这项工作由国家卫生研究院 (ES023350) 资助。丽莎 Chesner 是由训练补助金5T32HL007741 支持的。我们感谢娜塔莉 Tretyakova (明尼苏达大学) 和阿希斯·南迪巴苏 (康涅狄格大学) 及其实验室成员在这项工作的早期和中期阶段提供支持和技术咨询。

1. 生成含 DPC 的质粒
<ol><li>将含有 8 oxoguanine 残渣 (20 µL) 的寡核苷酸 80 pmol 在10x 核苷酸缓冲器 (1 µL) 中, 10 个 T4 连接酶激酶 (5 µL)。加水达到总容积50µL 和孵化在37°c 30 分钟在一个热水浴。<ol>
<li>结合磷酸化寡核苷酸 (50 µL), 80 pmol 的单链 DNA (80 ul), 100 单位 Taq 聚合酶 (20 µL) 在 10x Taq 反应缓冲器 (25 µL), 100 毫米 ATP (20 µL), 10 毫米 dNTPs (20 µL), 纳布缓冲器 2 (25 µL), 8 µg BSA (20 µL) 到总共260µL. 孵化样品在一个 thermocycler 设置在75°c 为15分钟跟随37°c 5 分钟。接下来, 添加 60 u 的 T4 聚合酶 (20 µL), 8000 u T4 连接酶 (20 µL), 100 毫米 ATP (20 µL), 10 毫米 dNTPs (20 µL), 纳布缓冲器 2 (15 µL), 8 µg BSA (20 µL) 到总共375µL. 在37摄氏度 (图 1A) 上一夜之间孵化反应。</li>
	</ol>
</li>
	<li>创建50:50 缓冲饱和苯酚: 氯仿混合物。将每个组件的相等体积, 混合, 并旋转在一个台式离心机在 21130 x g 5 分钟. 氯仿将水从缓冲饱和苯酚中取出, 形成两层: 上部、水层和底部、有机层。添加375µL 的下部, 有机层的引伸反应, 混合, 并旋转在一个台式离心机在 21130 x g 5 分钟。 注意: 苯酚和氯仿是有害物质, 应采取预防措施, 避免接触眼睛或皮肤。<ol>
<li>离心后, 仔细提取顶层和混合醋酸铵添加到最终浓度为0.3 米, 其次是2容量100% 乙醇。将解决方案存储在-20 摄氏度, 最少30分钟或一夜。</li>
		<li>将样品在台式离心机上旋转 1.5万 x rpm, 在4摄氏度, 10 分钟. 取出上清液, 用1毫升的70% 乙醇清洗颗粒。将样品在台式离心机上旋转 1.5万 x rpm, 在4摄氏度, 5 分钟. 取出上清液, 在100µL 水中并用重悬颗粒。</li>
	</ol>
</li>
	<li>将前一步骤中的50µL DNA 与16µL 6x 凝胶加载染料和34µL 水相结合, 并在0.8% 个含有0.5 µg/毫升溴溴的低熔点琼脂糖凝胶上进行电泳。在1x 泰式缓冲器中, 以10厘米的凝胶为6小时, 在2伏/厘米处运行凝胶。使用刀片超螺旋带, 并权衡凝胶切片 (图1A)。运行一个积极的控制, 作为一个标记的共价键关闭, 超螺旋 DNA 迁移。 注意: 溴溴化铵是一种诱变, 应采取预防措施, 避免接触皮肤。同时, 为了减少紫外线 photoproducts 的形成, 必须尽量减少质粒样品暴露于紫外线下的时间。<ol>
<li>通过添加10% β agarase 反应缓冲剂来消化凝胶切片, 每毫克凝胶重量。孵育在65°c 为10分钟和凉快到42°c。添加 10 U 的β agarase 和孵化在42°c 1 小时。</li>
		<li>孵化后, 测量体积, 并添加醋酸铵到最后浓度为0.3 米, 并在冰上冷却15分钟离心机在 1.5万 x g 在室温下15分钟, 收集上清, 并添加2卷异丙醇。晚上-20 摄氏度的寒意。</li>
		<li>离心机纯化, 超螺旋 DNA 10 分钟在 1.5万 x rpm 在一个台式离心机在4°c 和并用重悬的颗粒在40µL 的水。</li>
	</ol>
</li>
	<li>交联 12 pmol (15 µL) 的 DNA 到 36 pmol oxoguanine glycosylase (1 µL) 在缓冲含100毫米氯化钠 (3 µL), 1 毫米氯化镁2 (3 µL), 20 毫米三盐酸 pH 值 7.0 (6 µL) 和10毫米钠 cyanoborohydride (2 µL) 到达最后容量30µL 在37°c 为30分钟26。去除1µL 的交联样品, 稀释500µL 水, 作为 0 h 数据点, 并在步骤3中对 PCR 进行分析。</li>
</ol>2. 氯化钾/SDS 降水
<ol><li>为了可视化交联效率, 限制消化1µg 的交联质粒与 1 ul BspDI 在10x 缓冲区37°c 1 小时, 以产生两个不同大小的 DNA 片段。 注意: 一个片断将被交联到蛋白质 (4.4 kb), 另一个将不会 (2.8 kb) (图 1B)。<ol>
<li>将样品分成两半, 将 SDS 加到最后浓度为 0.5%, 在65摄氏度孵育10分钟。</li>
		<li>将氯化钾 (最终浓度100毫米) 添加到其中一个样品中, 在冰上孵育5分钟。</li>
		<li>离心机两个样品在 1.2万 x g 5 分钟在4摄氏度和运行上清液在琼脂糖凝胶, 以估计蛋白质共轭蛋白到DNA12。 注: 氯化钾/SDS 沉淀用于质量控制, 而不是基质的制备。</li>
	</ol>
</li>
</ol>3. 转染哺乳动物细胞
<ol><li>转染前1天, 板 0.5 x 106单元格/井在6井板中。第二天, 混合1.5 µg (30 µL) 的 DPC 含有质粒 (从步骤 1.6) 与300µL 的无血清培养基。在另一管中, 混合12µL 的转染试剂和300µL 的无血清培养基。将稀释后的 DNA 300 µL 与300µL 稀释转染试剂结合, 室温孵育5分钟。增加250µL 的配合物, 每两个水井和孵化至少1小时, 在37摄氏度。</li>
	<li>孵化后, 去除培养基, 添加1毫升 0.6% SDS/0.01 米 EDTA, 并在室温下孵育 10-15 分钟. 用橡皮警察刮破细胞, 然后转移到1.5 毫升的离心管。添加200µL 5 米氯化钠 (最后浓度1米), 轻轻反转5次, 并孵化在4°c 隔夜27。</li>
	<li>将样品在台式离心机的 21130 x g 处离心为30分钟, 收集上面所述的上清、乙醇沉淀, 并在50µL 水中并用重悬。</li>
</ol>4. SSPE-qPCR
<ol><li>将1µL 的 DNA 从每个时间点 (包括0小时)、2x SYBR 绿色 PCR 主混合 (30 µL)、27µL 水和1µL (100 pMol) 与质粒的损伤链 (底漆 R、图 2) 结合在一起。使用以下条件进行 PCR: 初始预熔10分钟在90°c, 其次8周期:90 °c, 十五年代, 65 °c, 1 分钟。在完成循环8中, 将第二个底漆的1µL (100 pMol) 添加到总计60µL (底漆 L、图 2)28 (参见材料表中的试剂详细信息)。</li>
	<li>混合1µL 的 unamplified 回收的 DNA 从每个时间点 (包括0小时), 2x 主混合 (30 µL), 27 µL 的水, 和1µL 的每个底漆 (100 pMol) 到总60µL。</li>
	<li>从步骤4.1 和 4.2 (20 µL/井, 三个) 中加载一个96井 PCR 板, 并使用上面描述的条件对30个循环执行 qPCR。</li>
	<li>平均每组三份样品的 CT 值。减去步骤4.1 中从步骤4.2 中生成的 ct 值, 以获取每个样本的增量 ct 值。从增量 CT 值中减去 0 h 时间点以删除任何背景 (有关详细说明, 请参见代表结果一节)。</li>
	<li>使用公式将增量 CT 值转换为百分比修复: <img alt="Equation" src="/files/ftp_upload/57413/57413eq1v2.jpg">
