在这个手稿的工作是由放射肿瘤学系和Huntsman癌症研究所和卫生拨款的国家研究所（R01-CA188520），以SB的资金支持。我感谢丹尼尔·约翰逊和史蒂芬·贝内特在我的实验室演示的协议和解释它的好处。我很感谢马赫什Chandrasekharan博士（Huntsman癌症研究所）对稿件批评意见。

1.染色质免疫沉淀（ChIP）以下BrdU标记<ol><li>培养NIH3T3细胞在DMSO或HDAC1,2选择性抑制剂的存在下如前面6所述。 </li><li>以下用DMSO或HDAC1,2选择性抑制剂治疗，在无菌组织培养罩添加BrdU的细胞中。孵育2×10 6个NIH3T3细胞接种在10ml的NIH3T3介质溴脱氧尿苷的20μM的最终浓度（BrdU）标记60分钟在无菌37℃组织培养箱中。 </li><li>交联细胞内蛋白质的DNA直接添加甲醛到培养基在1％的终浓度。孵育2×10 6的NIH3T3细胞与甲醛在室温在振荡器上10分钟。 </li><li>淬火通过加入甘氨酸至125 1mM的终浓度的交联。在室温下孵育5分钟，在摇动器上。 </li><li>取出纸张，收集细胞在PBS在15毫升离心管中。自旋CELLS在956 XG 5分钟，在4℃。用冰冷的PBS洗两次细胞沉淀。从管中取出PBS中。存储而不PBS（上清液）的交联的细胞沉淀在-80℃下直到使用。 </li><li>通过加入500微升FA140缓冲器准备从交联细胞沉淀的裂解物（50 HEPES的KOH毫pH为7.5，140 mM氯化钠的，1毫摩尔EDTA（pH8.0）中，1％的Triton-X-100和0.1％脱氧胆酸钠）补充有蛋白酶抑制剂混合物。 </li><li>通过超声处理样品5次35％的电力，并用15秒脉冲，0.9秒上和0.1秒关闭设定剪切染色质。保持在冰上的管中每一轮超声处理后，以避免样品的加热。 <ol><li>检查剪切的DNA凝胶作为剪切效率会不同sonicators之间变化上。要检查超声，反向交联加入NaCl至0.16米的浓度和执行步骤1.17.1。 - 1.22。运行洗脱10微升的DNA在1.5％琼脂糖凝胶上以检查是否剪切是之间的300 - 700个碱基，在500 bp的平均强度。 </li></ol></li><li>以下超声处理，离心样品10分钟，在17949×g离心在4℃下，将上清液转移到一个新试管中。 <ol><li>使用根据制造商的协议商用蛋白测定试剂盒进行蛋白估计。用裂解液等量的对照和实验样品的沉淀。一般情况下，使用1毫克每芯片的反应总裂解液。 </li></ol></li><li>准备使用含有蛋白酶抑制剂FA140缓冲蛋白A琼脂糖50％的浆液。加入1/10体积的蛋白酶抑制剂的鸡尾酒。计算所需珠基于在总的样本数的体积。 <ol><li>洗涤蛋白A琼脂糖珠FA140缓冲两次以除去存储缓冲器和等体积FA140缓冲器添加到珠子上，使50％的浆液。 </li><li>以除去蛋白质的非特异性结合的珠子，添加50μl的蛋白A琼脂糖的和在50％的淤浆cubate样品在4℃下以恒定的最终过端旋转旋转式烤架混合器30分钟。 </li></ol></li><li>旋转样品在956×g离心在4℃下进行一分钟。收集上清液，并通过添加任8微升H4K16ac抗体或5微升1mg / ml的SMARCA5抗体（5微克）在上清液进行免疫沉淀。