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DNA Fiber Assay upon Treatment with Ultraviolet Radiations
紫外线辐射处理后DNA纤维的检测   

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本实验方案简略版
Oncogene
Feb 2016

Abstract

Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV type C (UVC) exposure, the DNA fiber assay can be used to analyse the new origin firing and DNA synthesis rate (Jackson et al., 1998; Merrick et al., 2004; Alfano et al., 2016). The DNA fiber assay technique was conceived in the 90s and then further developed through the use of thymidine analogues (such as CldU and IdU), which are incorporated into the nascent DNA strands. By treating the cells in sequential mode with these analogues, which can be visualized through specific antibodies carrying different fluorophores, it is possible to monitor the replication fork activity and assess how this is influenced by UV radiations or others agents.

Keywords: Ultra Violet radiation (紫外线辐射), DNA damage (DNA损伤), Cell cycle checkpoints (细胞周期检查点), DNA fiber assay (DNA纤维检测试验), Halogenated pyrimidines (卤代嘧啶)

Background

Genomic instability is one of the hallmarks of cancer involved in both tumour development and progression (Hanahan and Weinberg, 2011). The preservation of genomic stability depends on a complex cascade of finely tuned events which are collectively known as the DNA damage response. This includes the activation of cell cycle checkpoints, which stall cell cycle to allow the cellular repair machinery to mend the damage. The identification of novel druggable proteins, involved in the DNA repair mechanisms, is part of modern cancer therapy. In this context, the DNA fiber assay can be used as a readout of replication fork activity to identify new potential players in the regulation of the intra-S phase checkpoint, which is triggered upon exposure to various chemotherapeutic drugs.

The unique chemistry of DNA, of its constituents and its chemical-physical properties allowed pioneering scientists to visualize the whole length of DNA fibers, first by labelling DNA with tritiated thymidine followed by detection through autoradiography (Cairns, 1963). Then Bensimon and colleagues showed that it was possible to align and ‘comb’ the DNA fibers on a solid matrix achieving a uniform stretching and an easy access for specific hybridizations (Bensimon et al., 1994). Jackson and Pombo were the first to use sequential labelling with halogenated pyrimidines, such as bromo-deoxyuridine (BrdU) and iodo-deoxyuridine (IdU), which are incorporated into DNA as thymidine analogues (Jackson and Pombo, 1998). This allowed them to assess qualitatively and quantitatively the activity of replicon clusters in HeLa cells at different times during S phase. Afterwards another study (Merrick et al., 2004) described a modified DNA fiber labelling, which was adapted by a classical DNA fiber autoradiography (Huberman and Riggs, 1968). Diffley and colleagues used pulse labelling with two halogenated nucleotides: chloro-deoxyuridine (CldU) and IdU, which could be differentially detected through specific antibodies, each carrying a different fluorescent dye. This allowed to visualize through fluorescent microscopy the effect of a specific cell treatment on the dynamics of the DNA synthesis process. The protocol described below was made as described by the two previously cited protocols with some modifications (Jackson and Pombo, 1998; Merrick et al., 2004).