</li>
</ol>

<ol>
	<li>Tretyakova, N. Y., Groehler, A., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-Protein+Cross-links:+Formation,+Structural+Identities,+and+Biological+Outcomes.">DNA-Protein Cross-links: Formation, Structural Identities, and Biological Outcomes.</a> Accounts of chemical research. 48 (6), 1631-1644 (2015).</li><li>Barker, S., Weinfeld, M., Murray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-protein+crosslinks:+their+induction,+repair,+and+biological+consequences.">DNA-protein crosslinks: their induction, repair, and biological consequences.</a> Mutat Res. 589 (2), 111-135 (2005).</li><li>At Groehler, A. t., Degner, A., Tretyakova, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry-Based+Tools+to+Characterize+DNA-Protein+Cross-Linking+by+Bis-Electrophiles.">Mass Spectrometry-Based Tools to Characterize DNA-Protein Cross-Linking by Bis-Electrophiles.</a> Basic Clin Pharmacol Toxicol. 121, 63-77 (2016).</li><li>Barker, S., Weinfeld, M., Zheng, J., Li, L., Murray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mammalian+proteins+cross-linked+to+DNA+by+ionizing+radiation.">Identification of mammalian proteins cross-linked to DNA by ionizing radiation.</a> J Biol Chem. 280 (40), 33826-33838 (2005).</li><li>Dizdaroglu, M., Gajewski, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanism+of+hydroxyl+radical-induced+formation+of+a+DNA-protein+cross-link+involving+thymine+and+lysine+in+nucleohistone.">Structure and mechanism of hydroxyl radical-induced formation of a DNA-protein cross-link involving thymine and lysine in nucleohistone.</a> Cancer Res. 49 (13), 3463-3467 (1989).</li><li>Chvalova, K., Brabec, V., Kasparkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+formation+of+DNA-protein+cross-links+by+antitumor+cisplatin.">Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin.</a> Nucleic Acids Res. 35 (6), 1812-1821 (2007).</li><li>Speit, G., Schutz, P., Merk, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+repair+of+formaldehyde-induced+DNA-protein+crosslinks+in+repair-deficient+human+cell+lines.">Induction and repair of formaldehyde-induced DNA-protein crosslinks in repair-deficient human cell lines.</a> Mutagenesis. 15 (1), 85-90 (2000).</li><li>Stingele, J., Bellelli, R., Boulton, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+DNA-protein+crosslink+repair.">Mechanisms of DNA-protein crosslink repair.</a> Nat Rev Mol Cell Biol. , (2017).</li><li>Kohn, K. W., Erickson, L. C., Ewig, R. A., Friedman, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+DNA+from+mammalian+cells+by+alkaline+elution.">Fractionation of DNA from mammalian cells by alkaline elution.</a> Biochemistry. 19 (15), 4629-4637 (1976).</li><li>Ewig, R. A., Kohn, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+and+repair+in+mouse+leukemia+L1210+cells+treated+with+nitrogen+mustard,+1,3-bis(2-chloroethyl)-1-nitrosourea,+and+other+nitrosoureas.">DNA damage and repair in mouse leukemia L1210 cells treated with nitrogen mustard, 1,3-bis(2-chloroethyl)-1-nitrosourea, and other nitrosoureas.</a> Cancer Res. 37, 2114-2122 (1977).</li><li>Ewig, R. A., Kohn, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-protein+cross-linking+and+DNA+interstrand+cross-linking+by+haloethylnitrosoureas+in+L1210+cells.">DNA-protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells.</a> Cancer Res. 38, 3197-3203 (1978).