment.
    Note: To determine the intensity of the lasers to be used in each different microscope it may be useful to monitor the recruitment of proteins involved in Single Strand Break Repair (SSBR) such as XRCC1 and proteins involved in the repair of Double Strand Breaks (DSBR), such as 53BP1 or NBS1 and keep the conditions that induce recruitment of XRCC1 but not of the DSBR proteins. In our case, only after addition of the DNA intercalant Hoechst 33342 we could observe the recruitment of DSBR proteins so we could validate that in our conditions we were mainly inducing SSB.

Analysis and quantification

  1. The fluorescence intensity at the micro-irradiated region is measured for each time point. Six images are taken before the micro-irradiation in order to quantify a mean of fluorescence that will be considered as the basal level of the protein in the region and used to normalize the measurements (this value is set to 1).
  2. In order to quantify the enrichment factor of XRCC1 in the micro-irradiation region the intensity observed for each time point is divided by the mean intensity measured before the micro-irradiation.
  3. The mean of at least 10 cells is displayed in the graph. Error bars represent the SEM.
  4. It is important to check if there is bleaching during the acquisition process and correct for that if necessary. In order to do that we define a Standard Region of Interest (ROI-2) in another cell expressing similar amounts of XRCC1-GFP that is in the same scan area. Fluorescence intensity is monitored both in the micro-irradiated cell and in the non micro-irradiated one during both the stimulation and the acquisition period. If there is bleaching, we will observe a decrease in the fluorescence in the non micro-irradiated cell. It was not the case in our system, so no further corrections were required. If bleaching is observed, it is recommended to adjust the acquisition conditions in order to reduce that problem.

Representative data


Figure 1. HeLa cell expressing the XRCC1-GFP or the mutant protein XRCC1(L360D) were microirradiated with a 405 nm laser. A) Presented images correspond to the cell before microirradiation and 1 min after microirradiation. The microirradiation region is indicated by a dashed line. The mutant protein XRCC1(L360D), that has not any more the ability to be recruited to SSB was used as a control. B) Fluorescence intensity corresponding to XRCC1-GFP at the microirradiated region was quantified and displayed in the graph. Error bars represent the SEM of 10 independent cells.

Video 1. HeLa cell expressing the XRCC1-GFP protein was microirradiated with a 405 nm laser

Acknowledgments

We thank the IRCM microscopy facility. This work was funded by INSERM and grants from the Association pour la Recherche sur le Cancer (PJA 20131200165) and the CEA Radiobiology program.

References

  1. Campalans, A., Kortulewski, T., Amouroux, R., Menoni, H., Vermeulen, W. and Radicella, J. P. (2013). Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair. Nucleic Acids Res 41(5): 3115-3129.

简介

DNA分子暴露于多种损害剂,其可损害其完整性:单链(SSB)和双链断裂(DSB),链内或链间交联,碱基丢失或修饰等。 >许多不同的DNA修复途径共存于细胞中以确保DNA分子的稳定性。 DNA损伤的性质将决定重组完整的双链DNA分子需要哪组蛋白质。需要多个和顺序的酶活性,负责那些活性的蛋白质不仅需要在形成基因组DNA的数百万和数百万个完整碱基对中找到待修复的损伤,而且它们的活性必须被协调以避免毒性修复中间体。例如在单链断裂(SSB)的修复中,将需要蛋白质PARP1,XRCC1,聚合酶β和连接酶III,并且它们的活性协调以确保损伤的正确修复。
此外,DNA不是自由的在核中但组织在具有不同压实水平的染色质。因此DNA修复蛋白质处理这种核组织以确保有效的DNA修复。在损伤诱导后研究核中DNA修复蛋白质的分布的一种方式是使用激光微辐射,其中可以在细胞核的局部区域中诱导特定类型的DNA损伤。所使用的激光的波长和强度将决定被诱导的主要类型的损伤。重要的是注意,在微照射部位也可以产生其他损伤。
用荧光蛋白XRCC1-GFP转染的活细胞在共聚焦显微镜下进行微照射,并在1分钟内跟踪荧光蛋白的募集动力学。在我们的协议中,使用405nm激光来诱导SSB。

关键字:DNA损伤, 单链DNA的旅行车, DNA修复, microirradiation, Live成像

材料和试剂

  1. 玻璃底35mm培养皿(MatTek,目录号:P35G-1.5-20-C)
  2. 培养中的HeLa贴壁细胞
  3. 编码荧光蛋白XRCC1-GFP的质粒
    注意:将XRCC1序列(NCBI P18887)克隆在Clontech pEGFP-N1载体中限制性位点EcoRI/ApaI处。
  4. 转染剂(LipoFectamine 2000)(Life Technologies,目录号:11668027)
    注意:目前,它是"Thermo Fisher Scientific,Invitrogen?,目录号:11668027"。
  5. 含有10%胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:16000-044)的DMEM [DMEM(1x)+ GlutaMAX TM(Thermo Fisher Scientific,Gibco TM,目录号:31966-021)

