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End-synapsis Assay
末端染色体结合试验

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Abstract

Many environmental agents induce double-strand breaks (DSBs) in DNA. Unrepaired or improperly repaired DSBs can lead to cell death or cancer. Nonhomologous end joining is the primary DNA double-strand break repair pathway in eukaryotes. During NHEJ pathway, several proteins recognize and bind DNA ends, bring the ends in a synaptic complex and, finally, process and ligate the ends.
Briefly, NHEJ starts with Ku protein. Ku binds the broken DNA ends and recruits the catalytic subunit of DNA dependent protein kinase (DNA-PKcs) forming DNA-PK. After processing, the XRCC4/Ligase IV complex executes the final ligation stimulated by Cernunnos-XLF.
Here, we describe an end-synapsis assay. This assay can be used in order to delineate which proteins are necessary to bring the DNA ends in a stable synaptic complex during NHEJ. Briefly, NHEJ competent extracts from human cells were incubated with both a double-stranded DNA fragment bound to streptavidin-coated magnetic beads and the same soluble radio-labeled fragment. The beads were then washed in mild salt buffer and the radioactivity recovered with the beads was measured by scintillation counting. Control experiments without extracts or with DNA-free beads were run in parallel to determine unspecific background.

Keywords: DNA double-strand breaks(DNA双链打破), Non homologous end-joining(非同源结束加入), DNA repair(DNA修复)

Materials and Reagents

  1. NHEJ competent human cells (e.g. AHH1 lymphoblastoid cells, Nalm6 pre-B cells, HeLa epithelial cells, MRC5SV fibroblasts, etc.)
  2. ~500 bp double-stranded DNA fragments amplified by PCR, biotinylated at one end or non-biotinylated
  3. [32P]-ATP
  4. T4 polynucleotide kinase
  5. Streptavidin paramagnetic beads (Dynabeads M280 streptavidin) (Life Technologies, Invitrogen™, catalog number: 112.06D )
  6. Glucose
  7. Hexokinase (Calbiochem, catalog number: 376811 )
  8. PBS
  9. Triethanolamine
  10. Magnesium acetate
  11. Dithiothreitol
  12. BSA (e.g. enzymatic restriction reaction grade)
  13. Potassium acetate
  14. EJ buffer (see Recipes)

Equipment

  1. PCR thermal cycler
  2. Scintillation counter
  3. Heat block (Eppendorf Thermomixer® comfort)

Procedure

  1. First, PCR is used to synthesize a ~500 bp dsDNA fragment (e.g. from pBluescript plasmid) with a non-biotinylated or biotinylated reverse primer and a non-biotinylated forward primer, producing a fragment biotinylated at one end (500 bio) or not. The non biotinylated fragment was then radiolabeled with T4 polynucleotide kinase in the presence of [32P]-ATP (500*).
  2. Next, beads associated with biotinylated DNA fragments were prepared: per point, 0.5 pmol of 500 bio dsDNA fragment were immobilized on 10 μl streptavidin paramagnetic beads as recommended by the manufacturer.
  3. In parallel, NHEJ competent cells extracts from human cells were used (Bombarde et al., 2010). Briefly, exponentially growing cells were lysed through three freeze/thaw cycles in lysis buffer containing protease and phosphatase inhibitor cocktail, then lysates were incubated at 4 °C for 20 min, cleared by centrifugation, and dialyzed against dialysis buffer as described (Bombarde et al., 2010). Protein concentration was determined using the Bradford assay and end-joining extracts were stored at -80 °C. Here, 40 μg extracts were incubated for 10 min at 30 °C with 2 mM glucose and 0.2 U hexokinase. Glucose and hexokinase is an ATP consuming system used to prevent any ligation activity (Calsou et al., 2003).
    Note: DNA end-synapsis is an early step of NHEJ which relies on protein/DNA interactions and is reversible (e.g. by washing with high salts or detergent) while ligation is the final step and is irreversible (resistant to harsh washes). Ligation has to be prevented to focus the analysis on synapsis.
  4. 10 μl of mocked (control) or DNA-treated beads were washed twice in 100 μl of 0.5x PBS.
  5. Wet mocked or DNA-treated beads were mixed at 16 °C for 30 min in 10 μl EJ buffer containing 0.1 pmol of radioactive 500* DNA fragment and 40 μg of NHEJ competent cell extracts pre-incubated as above. The beads were gently hand-agitated by every 5 min.
  6. After incubation, supernatant was removed for storage and wet beads were washed twice in 50 μl 0.5x PBS.
  7. The washes were pooled with the supernatant.
  8. Radioactivity associated with supernatants and beads were measured in a scintillation counter.
  9. Results are expressed as the % of radioactivity pulled down after subtraction of the counts in the sample without 500 bio on the beads (Figure 1).