</li><li>Zhitkovich, A., Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+sensitive+assay+to+detect+DNA-protein+crosslinks+in+intact+cells+and+in+vivo.">A simple, sensitive assay to detect DNA-protein crosslinks in intact cells and in vivo.</a> Carcinogenesis. 13 (8), 1485-1489 (1992).</li><li>Chiu, S. M., Sokany, N. M., Friedman, L. R., Oleinick, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+processing+of+ultraviolet+or+ionizing+radiation-induced+DNA-protein+cross-links+in+Chinese+hamster+cells.">Differential processing of ultraviolet or ionizing radiation-induced DNA-protein cross-links in Chinese hamster cells.</a> Int J Radiat Biol Relat Stud Phys Chem Med. 46 (6), 681-690 (1984).</li><li>Merk, O., Reiser, K., Speit, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chromate-induced+DNA-protein+crosslinks+with+the+comet+assay.">Analysis of chromate-induced DNA-protein crosslinks with the comet assay.</a> Mutat Res. 471 (1-2), 71-80 (2000).</li><li>Loeber, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+DNA-protein+cross-linking+by+antitumor+nitrogen+mustards.">Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards.</a> Chem Res Toxicol. 22 (6), 1151-1162 (2009).</li><li>Gherezghiher, T. B., Ming, X., Villalta, P. W., Campbell, C., Tretyakova, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1,2,3,4-Diepoxybutane-induced+DNA-protein+cross-linking+in+human+fibrosarcoma+(HT1080)+cells.">1,2,3,4-Diepoxybutane-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells.</a> J Proteome Res. 12 (5), 2151-2164 (2013).</li><li>Groehler, A. t., Villalta, P. W., Campbell, C., Tretyakova, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+DNA-Protein+Cross-Linking+by+Phosphoramide+Mustard+and+Nornitrogen+Mustard+in+Human+Cells.">Covalent DNA-Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells.</a> Chem Res Toxicol. 29 (2), 190-202 (2016).</li><li>Kiianitsa, K., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+assay+for+DNA-protein+covalent+complexes+in+living+cells.">A rapid and sensitive assay for DNA-protein covalent complexes in living cells.</a> Nucleic Acids Research. 41 (9), e104 (2013).</li><li>Kiianitsa, K., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+isolation,+identification+and+quantification+of+DNA-protein+adducts+by+ELISA-based+RADAR+assay.">Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.</a> Nucleic Acids Research. 42 (13), e108 (2014).</li><li>Stingele, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+Regulation+of+DNA-Protein+Crosslink+Repair+by+the+DNA-Dependent+Metalloprotease+SPRTN.">Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN.</a> Molecular Cell. 64 (4), 688-703 (2016).</li><li>Vaz, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloprotease+SPRTN/DVC1+Orchestrates+Replication-Coupled+DNA-Protein+Crosslink+Repair.">Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair.</a> Mol Cell. 64, 704-719 (2016).</li><li>Maskey, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spartan+deficiency+causes+accumulation+of+Topoisomerase+1+cleavage+complexes+and+tumorigenesis.">Spartan deficiency causes accumulation of Topoisomerase 1 cleavage complexes and tumorigenesis.</a> Nucleic Acids Res. 45 (8), 4564-4576 (2017).</li><li>Lessel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+SPRTN+cause+early+onset+hepatocellular+carcinoma,+genomic+instability+and+progeroid+features.">Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.