Materials and Reagents

  1. 60 mm cell culture dishes (Sigma-Aldrich, catalog number: CLS430166 )
    Manufacturer: Corning, catalog number: 430166.
  2. Silane-Prep Slides: glass slides coated with silane (aminoalkylsilane) (Sigma-Aldrich, catalog number: S4651 )
  3. HeLa cells (ATCC, catalog number: CCL-2 )
  4. Cell culture
    1. Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 61870010 )
    2. 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
    3. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
    4. 0.25% 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 (defined as ‘complete medium’).
  5. 5-Iodo-2’-deoxyuridine (IdU) (Sigma-Aldrich, catalog number: I7125 )
  6. 5-Chloro-2’-deoxyuridine (CldU) (Sigma-Aldrich, catalog number: C6891 )
  7. Chemicals of analytical grade
    1. Methanol (CARLO ERBA Reagents, catalog number: 414814 )
    2. Acetic acid glacial (Fisher Scientific, catalog number: A38-212 )
    3. Ethanol absolute (CARLO ERBA Reagents, catalog number: 308605 )
    4. 39.5% hydrochloric acid (HCl) (CARLO ERBA Reagents, catalog number: 403878 )
    5. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
    6. Tween® 20 (Sigma-Aldrich, catalog number: P9416 )
    7. ProLong® Gold anti-fade reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36935 )
    8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    9. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    10. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
    11. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 795488 )
    12. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 71725 )
    13. Tris (Roche Diagnostics, catalog number: 10708976001 )
    14. 0.5 M EDTA (pH 8.0) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9262 )
    15. NP-40 (Thermo Fisher Scientific, catalog number: 85124 )
  8. Antibodies
    1. Anti-BrdU clone BU1/75 (ICR1) (detects CldU) (Bio-Rad Laboratories, catalog number: OBT0030CX )
    2. Anti-BrdU clone B44 (detects IdU) (BD, BD Biosciences, catalog number: 347580 )
    3. Alexa Fluor 488-conjugated chicken anti-rat (Thermo Fisher Scientific, Invitrogen, catalog number: A21470 )
    4. Alexa Fluor 594-conjugated rabbit anti-mouse (Thermo Fisher Scientific, Invitrogen, catalog number: A-11062 )
  9. 1x phosphate buffered saline (PBS) (see Recipes)
  10. Spreading buffer (see Recipes)
  11. Stringent buffer (see Recipes)

Equipment

  1. Incubator
  2. UVC 500 UV Crosslinker (GE Healthcare, model: UVC 500 )
  3. Centrifuge
  4. Confocal microscope (e.g., Carl Zeiss, model: Zeiss LSM100 )
  5. Fume hood

Software

  1. GraphPad software

Procedure

See Figure 1 for an overview of the protocol.


Figure 1. Schematic overview of the protocol

  1. Culture HeLa cells in RPMI 1640 medium supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin (defined as ‘complete medium’).
  2. Treat a 60 mm dish of 80% confluent HeLa cells with CldU (20 μM final concentration) in complete RPMI 1640 medium (these cells are defined as ‘labelled cells’); whereas leave untreated another 60 mm dish of the same cells in the same conditions (these cells are defined as ‘unlabelled cells’). Incubate both dishes at 37 °C in a 5% CO2 incubator for 20 min (see Note 1).
  3. Wash twice with 1x PBS.
  4. Remove all the excess 1x PBS.
  5. Treat labelled HeLa cells with UVC at a dose of 10 J/m2 (see Note 2).
  6. Add to labelled cells IdU (100 μM final concentration) in fresh complete RPMI 1640 medium (see Note 1).
  7. Incubate at 37 °C in a 5% CO2 incubator for 40 min.
  8. Trypsinize both labelled and unlabelled HeLa cells, then harvest them through centrifugation (800 x g for 5 min).
  9. Wash the cell pellet with ice-cold (4 °C) 1x PBS twice.
  10. Resuspend HeLa cells with the ice-cold 1x PBS at 2.5 x 105 cells/ml.
  11. Dilute labelled cells 1:8 with unlabelled cells (vol/vol).
  12. Spot onto the Silane Prep slides 2.5 μl of this labelled/unlabelled cell solution. Allow the 2.5 μl sample to evaporate not completely. (see Note 3)
  13. Mix the cells with 7.5 μl of spreading buffer directly on the Silane Prep slide.
  14. Incubate at room temperature (RT) for 10 min.
  15. Tilt the slides to 15° to spread DNA fibers along their length.
  16. Air-dry the DNA spreads. (see Note 3)
  17. Fix DNA fibers (which are now spread onto the Silane Prep slide) in 3:1 methanol/acetic acid in a plastic or glass container at -20 °C for 15 min.
  18. Wash slides in 1x PBS twice.
  19. Incubate DNA fibers in 70% ethanol at 4 °C for one hour (see Note 4).
  20. Treat the slides with 2.5 N of HCl at 37 °C for one hour.
  21. Wash slides with 1x PBS twice.
  22. Block DNA fibers with 1x PBS + 0.5% BSA for 30 min at RT.
  23. Incubate slides with 1:500 Anti-BrdU clone BU1/75 (ICR1) (which detects CldU) and 1:300 Anti-BrdU clone B44 (which detects IdU) together in 1x PBS + 0.5% BSA for one hour at 37 °C.
  24. Wash DNA fibers twice for 5 min at RT and once for 15 min at RT with stringent buffer (no shaking required).
  25. Incubate Silane Prep slides with 1:300 Alexa Fluor 488-conjugated chicken anti-rat and 1:400 Alexa Fluor 594-conjugated rabbit anti-mouse together in 1x PBS + 0.5% BSA for 30 min at 37 °C. Both antibodies were tested singularly to detect possible cross reactivity in HeLa cells and were previously successfully used for this technique (Alfano et al., 2016).
  26. Wash Silane Prep slides with 1x PBS + 0.5% Tween 20 for 5 min at RT for three times.
  27. Mount the slides with ProLong Gold anti-fade reagent.
  28. Analyse the slides through confocal fluorescent microscopy in sequential scanning mode.