    Figure 1. Quantification of the specific radioactivity pulled-down under synapsis conditions in vitro with extracts of Nalm6 or N114 cells. N114 cells have a defect in Lig4 expression which impacts on synapsis formation.

Recipes

  1. EJ buffer
    50 mM Triethanolamine (pH 8.0)
    0.5 mM magnesium acetate
    1 mM dithiothreitol
    0.1 mg/ml BSA
    60 mM potassium acetate

Acknowledgments

The end-synapsis protocol was adapted from a reported assay (DeFazio et al., 2002) This work was partly supported by grants from La Ligue Nationale Contre le Cancer (Equipe labellisée), Electricité de France (EDF, Conseil de Radioprotection) and the Institut National Contre le Cancer (XXL-screen program). J. Cottarel was supported by a PhD fellowship from La Ligue Nationale Contre le Cancer. P. Calsou is a scientist from INSERM, France.

References

  1. Bombarde, O., Boby, C., Gomez, D., Frit, P., Giraud-Panis, M. J., Gilson, E., Salles, B. and Calsou, P. (2010). TRF2/RAP1 and DNA-PK mediate a double protection against joining at telomeric ends. EMBO J 29(9): 1573-1584. 
  2. Calsou, P., Delteil, C., Frit, P., Drouet, J. and Salles, B. (2003). Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment. J Mol Biol 326(1): 93-103. 
  3. Cottarel, J., Frit, P., Bombarde, O., Salles, B., Negrel, A., Bernard, S., Jeggo, P. A., Lieber, M. R., Modesti, M. and Calsou, P. (2013). A noncatalytic function of the ligation complex during nonhomologous end joining. J Cell Biol 200(2): 173-186. 
  4. DeFazio, L. G., Stansel, R. M., Griffith, J. D. and Chu, G. (2002). Synapsis of DNA ends by DNA-dependent protein kinase. EMBO J 21(12): 3192-3200.

简介

许多环境因素诱导DNA中的双链断裂(DSB)。未修复或不正确修复的DSB可导致细胞死亡或癌症。非同源末端连接是真核生物中的主要DNA双链断裂修复途径。在NHEJ途径期间,几种蛋白质识别和结合DNA末端,将末端引入突触复合物,最后进行处理并连接末端。
简言之,NHEJ起始于Ku蛋白。 Ku结合断裂的DNA末端,并招募形成DNA-PK的DNA依赖性蛋白激酶(DNA-PKcs)的催化亚基。处理后,XRCC4 /连接酶IV复合物执行Cernunnos-XLF刺激的最终连接。
在这里,我们描述一种末端突触检测。可以使用该测定法来描绘在NHEJ期间将DNA末端置于稳定突触复合物中所必需的蛋白质。简言之,来自人细胞的NHEJ感受态提取物与结合链霉抗生物素蛋白包被的磁珠和相同的可溶性放射性标记片段的双链DNA片段一起温育。然后将微珠在温和的盐缓冲液中洗涤,并通过闪烁计数测量用珠回收的放射性。没有提取物或无DNA珠的对照实验平行进行以确定非特异性背景。

关键字:DNA双链打破, 非同源结束加入, DNA修复

材料和试剂

  1. NHEJ感受态人细胞(例如AHH1淋巴母细胞样细胞,Nalm6前B细胞,HeLa上皮细胞,MRC5SV成纤维细胞等)。
  2. 通过PCR扩增的〜500bp的双链DNA片段,在一端生物素化或非生物素化的
  3. [ 32 P] -ATP
  4. T4多核苷酸激酶
  5. 链霉亲和素顺磁珠(Dynabeads M280链霉亲和素)(Life Technologies,Invitrogen TM,目录号:112.06D)
  6. 葡萄糖
  7. 己糖激酶(Calbiochem,目录号:376811)
  8. PBS
  9. 三乙醇胺
  10. 醋酸镁
  11. 二硫苏糖醇
  12. BSA(例如酶限制反应级)
  13. 乙酸钾
  14. EJ缓冲区(参见配方)