孵育在4°CO / N在一个烤肉店搅拌机恒端过端的转动。 </li><li>保存提取物"输入"控制的1/10，并设置为预留在-20℃。输入需要反向以及IP样品交联的。 </li><li>使用等量兔IgG（5微升1mg / ml的）作为阴性对照免疫沉淀的样品。 </li><li>次日，添加50微升50％的蛋白A琼脂糖浆提取物，孵育1小时，在4℃下用旋转。 </li><li>在4℃颗粒琼脂糖珠离心956 XG 2分钟，小心地取出上清，WAsh的珠粒与顺序如下缓冲液。在缓冲制剂，使用前右侧添加蛋白酶抑制剂。 <ol><li>低盐免疫复合物的洗涤缓冲液洗（FA140; 50mM的HEPES的KOH pH为7.5的终浓度，140mM的氯化钠，1mM EDTA中（pH为8.0），1％的Triton-X-100和0.1％脱氧胆酸钠的），用于5分钟，在4℃下与最终过端旋转。 </li><li>用高盐免疫复合物的洗涤缓冲液洗（FA500; 50mM的HEPES的KOH pH为7.5的终浓度，500mM的氯化钠，1mM EDTA中（pH为8.0），1％的Triton-X-100和0.1％脱氧胆酸钠的），用于5分钟，在4℃下与最终过端旋转。 </li><li>同的LiCl免疫复合物的洗涤缓冲液进行5分（三Cl pH值8.0，250mM的氯化锂，0.5％NP40和0.5％脱氧胆酸钠的10毫摩尔终浓度）在4℃下与最终过端旋转清洗。 </li></ol></li><li>如上所述下列洗涤，加入200微升的洗脱溶液和incubat（1％SDS和100mM碳酸氢钠的终浓度）Ë在室温为15分钟，在室温月底环比端的转动。 </li><li>旋转样品在956×g离心在RT和洗脱液转移到一个新的1.5毫升管。额外的200微升洗脱液重复步骤1.15。 </li><li> 400微升洗脱液，氯化钠加入到了0.16米的浓度，搅拌均匀。 <ol><li>此外，新增400微升洗脱的解决方案被搁置10微升输入（步骤1.11），并添加氯化钠，以0.16米的浓度，以扭转交联输入样本为好。扭转孵育在65℃水浴中5小时交联的样品。 </li></ol></li><li>加入1毫升100％乙醇到反向交联洗脱液。拌匀，孵育在-80℃下2 - 3小时，或在-20°CO / N。旋转样品在17949×g离心15分钟并弃去乙醇。 </li><li>添加800微升70％乙醇洗涤沉淀。旋在17949×g离心15分钟，在4℃并丢弃乙醇。旋简要地，以去除任何残留的乙醇。空气干燥样品，在37℃下进行10分钟以去除残留的乙醇。 </li><li>溶解的DNA在90微升RNA酶和DNA酶的水和加入2微升10毫克/毫升RNA酶在0.2毫克/毫升的最终浓度。在水浴在37℃进行30分钟。 </li><li>添加10微升10X蛋白酶K缓冲液（100mM的三Cl pH为8.0，50毫摩尔EDTA pH为8.0，5％SDS）中。搅拌均匀，加入1μl蛋白酶K.孵育42℃1小时。 </li><li>使用根据商用PCR纯化试剂盒制造商的协议净化输入和沉淀的DNA并在50μl水中洗脱的DNA。商店的DNA在-20℃直至进一步使用。 </li></ol> 2.