设备

  1. 细胞培养箱(Thermo Fisher Scientific,型号:NAPCO 5400)
    注意:目前,它是"BioSurplus,目录号:NAPCO 5400"。
  2. 尼康A1倒置共聚焦显微镜配备环境室,允许控制温度,湿度和气体混合物 注意:使用的激光器如下:用于微照射的405nm激光器和用于可视化GFP荧光的488nm氩激光器。使用目标63x。

软件

  1. NIS软件
    注意:它用于使用时间测量功能定量微照射区域的荧光强度。

程序

  1. 将HeLa细胞接种在35mm玻璃底培养皿(250,000个细胞)中 ?每皿),并用XRCC1-GFP质粒转染24小时后 脂质体2000根据制造商的建议。
  2. 转染后24小时,用405nm进行显微照射 二极管激光器设置为5%功率。激光功率在光纤出口处 是4mW,并且在10x目标的出口处大约1mW瓦特。
  3. 通过将激光器固定在100%的激光下进行测量 点漂白与数字手持光功率PM100D从 THORLABS。
    1. 使用512×512的图像大小以60倍物镜以4的放大率进行刺激和获取。
    2. 定义5μm的刺激线并进行微辐射 6秒。在微照射之前拍摄六幅图像以进行计算 基础荧光水平。在微照射后,形成图像 每2秒进行1分钟。
    3. 在每个实验中微生物照射10至20个细胞。
    注意:为了确定在每个不同的显微镜中使用的激光器的强度,可能有用的是监测参与单链断裂修复(SSBR)的蛋白质的募集,例如XRCC1和参与双链断裂修复的蛋白质(DSBR ),如53BP1或NBS1,并保持诱导XRCC1而非DSBR蛋白募集的条件。在我们的情况下,只有在加入DNA插入剂Hoechst 33342之后,我们可以观察到DSBR蛋白的募集,所以我们可以验证在我们的条件下,我们主要诱导SSB。

分析和定量

  1. 测量微照射区域的荧光强度 为每个时间点。在微照射之前拍摄六个图像 以便量化将被视为的荧光的平均值 该区域的蛋白质的基础水平,并用于标准化 测量(此值设置为1)。
  2. 为了量化XRCC1的富集因子 微照射区域观察到的每个时间点的强度 除以在微照射之前测量的平均强度。
  3. 在图中显示至少10个细胞的平均值。误差棒表示SEM。
  4. 重要的是检查在采集过程中是否有漂白,如果需要,是否正确。为了做到这一点,我们在表示相同量的XRCC1-GFP的另一个细胞中定义了在相同扫描区域中的标准目标区域(ROI-2)。在刺激和采集期间,在微辐射的细胞和非微辐射的细胞中监测荧光强度。如果存在漂白,我们将观察到非微辐照细胞中荧光的减少。在我们的系统中不是这样,因此不需要进一步修正。如果观察到漂白,建议调整采集条件以减少该问题。

代表数据


图1.用405nm激光对表达XRCC1-GFP或突变蛋白XRCC1(L360D)的HeLa细胞进行显微照射。 A)呈现的图像对应于微辐射之前的细胞和微辐射之后的1分钟。微辐射区域由虚线表示。突变蛋白XRCC1(L360D),其不再具有被招募到SSB的能力被用作对照。 B)在微照射区域对应于XRCC1-GFP的荧光强度被定量并显示在图中。误差条表示10个独立细胞的SEM。

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视频1.表达XRCC1-GFP蛋白的HeLa细胞用405nm激光
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致谢

我们感谢IRCM显微镜设施。这项工作由INSERM和来自癌症协会(PJA 20131200165)和CEA放射生物学计划的资助。

参考文献

  1. Campalans,A.,Kortulewski,T.,Amouroux,R.,Menoni,H.,Vermeulen,W.and Radicella,J.P。 XRCC1募集对单链断裂和碱基切除修复的不同时空模式和PARP依赖性。 a> Nucleic Acids Res 41(5):3115-3129。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Campalans, A. and Radicella, J. P. (2016). Laser Microirradiation and Temporal Analysis of XRCC1 Recruitment to Single-strand DNA Breaks. Bio-protocol 6(5): e1746. DOI: 10.21769/BioProtoc.1746.
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