Data analysis

To process the images obtained from the DNA fiber assay (Figure 2) one can use either manual counting or an automatic analysis by using a specific software. To analyze the frequency of the various DNA fiber types (ongoing, newly fired, or others) we manually counted 600 fibers for each experiment of three independent ones. To evaluate the statistical significance among different conditions or treatments assessed through DNA fiber assay, we applied the appropriate statistical test through the GraphPad software. To measure the length of DNA spreads to estimate the replication speed (1 μm corresponds to 2.59 kb), we used the specific image analysis software ImageJ (https://imagej.nih.gov/ij/). The University of North Carolina developed a software for the automatic analysis of DNA spreads (http://dnafiberanalysis.com/).


Figure 2. Representative kinds of DNA fibers identified by using this protocol. The sequential labelling provides a snapshot of different DNA replication steps: 1) Ongoing forks can appear as in a (in the case of unidirectional progression through the replication fork) or as in b (in the case of bidirectional progression through the replication fork). 2) Termination events can appear as in a (as an only-green line in the case of a replication origin in which DNA synthesis terminated while the cells were still undergoing incubation with the first label), or as in b (as adjoined red-green-red signals with divergent trend). 3) New fired origins appear as shown in the lower panel of the figure. These origins were fired during incubation with the second label (consisting of an only-red line, according to the protocol herein described).

Notes

  1. The pulse time and the working concentration of CldU and IdU might have to be adapted depending on the different type of cells under study.
  2. Prior to exposing the cells to the UV bulb remove the plate cap and make sure to aspirate completely any residual cell growth medium or 1x PBS so as not to shield the UV radiations.
  3. Do not evaporate the samples in the fume hood.
  4. At this step, the slides can be stored at 4 °C in 70% ethanol overnight, but we prefer to process the fibers directly.

Recipes

  1. 1x phosphate buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Adjust to a final pH of 7.4
  2. Spreading buffer
    0.5% sodium dodecyl sulfate (SDS)
    200 mM Tris-HCl pH 7.4
    50 mM EDTA pH 8
  3. Stringent buffer
    10 mM Tris-HCl pH 7.4
    400 mM NaCl
    0.2% Tween 20
    0.2% NP-40

Acknowledgments

We are grateful to the Sbarro Health Research Organization (http://www.shro.org), the Human Health Foundation (http://www.hhfonlus.org), the Commonwealth of Pennsylvania and the Associazione Italiana per la Ricerca sul Cancro (IG 2014-15690) for their support. A.G. is also Director of the Cell Cycle and Cancer Research Line at CROM, Istituto Nazionale Per Lo Studio E La Cura Dei Tumori; Naples. F.P. is also Adjunct Associate Professor at Temple University, Department of Biology, Philadelphia, PA, USA.