插槽的ChIP的DNA印迹分析<ol><li>测量输入和沉淀的DNA在50μl水中洗脱的产率作为用分光光度计在280 nm处，根据制造商的协议，在步骤1.22中描述。 </li><li>取输入的是检测的线性范围内的等体积（50微升）或免疫沉淀的DNA。 <ol><li>例如，如果输入DNA产量为1.5纳克/微升，然后取30微升（或45 ng总DNA），并卷至50微升水。对于要素沉淀，需要从步骤2.1整个50微升洗脱液，并继续执行步骤2.3。 </li></ol></li><li>变性50微升输入或免疫沉淀的DNA中加入2.5体积的0.4的1N NaOH，短暂涡旋，离心956×g离心10秒，并在室温下孵育30分钟。 </li><li>通过加入175微升的1M Tris-HCl中，pH值6.8，涡旋，离心956×g离心10秒中和变性的DNA，然后将样品在冰上。 </li><li>制备系列稀释的输入和免疫沉淀的DNA用于插槽印迹测定法，如下所述。 <ol><li>按照步骤2.3 - 2.4得到的350微升总样本量（ 即 50微升输入/芯片DNA，125微升0.4 1N氢氧化钠，175微升的1M的Tris-HCl，pH值6.8）。 注:对于上面的例子说明，45纳克的DNA模板，都会变成0.129纳克/微升。 </li><li>对于输入DNA，标签三管小号为100，50和25，并添加100微升，50微升和25微升变性和中和的DNA样品。使终体积为100微升用无核酸酶水。芯片的DNA，标记三个管200，100和50，并添加200微升，100微升和50微升变性和中和的DNA样品。使终体积为200微升用不含核酸酶的水。 </li></ol></li><li>切膜到达槽印迹装置的尺寸。预润湿的膜和吸油纸在20×SSC之前和之前添加的DNA。根据制造商的方案将它们组装成的槽印迹装置。 </li><li>吸取DNA步骤2.5.2为适当的插槽。适用于真空慢慢拉DNA到泽塔探头膜。停止真空后所有样品都通过装置。 <ol><li>拆卸该装置，并立即将DNA固定到使用UV交联剂膜。有该DNA的一面朝上在UV交联剂和使用盟tocrosslink在交联剂设置。按照制造商的协议。 </li></ol></li><li>检测的BrdU的插槽印迹通过执行印迹。 <ol><li>方框用5％无脂干牛奶的PBS含有0.1％Tween-20（PBST）中进行1小时，在室温，以防止抗体的非特异性结合到所述膜的膜。 </li><li>添加初级抗BrdU抗体（1:500稀释度）稀释于1％非脂肪干乳在PBST中孵育与第一抗体的印迹3小时在室温在振荡器上。第一抗体温育后用PBST洗膜三次。 </li><li>添加HRP偶联的抗小鼠二抗的印迹（1:5000稀释）和在室温下孵育1小时。第二抗体培养后用PBST洗膜三次。 注:使用足够量的抗体，以完全覆盖在初级和二级抗体孵育膜。 </li></ol></li><li>制备ECL混合并把它添加到印迹交流盘带制造商的协议。孵育用ECL混合膜5分钟，室温。 <ol><li>将膜在X射线盒，暴露在膜的X射线胶片和开发使用制造商的方案的印迹。 </li><li>量化使用根据制造商的协议的的Image J软件扫描印迹后所获得的输入和ChIPed DNA中的信号。用于定量，使用是检测的线性范围内，以确保测量的精确性的信号。 </li></ol></li></ol>