</a> Nature Genetics. 46 (11), 1239-1244 (2014).</li><li>Baker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+excision+repair+eliminates+unique+DNA-protein+cross-links+from+mammalian+cells.">Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells.</a> J Biol Chem. 282 (31), 22592-22604 (2007).</li><li>Ahn, B., Kang, D., Kim, H., Wei, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+mitomycin+C+cross-linked+DNA+in+mammalian+cells+measured+by+a+host+cell+reactivation+assay.">Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay.</a> Mol Cells. 18 (2), 249-255 (2004).</li><li>Fromme, J. C., Bruner, S. D., Yang, W., Karplus, M., Verdine, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product-assisted+catalysis+in+base-excision+DNA+repair.">Product-assisted catalysis in base-excision DNA repair.</a> Nat Struct Biol. 10 (3), 204-211 (2003).</li><li>Hirt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures.">Selective extraction of polyoma DNA from infected mouse cell cultures.</a> J Mol Biol. 26 (2), 365-369 (1967).</li><li>Lee, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+repair+of+DNA+damage+in+cell+lines+and+human+peripheral+blood+mononuclear+cells.">Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells.</a> Anal Biochem. 365 (2), 246-259 (2007).</li><li>Sikorsky, J. A., Primerano, D. A., Fenger, T. W., Denvir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+reduces+Taq+DNA+polymerase+fidelity+and+PCR+amplification+efficiency.">DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency.</a> Biochemical and biophysical research communications. 355 (2), 431-437 (2007).</li><li>Wickramaratne, S., Mukherjee, S., Villalta, P. W., Scharer, O. D., Tretyakova, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+sequence-specific+DNA-protein+conjugates+via+a+reductive+amination+strategy.">Synthesis of sequence-specific DNA-protein conjugates via a reductive amination strategy.</a> Bioconjug Chem. 24 (9), 1496-1506 (2013).</li><li>George, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+nucleotide+excision+repair+in+a+helicase-deficient+XPD+mutant+from+intragenic+suppression+by+a+trichothiodystrophy+mutation.">Restoration of nucleotide excision repair in a helicase-deficient XPD mutant from intragenic suppression by a trichothiodystrophy mutation.</a> Mol Cell Biol. 21 (21), 7355-7365 (2001).</li><li>Minko, I. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+Repair+of+DNA-Polypeptide+Cross-Links+by+the+UvrABC+Nuclease.">Initiation of Repair of DNA-Polypeptide Cross-Links by the UvrABC Nuclease.</a> Biochemistry. 44 (8), 3000-3009 (2005).</li><li>Minko, I. G., Zou, Y., Lloyd, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incision+of+DNA-protein+crosslinks+by+UvrABC+nuclease+suggests+a+potential+repair+pathway+involving+nucleotide+excision+repair.">Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair.</a> Proc Natl Acad Sci U S A. 99, (2002).</li><li>Reardon, J. T., Sancar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA-polypeptide+crosslinks+by+human+excision+nuclease.">Repair of DNA-polypeptide crosslinks by human excision nuclease.</a> PNAS. 103, 4056-4061 (2006).</li><li>Lopez-Mosqueda, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPRTN+is+a+mammalian+DNA-binding+metalloprotease+that+resolves+DNA-protein+crosslinks.">SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks.</a> eLife. 5, e21491 (2016).</li><li>Stingele, J., Schwarz, M. S., Bloemeke, N., Wolf, P. G., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA-dependent+protease+involved+in+DNA-protein+crosslink+repair.">A DNA-dependent protease involved in DNA-protein crosslink repair.</a> Cell. 158 (2), 327-338 (2014).</li><li>Duxin, J. P., Dewar, J. M., Yardimci, H., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+a+DNA-Protein+Crosslink+by+Replication-Coupled+Proteolysis.">Repair of a DNA-Protein Crosslink by Replication-Coupled Proteolysis.</a> Cell. 159 (2), 346-357 (2014).</li><li>Stingele, J., Habermann, B., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-protein+crosslink+repair:+proteases+as+DNA+repair+enzymes.">DNA-protein crosslink repair: proteases as DNA repair enzymes.</a> Trends in Biochemical Sciences. 40 (2), 67-71 (2015).</li><li>Potter, H., Heller, R., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+by+Electroporation.">Transfection by Electroporation.</a> Current protocols in molecular biology. , (2003).</li></ol>

作者没有什么可透露的。

SSPE-qPCR 方法通过检查含有单一的、定义的 DPC 病变的同质种群的修复, 提供了许多优于其他方法的优势。值得注意的是, 除了控制蛋白质的特性和用于将蛋白质与 DNA 连接的化学交联类型外, 还可以很容易地操作引入 DPC 损伤的序列上下文。我们已经探讨了对 DPC 修复的影响, 介绍了在模板或编码链的一个质粒下游的活动助剂轨迹。同样, 我们正在调查的影响, DPC 修复复制使用一个 M13 质粒, 其中含有 SV40 的复制的起源转染成 HEK293T 细胞。此处描述的检测直接测量 DPC 修复, 而不是其他策略, 如间接估计修复活动24、25的主机单元重新激活。此外, 该系统是稳健的, 敏感的和定量的。与其他测量 DPC 清除的系统不同, 此检测仅检测到完整的修复事件,即, 它不仅要求删除 DPC 病变, 而且还需要完全恢复双相 DNA 的完整性17。这是因为 abasic 的站点和磷酸二酯骨干的缺口或断裂会有效地阻止检测, 如原始 DPC病变29。
虽然本报告的重点是一种特殊类型的 DPC, 这是由硼氢化捕获的酶反应中间体, 我们目前正在开发的方法来研究修复 DPCs 涉及其他蛋白质和病变, 其中蛋白质链接到DNA 发生在核苷基上, 而不是核糖位置。使用还原胺化, 我们创造了蛋白质和肽 crosslinks 附着在鸟嘌呤或胞嘧啶基的 DNA 底漆30。这些寡核苷酸被纯化为均匀性, 用于生成含有 dna 蛋白和 dna 肽 crosslinks 的超螺旋质粒。虽然这些反应的效率相对于上文详细描述的 oxoguanine glycosylase 交联方法有所减少, 但它们是成功的, 并允许在野生型的情况下检查这些基底的修复和核苷酸切除修复缺陷的哺乳动物细胞系。我们还使用了 SSPE qPCR 试验研究 oxoguanine 病变的修复和合成核糖-胆固醇共轭, 这是以前证明是通过细胞核苷酸切除修复机械31修复。正如图 2图形描述的那样, 在原则上, 用 SSPE-qPCR 检测方法来测量阻止 Taq 聚合酶引伸的任何病变。
目前的 DPC 修复模型表明, 更大的 DPCs (> 10 kDa) 在删除之前会被蛋白水解处理到更小的肽类病变 (32,33,34。最有可能的候选人负责这一蛋白水解是在人类细胞的蛋白酶体或特定的蛋白酶, 名为斯巴达20,21,35,36,37,38. 对这些蛋白酶作用的进一步调查可以使用 SSPE-qPCR 法进行。蛋白酶体抑制剂可用于预处理细胞在转染含 DPC 的基质之前。或者, 斯巴达的击倒细胞系可以被受损的质粒转染, 以阐明其在大 DPCs 的蛋白质水解中的作用。
无论使用何种方法来创建交联或 DNA 损伤的性质, 值得强调的是, SSPE-qPCR 方法是严重依赖于能够产生大量的均质的 DPC-质粒基质。这一分析的关键步骤包括纯化的共价键, 封闭圆形质粒后底漆延伸, 以消除任何缺口或线性质粒分子。必须消除这些污染物, 以确保任何后续的 DPC 修复都不是由于尼克定向或双链断裂定向修复过程。通过将寡核苷酸与单链 dna 的比值变化, 可以克服在引物延伸反应后未能获得足够数量的超螺旋 dna。SSPE-qPCR 方法的另一个重要步骤是蛋白质对质粒的交联效率。在本研究中, 一种高效的酶反应被用来将 oxoguanine glycosylase 蛋白与 8-氧鸟嘌呤的病变交联。通过改变添加到反应中的蛋白质量, 可以改善次优交联。
虽然本报告中描述的结果依赖于 lipofection 将 DPC 修复基板引入接收单元, 但没有任何理由,先验, 其他 transfections 方法无法使用。我们进行了初步研究, 并观察到电穿孔39也可以使用。然而, 值得注意的是, 在我们的经验中, 电穿孔的转染效率与 lipofection 相比降低, 我们发现除了1.5 µg 的 DPC 基板获得修复数据外, 还有必要使用载体 DNA (最多5µg)。总的来说, 上述 SSPE qPCR 方法提供了一种创新的方法, 专门检查 DPC 修复的质粒 dna, 并产生新的洞察力的 dna 损伤反应。

本文描述的是一种 pcr 检测方法, 称为 SSPE-qPCR。该方法的目的是对转染修复缺陷和精通哺乳动物细胞的质粒 DNA 的 DPC 修复进行量化。这种检测方法快速、定量、极具灵活性, 并可直接测量修复活动。虽然本报告的重点是使用这种方法来研究修复 DPCs, 下面的结果表明, 修复任何损害, 阻止 Taq 聚合酶可以研究使用这种方法。
这一方法的发展背后的基本原理是深入了解哺乳动物细胞修复 DPCs 的机制。与其他类型的 DNA 损坏不同, DPCs 具有大量不同的1、2。研究表明, 数以百计的细胞蛋白可以与 DNA 交联, 对于每种蛋白质, 原则上, 许多氨基酸侧链可能成为共价键附着在细胞 DNA3。此外, 有许多化学附着点的蛋白质到 DNA 主干, 包括几个位置上的核苷酸基地以及在核糖糖4,5。这种化学多样性使人们有可能依靠不同的生物化学途径来修复不同类型的 DPCs。考虑到这一问题, SSPE-qPCR 的测定被开发出来了。
为了深入了解细胞 DPC 修复的分子生物学, 开发了几种技术。下面概述了已开发的主要方法, 并总结了各自拥有的主要长处和弱点。值得强调的是, 虽然本综述的重点是在哺乳动物细胞培养系统中 dpc 修复的研究, 但对目前的 dpc 修复模型作出了重大贡献, 使用的微生物和无细胞系统, 在本手稿.