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. Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F. and Bensimon, D. (1994). Alignment and sensitive detection of DNA by a moving interface. Science 265(5181): 2096-8.
  3. Branzei, D. and Foiani, M. (2009). The checkpoint response to replication stress. DNA Repair (Amst) 8(9): 1038-1046.
  4. Cairns, J. (1963). The bacterial chromosome and its manner of replication as seen by autoradiography. J Mol Biol 6: 208-13.
  5. Hanahan, D. and Weinberg, B. (2011). Hallmarks of Cancer: The next generation. Cell 144(5): 646-674.
  6. Huberman, J. A. and Riggs, A. D. (1968). On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol 32(2):327-41.
  7. 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.
  8. Kaufmann, W. K. (2010). The human intra-S checkpoint response to UVC-induced DNA damage. Carcinogenesis 31(5): 751-765.
  9. Merrick, C. J., Jackson, D. and Diffley, J. F. (2004). Visualization of altered replication dynamics after DNA damage in human cells. J Biol Chem 279(19): 20067-20075.

简介

基因组稳定性不断受到DNA损伤因素的广泛影响。为了促进正确的DNA修复和细胞存活,细胞协调统一称为DNA损伤反应(DDR)的协调和精细调整的事件级联。超紫外线(UV)是DNA损伤的主要环境来源之一,也是公认的癌症危险因素。紫外线诱导形成毒性环丁烷型嘧啶二聚体(CPD)和[6-4]嘧啶嘧啶酮(6-4PP)光产物,其触发S期细胞周期检查点的活化(Kaufmann,2010),目的在于防止复制叉崩溃,晚期起火和稳定脆弱场所(Branzei和Foiani,2009)。为了监测响应于UV型C(UVC)暴露的S-S相检查点的激活,DNA纤维测定可用于分析新的起始点和DNA合成速率(Jackson等, ,1998; Merrick等人,2004; Alfano等人,2016)。 DNA纤维测定技术在90年代被设想,然后通过使用并入新生DNA链中的胸苷类似物(如CldU和IdU)进一步开发。通过用这些类似物以连续模式处理细胞,可以通过携带不同荧光团的特异性抗体来观察细胞,可以监测复制叉活性并评估其如何受到紫外辐射或其他试剂的影响。

背景 基因组不稳定是涉及肿瘤发展和进展的癌症的标志之一(Hanahan和Weinberg,2011)。基因组稳定性的保存取决于被统称为DNA损伤反应的精细调节事件的复杂级联。这包括激活细胞周期检查点,这阻止细胞周期,以允许细胞修复机械修复损伤。涉及DNA修复机制的新型可药用蛋白质的鉴定是现代癌症治疗的一部分。在这种情况下,DNA纤维测定可用作复制叉活性的读数,以鉴定在暴露于各种化学治疗药物时触发的S-S期检查点调节中的新潜在参与者。
  DNA的独特化学成分及其化学物理性质使得先驱科学家可以看出DNA纤维的全长,首先用氚标记的胸苷标记DNA,然后通过放射自显影检测(凯恩斯,1963)。然后Bensimon及其同事表明,有可能使固定基质上的DNA纤维对准和“梳理”,实现均匀的拉伸和容易进入特异性杂交(Bensimon等,1994)。 Jackson和Pombo是第一个使用卤代嘧啶(如溴脱氧尿苷(BrdU)和碘 - 脱氧尿苷(IdU))进行顺序标记,它们作为胸苷类似物掺入DNA中(Jackson和Pombo,1998)。这允许他们在S期的不同时间定性和定量地评估HeLa细胞中复制子簇的活性。之后另一项研究(Merrick等人,2004)描述了经修饰的DNA纤维标记,其通过经典的DNA纤维放射自显影进行了改造(Huberman和Riggs,1968)。 Diffley及其同事使用脉冲标记两个卤代核苷酸:氯 - 脱氧尿苷(CldU)和IdU,可通过特异性抗体差异检测,每个携带不同的荧光染料。这允许通过荧光显微镜观察特定细胞处理对DNA合成过程的动力学的影响。下面描述的方案是由以前引用的两个方案描述的,具有一些修改(Jackson和Pombo,1998; Merrick等人,2004)。