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I have no competing financial interests.

在这个手稿中描述的协议是一个相对快速的方法以证明对新复制或新生的DNA的蛋白质或它们的翻译后修饰的形式的存在。此外，这种技术允许一个以测量的蛋白质或它与新生的DNA修饰形式的关联-解离动力学。这种技术是相辅相成的优雅iPOND技术13。在iPOND技术，新合成的DNA标记用乙酸乙酯脱氧尿苷（EDU）。生物素结合物，然后用点击化学添加到EDU。生物素标记的新​​生DNA，然后用链霉珠?...

有缺陷的DNA修复和/或DNA复制是基因组不稳定性，这可以触发细胞死亡的一个主要原因。单个未修复的双链断裂足以导致细胞死亡1。染色质组织都复制期间瞬时改变和修2,3，和故障维护表观遗传信息在这些过程将导致威胁基因组的完整性。 HDAC3或HDAC1,2功能丧失阻碍S期的进展，DNA复制和修复，导致基因毒性应激（DNA损伤）和细胞死亡4-9。因此，它是一个实际的策略，利用选择性HDAC抑制剂破坏复制，修复和染色质在癌细胞和引起DNA损伤，这反过来又可以停止生长，诱导细胞死亡选择性地迅速增长的癌细胞。 期间DNA复制，染色质快速拆卸和重新组装下列DNA重复。新学年nthesized组蛋白H4设置在K5和K12残基（H4K5K12ac）和以下沉积乙酰到染色质，它们被迅速由组蛋白脱乙酰酶（HDACs）10,11脱乙酰。细胞未能保持由组蛋白去乙酰化新生染色质完整性可导致叉崩溃，这反过来可导致DNA损伤和细胞死亡。我们最近发现，HDAC1,2选择性抑制增加组蛋白乙酰化（H4K5ac，H4K12ac和H4K16ac）并抑制SMARCA5染色质remodeler活动上新生染色质，这与减少的复制叉进展，增加的叉崩溃和增加的复制压力诱导的DNA损伤6 。因此，HDAC抑制剂治疗可以改变新生染色质结构，以非常快速地触发DNA损伤和死亡癌细胞中，因为这些细胞周期迅速并通过S相通许多次。据了解的HDAC如何发挥作用来调节组蛋白乙酰化和蛋白质宾迪因此，重要纳克维持在哺乳动物细胞中DNA的复制。 定量地测量组蛋白乙酰化和DNA复制过程中与新生的DNA分子上SMARCA5染色质remodeler的量，我们设计了一种改性ChIP实验称为BrdU的芯片插槽西方技术。使用槽孔以下的所希望的蛋白质或组蛋白修饰染色质免疫沉淀（ChIP），新生的DNA的量的沉淀样品中可以使用的BrdU标记的ChIP的DNA印迹分析来检测（使用胸苷类似物，BrdU阳性）转移到膜印迹装置。使用这种技术，我们发现，H4K16ac（参与染色质包装的标记）和ISWI家族成员SMARCA5（染色质SWI / SNF相关的基质相关的肌动蛋白依赖调节剂）的染色质remodeler与在S期细胞新生DNA的关联6。我们还发现，新生H4K16ac标记通过HDAC1,2 DNA复制6中脱乙酰。 H4K16ac抑制在ISWI家族成员SMARCA5 12的染色质remodeler活动。因而，使用的BrdU片插槽西方技术中，我们可以连接为HDAC1,2的功能在DNA复制过程中调节染色质重塑。因此，尿嘧啶芯片插槽，Western印迹技术是一种有效的方法来定量测量绑定到新生的DNA或蛋白质的翻译后修饰的结合和解离动力学。