也许, 可以采取最简单的策略, 以了解 DPC 修复的遗传学是评估各自对细胞死亡的敏感性观察野生类型和突变细胞暴露在诱导 DPCs6,7。这种策略相对较快, 成本低廉, 而且不需要专门的专门知识才能完成基本的细胞培养技术。制衡这些优点对此方法有许多限制, 包括以下内容。首先, 该检测不能直接测量 DNA 的修复。这一策略的工作假设是, 在编码相关 dna 修复蛋白的基因中, 失活突变会导致 dna 损伤的累积, 触发程序性细胞死亡。然而, 编码非 DNA 修复蛋白的基因突变可以在主, 增强 (或减少) 细胞对 xenobiotic 诱导的细胞死亡的敏感性。其次, 创建 DPCs 的代理总是诱发其他类型的 DNA 损伤 (一个例外是 5-aza-2 '-deoxycytadine, 但此代理也耗尽了细胞甲基水平8)。因此, 可以想象, 增强的细胞对该制剂的过敏反应可能反映修复 interstrand crosslinks 或其他病变的缺陷。第三, 如上所述, DPCs 代表了一个巨大的异构类组成的不同类型的化学 crosslinks, 涉及不同的蛋白质合作伙伴。可能在特定的遗传背景下, 修复一个或多个子类型的病变可能会改变, 这种差异可能不足以显著改变细胞对死亡的过敏反应。总之, 虽然这一战略是一个有吸引力的出发点, 上面概述的限制强调了寻求其他更直接的方法来研究 DPC 修复动力学的重要性。
为了实现这一目标, 已经制定了若干相关的办法。例如, 调查人员已经开发出方法来区分 ' 自由 ' dna 和 ' 蛋白质绑定 ' dna9,10,11。利用这些方法, 可以比较 DPCs 的稳态水平或在不同遗传背景下暴露于 DPC 生成剂的情况下。最广泛使用的两种策略涉及使用硝化膜结合策略或氯化钾/SDS 沉淀12、13, 从游离 dna 中分离含 DPC dna。在前处理细胞是裂解和通过硝化棉过滤器。由于硝化纤维素结合蛋白质, 过滤器保留蛋白质链接的 dna, 允许游离 DNA 通过。在后一种战略中, 蛋白质绑定 dna 与游离 dna 分离, 其基础是 SDS 与蛋白质而不是 DNA 结合, 并且可以通过添加氯化钾来沉淀。因此, 蛋白质链接的 dna 变得不溶, 而未绑定的 dna 仍然在溶液中。量化的 dna 可以使用核素胸苷 (如果细胞最初新陈代谢标记) 或 dna 选择性荧光染料如赫斯特33258。这些方法是可重现的, 需要少量的步骤。然而, 它们并没有提供有关蛋白质附着在 DNA 上的化学交联性质的信息。此外, 重要的是要注意, 这些化验可能过度估计 DPC 修复的错误评分不完全修复,即, 蛋白水解处理, 以较小的 dna 肽 crosslinks, 可能不容易被捕获或沉淀, 作为真正的 DNA修复.
彗星的化验可以用来可视化 DPC 形成在细胞14。在这些实验中, DPCs 的存在减少了 DNA 的迁移, 从而可以通过对蛋白酶 K 的预处理来逆转。因此, 尾部的长度可以用来估计 DPC 的形成。然而, 如上所述, DPC 形成的药物产生其他类型的 DNA 损伤, 可能改变尾部长度。该协议还具有高度技术性, 需要在共聚焦成像方面的专门知识和培训。
质谱法可用于研究 DPC 修复动力学后的交联剂的治疗,15,16,17。这些实验用 DPC 形成剂处理细胞, 通过生物素捕获或苯酚分离 DPCs: 氯仿 (1:1) 萃取。质谱可以用来识别交联蛋白或定量 DPCs 的数量。这种方法的主要优点是生成的数据的性质。有可能精确地编目在接触到 xenobiotic 后交联的蛋白质的类型, 但是, 这个协议是昂贵的, 耗时的, 并受可检测的交叉的类型的限制。
Maizels et . 开发了一种敏感的 "雷达" (dna 加合恢复的快速方法), 用于定量 immunodetection dna 蛋白加合物, 以及一种基于 ELISA 的雷达检测18, 19.这些化验方法对于诱捕细胞中瞬时形成的 DNA 蛋白中间体和生成适合于质谱的样品来识别新的蛋白质加合物特别有用。这种 immunodetection 的检测依赖于是否有抗体来捕获 DNA 蛋白交联, 因此, 可能无法检测在修复过程中形成的降解 DNA-肽加合物。最近, 一个特定的 DPC 修复通路链接到 dna 复制和 dna 依赖金属蛋白酶斯巴达被发现, 其中 DPCs 是 proteolyzed 小肽在修复期间 20, 21.遗传突变的基因是与 Ruijs-Aalfs 综合征的人, 一种疾病的特点是基因组不稳定, 过早衰老, 和肝癌22。基因设计的斯巴达基因缺陷小鼠显示类似的表型23。
宿主细胞重新激活转录活性已被用来研究修复已转染的质粒 DNA 基板上的定义病灶24,25。在这些实验中, 含有 DPCs (或其他类型的 DNA 损伤) 的质粒, 如荧光素酶, 被转染成细胞。24-72 小时后的发光测量结果与 DPC 修复相关。然而, 这些间接的修复化验没有能力检测的修复事件早于24小时后转染, 不能区分 RNA 聚合酶旁路部分修复基底和完全修复。
上述每种方法都有其优点, 并为 DPC 修复的当前模型做出了贡献。然而, SSPE-qPCR 检测绕过了与这些其他方法相关的一些限制, 因此可以更具体地了解 DPC 修复机制。例如, SSPE-qPCR 检测可以直接测量完整哺乳动物细胞 DNA 中特定部位 DPCs 的修复。这种方法是通用的, 并已被用于获得修复结果后, 转染仓鼠和人的细胞线。在培养的哺乳动物细胞系中, 可以使用 lipofection 或电穿孔来进行质粒的转染。它还确保只测量定义的 DPCs 的修复, 而不是由大多数 DPC 形成的药物引起的其他类型的 DNA 损伤。SSPE-qPCR 易于执行, 价格低廉, 速度快。结果表明, 使用该方法检测的修复事件早在2小时后转染。使用这种方法, 可能影响 DPC 修复结果的变量可以以敏感和有效的方式进行研究。例如, 转录在 DPC 修复中的作用尚未得到严格评估。由于 SSPE-qPCR 试验的灵活性, 可以对 DPC 的交联部位进行处理, 以解决这个问题。此外, 引入一个复制到 dpc 轴承质粒的起源可以用来解决复制对 dpc 修复的影响。此外, 可以在质粒上创建多个 crosslinks, 以检查单个 DPC 与多 crosslinks 的修复差异。这些问题是很难回答使用染色体 DNA, 但可以很容易地解决使用 SSPE-qPCR 化验。总的来说, SSPE-qPCR 的检测要求纯化, 质粒 DNA 的微克数量包含一个已知位置的损害。加合物除 DPC 外, 可用于本试验, 但是, 病变必须能够阻止 Taq 聚合酶的扩展。