关键字:紫外线辐射, DNA损伤, 细胞周期检查点, DNA纤维检测试验, 卤代嘧啶

材料和试剂

  1. 60mm细胞培养皿(Sigma-Aldrich,目录号:CLS430166)
    制造:康宁,目录号:430166。
  2. 硅烷制备载玻片:涂有硅烷(氨基烷基硅烷)的玻璃载片(Sigma-Aldrich,目录号:S4651)
  3. HeLa细胞(ATCC,目录号:CCL-2)
  4. 细胞培养
    1. 罗斯韦尔公园纪念研究所(RPMI)1640培养基(Thermo Fisher Scientific,Gibco TM,目录号:61870010)
    2. 10%胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
    3. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
    4. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,目录号:25200056)
    注意:将HeLa细胞在补充有10%FBS,1μg/ ml青霉素和1μg/ ml链霉素(定义为“完全培养基”)的RPMI 1640中培养。
  5. 5-碘-2'-脱氧尿苷(IdU)(Sigma-Aldrich,目录号:I7125)
  6. 5-氯-2'-脱氧尿苷(CldU)(Sigma-Aldrich,目录号:C6891)
  7. 化学品分析等级
    1. 甲醇(CARLO ERBA试剂,目录号:414814)
    2. 乙酸冰川(Fisher Scientific,目录号:A38-212)
    3. 乙醇绝对(CARLO ERBA试剂,目录号:308605)
    4. 39.5%盐酸(HCl)(CARLO ERBA试剂,目录号:403878)
    5. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
    6. Tween 20(Sigma-Aldrich,目录号:P9416)
    7. ProLong ®金抗褪色试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:P36935)
    8. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
    9. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    10. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793)
    11. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:795488)
    12. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:71725)
    13. Tris(Roche Dignostics,目录号:10708976001)
    14. 0.5M EDTA(pH8.0)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9262)
    15. NP-40(Thermo Fisher Scientific,目录号:85124)
  8. 抗体
    1. 抗BrdU克隆BU1 / 75(ICR1)(检测CldU)(Bio-Rad Laboratories,目录号:OBT0030CX)
    2. 抗BrdU克隆B44(检测IdU)(BD,BD Biosciences,目录号:347580)
    3. Alexa Fluor 488缀合的鸡抗鼠(Thermo Fisher Scientific,Invitrogen,目录号:A21470)
    4. Alexa Fluor 594-缀合的兔抗小鼠(Thermo Fisher Scientific,Invitrogen,目录号:A-11062)
  9. 1x磷酸缓冲盐水(PBS)(见食谱)
  10. 扩展缓冲区(请参阅配方)
  11. 严格缓冲(见配方)

设备

  1. 孵化器
  2. UVC 500 UV交联剂(GE Healthcare,型号:UVC 500)
  3. 离心机
  4. 共焦显微镜(例如,卡尔蔡司,型号:Zeiss LSM100)
  5. 通风柜