<table><tbody><tr><td>Anti-BrdU (westerns)</td><td>BD Biosciences</td><td>B555627</td><td></td></tr><tr><td>Anti-SMARCA5</td><td>Abcam</td><td>ab3749</td><td></td></tr><tr><td>Anti-H4K16ac</td><td>Active Motif</td><td>39167</td><td></td></tr><tr><td>Zeta-Probe GT Membrane</td><td>Bio-Rad</td><td>162-0197</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>Protein A agarose</td><td>Millipore</td><td>16-156</td><td></td></tr><tr><td>Rnase A</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P4850</td><td></td></tr><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>BrdU</td><td>Sigma</td><td>B9285</td><td></td></tr><tr><td>Rabbit IgG&amp;nbsp;</td><td>Millipore</td><td>12-370</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S-3014</td><td></td></tr><tr><td>Triton-X-100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>NP40</td><td>USB Corporation</td><td>19628</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>D6750</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S-4019</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>04-355-222</td><td></td></tr><tr><td>DEPC-treated water</td><td>Sigma</td><td>95284</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>BP-152-5</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>SDS</td><td>Ambion</td><td>AM9820</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Mallinckrodt GenAR&amp;nbsp;</td><td>MAL7772-06</td><td></td></tr><tr><td>20x SSC</td><td>Life Technologies</td><td>15557-036</td><td></td></tr><tr><td>Non-fat dry milk</td><td>Lab Scientific Inc</td><td>M0841</td><td></td></tr><tr><td>ECL</td><td>Thermo Fisher</td><td>80196</td><td></td></tr><tr><td>X-ray film</td><td>Genesee Sci</td><td>30-101</td><td></td></tr><tr><td>Developer</td><td>Konica Minolta</td><td>SRX-101A</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>XLE-1000</td><td></td></tr><tr><td>Sonicator</td><td>Branson Sonifier&amp;nbsp; Digital Ultrasonic Cell Disruptor</td><td>Model: 450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Model: 5810R</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific Isotemp</td><td>Model: 2322</td><td></td></tr><tr><td>NanoDrop 1000 spectrophotometer</td><td>ThermoScientific</td><td>Model: 1000</td><td></td></tr><tr><td>Slot Blot apparatus&amp;nbsp;</td><td>Schleicher and Schuell Minifold II</td><td>44-27570</td><td></td></tr><tr><td>Tissue Culture Incubator</td><td>Thermo Scientific</td><td>Series II 3110 Water-Jacketed CO2 Incubators</td><td></td></tr></tbody></table>

examination of proteins bound to nascent dna in mammalian cells using  brdu-chip-slot-western technique

组蛋白脱乙酰1和2（HDAC1,2）定位于DNA复制的位点。在以前的研究中，使用的选择性抑制剂和遗传击倒系统，我们在复制叉进展和在哺乳动物细胞中染色质新生维护表明新颖功能HDAC1,2。此外，我们使用了结合的溴脱氧尿苷（尿嘧啶）标记的DNA染色质免疫沉淀（ChIP）与狭槽印迹和西方分析定量测量与新生的DNA结合蛋白或组蛋白修饰的BrdU芯片插槽西方的技术。 活跃分裂细胞用HDAC1,2选择性抑制剂或转染的抗HDAC1和 HDAC2的siRNA，然后新合成的DNA进行标记与胸苷类似物溴脱氧尿苷（BrdU）标记。 BrdU标记做在一个时间点没有显著细胞周期阻滞或凋亡因HDAC1,2功能的丧失。用L细胞用BrdU的组蛋白乙酰化的痕迹，染色质免疫沉淀或染色质remodeler的abeling用特异性抗体进行。 BrdU标记输入DNA和免疫沉淀（或ChIPed）DNA随后点样到使用狭缝印迹技术的膜和使用UV固化。新生DNA的每个时隙的数量，然后定量地使用Western分析用抗BrdU抗体进行评估。对新合成的DNA相关的组蛋白乙酰化的标记和染色质remodeler水平HDAC1,2功能损耗的效果然后通过从处理过的样品，以对照样品中获得的BrdU的芯片信号标准化来确定。

为了确定HDAC1,2选择性抑制剂的特异性，Hdac1,2 FL / FL和 HDAC3 FL / FL纤维肉瘤细胞。 Cre重组酶（AD-CRE）含有腺病毒是用来删除Hdac1,2和HDAC3在这些细胞中。以下广告Cre重组感染Hdac1,2 FL / FL的 细胞中，强劲增长H4K5ac进行了观察。 Hdac1,2敲除细胞用233或898的治疗不导致确认233和898抑制Hdac1,2和不HDAC3在这?...

Watch this Scientific Journal Video about Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique at JoVE.com

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

examination-proteins-bound-to-nascent-dna-mammalian-cells-using-brdu

Research

JoVE Journal

Biology

9.1K 次观看.  University of Utah School of Medicine. In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA. 

视频: Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

科研

遗传学

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

生物化学

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

生物学

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

使用BrdU-DNA标记结合细胞周期鉴别成像评估全球DNA双链末端切除术

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

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