<table border="0" cellpadding="0" cellspacing="0" width="657">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:215px;">8-oxoguanine modified oligo</td>
			<td style="width:157px;">Midland Certified Reagent Company</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;">Sequence: 5&#8217;AGGGTTTTCCA (8-oxo-dG)TCACGACGTT 3&#8217;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">T4 PNK</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">M0201S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Single-stranded M13</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">N4040S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Taq polymerase</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">M0267L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">ATP, 100mM</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:119px;">R0441</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">dNTP Mix, 10mM each</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:119px;">R0192</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">BSA</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">B9001S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">T4 polymerase</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">M0203L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">T4 ligase</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">M0202L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">&#946;-agarase</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">M0392S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Oxoguanine glycosylase 1</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">M0241S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">SYBR Green PCR Master Mix</td>
			<td style="width:157px;">Applied Biosystems</td>
			<td style="width:119px;">4309155</td>
			<td style="width:167px;">green PCR master mix</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Primer R</td>
			<td style="width:157px;">UMN Genomic Center</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;">5&#8217;CGGCTCGTATGTTGTGTG 3&#8217;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Primer L</td>
			<td style="width:157px;">UMN Genomic Center</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;">5&#8217; GCTGCAAGGCGATTAAGT 3&#8217;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Lipofectamine Reagent</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:119px;">18324-012</td>
			<td>transfection reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">BspD1</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">R0557S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NEB buffer 2</td>
			<td style="width:157px;">NEB</td>
			<td style="width:119px;">B7002S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">StepOnePlus Real Time PCR System</td>