软件

  1. GraphPad软件

程序

有关协议的概述,请参见图1

图1.协议原理图概述

  1. 在补充有10%FBS,1μg/ ml青霉素和1μg/ ml链霉素(定义为“完全培养基”)的RPMI 1640培养基中培养HeLa细胞。
  2. 在完整的RPMI 1640培养基(这些细胞被定义为“标记细胞”)处理具有CldU(20μM终浓度)的80%汇合HeLa细胞的60mm培养皿。而在相同条件下将未处理的相同细胞的另外60mm培养皿放置(这些细胞定义为“未标记细胞”)。在37℃下在5%CO 2培养箱中孵育两个培养皿20分钟(见注1)。
  3. 用1x PBS洗两次。
  4. 取出所有多余的1x PBS。
  5. 用UVC以10J / m 2的剂量处理标记的HeLa细胞(参见注2)。
  6. 在新鲜完整的RPMI 1640培养基中加入标记细胞IdU(终浓度为100μM)(参见注1)。
  7. 在37℃,5%CO 2培养箱中孵育40分钟
  8. 对标记的和未标记的HeLa细胞进行胰蛋白酶化,然后通过离心(800μg×5分钟)收获它们5分钟。
  9. 用冰冷(4℃)1x PBS洗涤细胞沉淀两次。
  10. 用2.5×10 5细胞/ ml的冰冷的1×PBS重悬细胞HeLa细胞。
  11. 用未标记的细胞稀释标记细胞1:8(vol / vol)。
  12. 点到硅烷准备载玻片上2.5μl该标记/未标记的细胞溶液。允许2.5μl样品不完全蒸发。 (见注3)
  13. 将细胞与7.5μl扩散缓冲液直接混合在硅烷准备载玻片上
  14. 在室温(RT)下孵育10分钟
  15. 将幻灯片倾斜至15°,沿着长度铺展DNA纤维。
  16. 空气干燥DNA扩散。 (见注3)
  17. 将DNA纤维(现在分散在硅烷制备载玻片上)在塑料或玻璃容器中的3:1甲醇/乙酸中,-20℃下固化15分钟。
  18. 在1x PBS中洗两次载玻片。
  19. 将DNA纤维在70%乙醇中于4℃孵育1小时(见附注4)
  20. 用2.5N HCl在37℃下处理载玻片1小时
  21. 用1x PBS洗涤载玻片两次。
  22. 在室温下用1x PBS + 0.5%BSA封闭DNA纤维30分钟。
  23. 使用1:500抗BrdU克隆BU1 / 75(ICR1)(其检测CldU)和1:300抗BrdU克隆B44(其检测IdU)在1×PBS + 0.5%BSA中在37℃孵育1小时。
  24. 在室温下洗涤DNA纤维两次5分钟,并在室温下用严格缓冲液(无需摇动)一次,持续15分钟
  25. 使用1:300的Alexa Fluor 488缀合的鸡抗鼠和1:400的Alexa Fluor 594-缀合的兔抗小鼠一起在1×PBS + 0.5%BSA中于37℃孵育Silane Prep载玻片30分钟。两种抗体均被单独测试以检测HeLa细胞中的可能的交叉反应性,并且之前成功地用于该技术(Alfano等人,2016)。
  26. 用1×PBS + 0.5%吐温20洗涤硅烷准备载玻片5分钟,室温3次
  27. 用ProLong Gold防褪色试剂装载幻灯片。
  28. 通过共焦荧光显微镜在顺序扫描模式下分析载玻片。

数据分析

为了处理从DNA纤维测定获得的图像(图2),可以使用手动计数或使用特定软件进行自动分析。为了分析各种DNA纤维类型(正在进行,新开发的或其他类型)的频率,我们为三个独立的每个实验手动计数600个纤维。为了评估通过DNA纤维测定评估的不同条件或治疗的统计学显着性,我们通过GraphPad软件进行了适当的统计检验。为了测量DNA扩展的长度以估计复制速度(1μm对应于2.59kb),我们使用特定的图像分析软件ImageJ( https://imagej.nih.gov/ij/ )。北卡罗来纳大学开发了一种用于DNA扩散自动分析的软件( http://dnafiberanalysis.com / )。