设备

  1. PCR热循环仪
  2. 闪烁计数器
  3. 热块(Eppendorf Thermomixer ®舒适)

程序

  1. 首先,使用PCR与非生物素化或生物素化的反向引物和非生物素化的正向引物合成〜500bp的dsDNA片段(例如来自pBluescript质粒的),产生在一端生物素化的片段(500生物)。然后在[32 P] -ATP(500)的存在下用T4多核苷酸激酶放射性标记非生物素化的片段。
  2. 接下来,制备与生物素化DNA片段相关的珠子:每点,将0.5pmol 500生物dsDNA片段固定在10μl链霉抗生物素蛋白顺磁珠上,如制造商所推荐。
  3. 同时,使用来自人细胞的NHEJ感受态细胞提取物(Bombarde等人,2010)。简言之,通过在含有蛋白酶和磷酸酶抑制剂混合物的裂解缓冲液中的三个冻/融循环裂解指数生长的细胞,然后裂解物在4℃孵育20分钟,通过离心澄清,并如所述的透析缓冲液透析(Bombarde > et al。,2010)。使用Bradford测定法测定蛋白质浓度,将末端连接提取物储存在-80℃。这里,将40μg提取物在30℃下用2mM葡萄糖和0.2U己糖激酶温育10分钟。葡萄糖和己糖激酶是用于预防的ATP消耗系统 任何连接活性(Calsou等人,2003)。
    注意:DNA末端突触是NHEJ的早期步骤,其依赖于蛋白质/DNA相互作用并且是可逆的(例如通过用高盐或洗涤剂洗涤),而连接是最后步骤并且是不可逆的(耐苛性洗涤) 。必须防止结扎以将分析集中在突触上。
  4. 将10μl模拟(对照)或DNA处理的珠子在100μl的0.5×PBS中洗涤两次
  5. 将湿的模拟或DNA处理的珠子在16℃下在含有0.1pmol放射性500×DNA片段和40μg如上所述预温育的NHEJ感受态细胞提取物的10μlEJ缓冲液中混合30分钟。每5分钟轻轻地手动搅拌小珠。
  6. 孵育后,取出上清液保存,并将湿珠在50μl0.5x PBS中洗涤两次。
  7. 洗涤液与上清液合并。
  8. 在闪烁计数器中测量与上清液和珠子相关的放射性。
  9. 结果表示为减去样品中的计数(在珠上没有500个生物)时下拉的放射性的%(图1)。

    图1.在突触条件下在体外用Nalm6或N114细胞的提取物下拉的比放射性的定量。 N114细胞在Lig4表达中具有影响突触的缺陷 形成

食谱

  1. EJ缓冲区
    50mM三乙醇胺(pH8.0) 0.5mM乙酸镁 1mM二硫苏糖醇 0.1mg/ml BSA
    60mM乙酸钾

致谢

末端突触协议改编自报道的测定(DeFazio等人,2002)。该工作部分地由来自La Ligue Nationale Contrele Cancer(Equipelabellisée),Electricitéde France(EDF ,Conseil de Radioprotection)和国家竞争研究所(XXL-屏幕计划)。 J. Cottarel获得了La Ligue Nationale Contrele Cancer的博士研究生支持。 P. Calsou是法国INSERM的科学家。

参考文献

  1. Bombarde,O.,Boby,C.,Gomez,D.,Frit,P.,Giraud-Panis,M.J.,Gilson,E.,Salles,B.and Calsou, TRF2/RAP1和DNA-PK介导对端粒末端连接的双重保护。 EMBO J 29(9):1573-1584。 
  2. Calsou,P.,Delteil,C.,Frit,P.,Drouet,J。和Salles,B。(2003)。 DNA末端DNA依赖性蛋白激酶的Ku和p460亚基的协调装配对于XRCC4是必需的 - 寡核苷酸IV募集。 J Mol Biol 326(1):93-103。 
  3. Cottarel,J.,Frit,P.,Bombarde,O.,Salles,B.,Negrel,A.,Bernard,S.,Jeggo,PA,Lieber,MR,Modesti,M.and Calsou, 。 非同源末端连接期间连接复合物的非催化功能。细胞 Biol 200(2):173-186。
  4. DeFazio,L.G.,Stansel,R.M.,Griffith,J.D.and Chu,G。(2002)。 DNA末端的突触由DNA依赖性蛋白激酶引起。 /em> 21(12):3192-3200。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cottarel, J. and Calsou, P. (2013). End-synapsis Assay. Bio-protocol 3(18): e900. DOI: 10.21769/BioProtoc.900.
  2. Cottarel, J., Frit, P., Bombarde, O., Salles, B., Negrel, A., Bernard, S., Jeggo, P. A., Lieber, M. R., Modesti, M. and Calsou, P. (2013). A noncatalytic function of the ligation complex during nonhomologous end joining. J Cell Biol 200(2): 173-186. 
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