图2.使用该方案鉴定的代表性的DNA纤维种类。顺序标记提供不同DNA复制步骤的快照:1)正在进行的叉可以如(在单向进展的情况下)复制分支)或b中(在通过复制分支的双向进度的情况下)。 2)终止事件可以如(在复制起点的唯一绿线,其中DNA合成终止,而细胞仍然与第一标记进行孵育)或b(作为邻接的红 - 绿红色信号具有不同的趋势)。 3)新出现的起始物如图所示,如下图所示。这些来源在与第二个标记(根据本文所述的方案由唯一的红线组成)的温育期间被烧制。

笔记

  1. CldU和IdU的脉冲时间和工作浓度可能需要根据研究中不同类型的细胞进行调整。
  2. 在将细胞暴露于紫外线灯泡之前,取下板盖,并确保完全吸出任何残留的细胞生长培养基或1x PBS,以免屏蔽UV辐射。
  3. 不要在通风橱中蒸发样品。
  4. 在该步骤中,载玻片可以在4℃下在70%乙醇中储存过夜,但是我们更喜欢直接处理纤维

食谱

  1. 1x磷酸缓冲盐水(PBS)
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH PO 4
    调整到7.4的最终pH值
  2. 扩展缓冲区
    0.5%十二烷基硫酸钠(SDS)
    200mM Tris-HCl pH 7.4
    50 mM EDTA pH 8
  3. 严格的缓冲区
    10mM Tris-HCl pH 7.4
    400 mM NaCl
    0.2%吐温20
    0.2%NP-40

致谢

我们感谢Sbarro健康研究组织( http://www.shro.org ),人类健康基金会( http://www.hhfonlus.org ) ,宾夕法尼亚州联邦和意大利人Asacciazione每la Ricerca sul Cancro(IG 2014-15690)的支持。 A.G.还是CROM的细胞周期和癌症研究线的主管,国立波斯尼亚人立陶宛工作室E La Cura Dei Tumori;那不勒斯。 F.P.也是美国宾夕法尼亚州宾夕法尼亚州费城大学生物学系Temple大学副教授。

参考

  1. Alfano,L.,Costa,C.,Caporaso,A.,Altieri,A.,Indovina,P.,Macaluso,M.,Giordano,A.and Pentimalli,F.(2016)。  NONO根据紫外线辐射来调节S相检查点。癌基因 35(5):567-576。
  2. Bensimon,A.,Simon,A.,Chiffaudel,A.,Croquette,V.,Heslot,F.and Bensimon,D。(1994)。  通过移动界面对DNA的对准和敏感检测。 科学 265(5181 ):2096-8。
  3. Branzei,D.和Foiani,M.(2009)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/19482564”target =“_ blank” >复制应力的检查点响应。 DNA修复(Amst) 8(9):1038-1046。
  4. 凯恩斯J.(1963)。细菌染色体和其复制方式如放射自显影所见。 J Mol Biol 6:208-13。
  5. Hanahan D.和Weinberg,B.(2011)。癌症的标志:下一代。 细胞 144(5):646-674。
  6. Huberman,JA和Riggs,AD(1968)。  On哺乳动物染色体中DNA复制的机制。 J Mol Biol 32(2):327-41。
  7. Jackson,DA和Pombo,A。(1998)。复制子簇是染色体结构的稳定单位:证据表明核组织有助于S细胞在人细胞中的有效活化和繁殖。 140(6):1285-1295。
  8. Kaufmann,WK(2010)。  人类S检查点对UVC诱导的DNA损伤的反应。致癌作用 31(5):751-765。
  9. Merrick,CJ,Jackson,D.and Diffley,JF(2004)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/14982920”target = “_blank”>在人类细胞中DNA损伤后可变化的复制动力学。 J Biol Chem 279(19):20067-20075。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Luigi, A., Giordano, A. and Pentimalli, F. (2017). DNA Fiber Assay upon Treatment with Ultraviolet Radiations. Bio-protocol 7(11): e2301. DOI: 10.21769/BioProtoc.2301.
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