			<td style="width:157px;">Applied Biosystems</td>
			<td align="right" style="width:119px;">4376600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">UltraPure Buffer-Saturated Phenol</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:119px;">15513-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Chloroform, reagent ACS, spectro grade</td>
			<td style="width:157px;">ACROS</td>
			<td style="width:119px;">40463-5000</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple, rapid, and quantitative assay to measure repair of dna-protein crosslinks on plasmids transfected into mammalian cells

该方法的目的是提供一个灵活, 快速, 定量的技术, 以检查 DNA 蛋白交联 (DPC) 修复哺乳动物细胞系的动力学。这项研究不是在全球范围内对 xenobiotic 诱导的或自发的染色体 DPC 去除进行检测, 而是检查在质粒 DNA 基底内的一个部位专门介绍的均匀的、化学定义的病灶的修复。重要的是, 这种方法避免了使用放射性材料, 不依赖昂贵的或高度专业化的技术。相反, 它依赖于标准的重组 DNA 程序和广泛可用的实时, 定量聚合酶链反应 (qPCR) 仪器。鉴于所采用的战略具有固有的灵活联蛋白的大小 , 以及化学联系的性质和附着地点的精确 DNA 序列上下文可以多种多样 , 以解决这些参数提高了 DPC 修复的整体效率。采用该方法, 将含有特定地点的 DPC 的质粒转染成细胞, 并在不同的时间后转化后恢复到低分子量的 DNA。恢复后的 DNA 会受到链特定的底漆延伸 (SSPE), 使用底漆补充的质粒受损的线。由于 DPC 病变阻断 Taq dna 聚合酶, 因此可以用 qPCR 定量评估修复 dna 的比例。周期门限 (CT) 值用于计算各个单元格行中不同时间点的百分比修复。这种 SSPE qPCR 方法还可用于定量地评估阻止 Taq 聚合酶的任何 DNA 加合物的修复动力学。

为了利用 SSPE qPCR 法评估细胞 dpc 修复, 必须准备足够数量 (µg) 高质量的 dpc 修复基质。为了获得这个产品, 一个含有 8-oxoguanine 残留的寡核苷酸被退火到互补的单链 DNA, 底漆延伸, 和凝胶纯化, 以确保只有共价键, 封闭循环产品用于 transfections (图1A). 本报告的重点是利用硼氢化陷印法在双链圆形质粒分子上创建 oxoguanine glycosylase 和核糖单元的共价键, 尽管类似的方法可用于获得化学上不同的 DPC 基板。由于交联反应的效率极高, oxoguanine glycosylase/硼氢化捕集策略特别具有吸引力。如图 1B所示, 在 dpc 基板上进行的氯化钾/SDS 沉淀是有选择地消化成两个碎片的, 几乎定量地耗尽了窝藏 dpc 的 DNA 片段。这些结果支持的结论是, 基本上100% 的质粒基质分子含有蛋白质交联。
SSPE-qPCR 检测在协议部分描述的 CT 值, 利用 qPCR 产生的百分比的 DPC 修复后转染哺乳动物细胞。如图 2所示, 蛋白质交联阻止 Taq 聚合酶将 "R" 底漆退火至含 DPC 的链。第二个关键特征是在进行 qPCR 试验之前, 将八轮 SSPE 反应 (使用 R 底漆) 纳入。虽然未损坏 (或修复) DNA 将在这些 SSPE 反应中延长, 为下游 ' L ' 底漆创建一个结合部位, 受损的 dna (或未完全修复的 dna) 将不会延长。因此, SSPE 增加了每一个未损坏 (或修复) 链的丰度八倍。这意味着, 在 qPCR 之前执行了 SSPE 步骤的修复示例将显示一个 CT 值, 该数值比在 qPCR (图 2) 之前未执行 SSPE 的相同样本所获得的三个单位低。两种治疗方法 (称为ΔCT) 观察到的 CT 值的三单位差异反映了 8 = 23的事实。此增量 CT 值可用于计算细胞 dna 修复活动的公式: dna 修复百分比 = (2ΔCT/23) x 100。控制实验证实, 当未损坏的基质质粒受到 SSPE-qPCR (未显示的数据) 时, 大约3的三角洲 CT 值一直被观察到。
表 1描述了从中国仓鼠肺成纤维细胞 3 h 和 8 h 后转染 (表 1A) 以及从时间零样本中恢复的 DPC 包含质粒基底产生的 CT 值, 即, 按照 "协议" 部分中所述编写的示例, 但未转到单元格中 (表1B)。该表还提供了ΔCT 和百分比修复值 (使用从这些示例获得的公式 2ΔCT/23 x 100) 进行计算。如表 1A所示, 从采集的3小时和8小时后转染的样本计算的百分比修复分别为66% 和93%。然后, 从 0 h 样本分析所得的值中减去这些数值, 以确定修复百分比。表 1B描述了两个不同的 0 h 样本的 CT 值, 在转染之前, 有效的交联是或未达到。有效交联样品导致三角洲 ct 值为 0.8, 而交联样品差, 导致三角洲 ct 值为2.5。迄今为止, 在有效交联的 0 h 样品中, 无法精确确定0.8 背景信号的来源。这种背景不太可能反映自发 dpc 去除的低水平, 或者是由于 Taq 聚合酶通过 DPC 的综合体旁路合成的低水平: 在高度纯化的单链 M13 基片上进行的广泛试验一直产生的三角洲 CT 值约 0.8,即, 基本上完全相同的 ' 背景 ' 扩展在含 DPC 基板上看到。因此, 在 SSPE 过程中, 可能会出现小程度的 "R" 底漆的错误启动, 从而产生少量的产品, 在添加 "L" 底漆时, 可以放大, 然后 qPCR 执行。迄今为止, 通过操纵 PCR 条件来消除这种背景的努力未能弥补这一缺陷。但是, 上述减法策略允许准确估计 DPC 修复活动。值得注意的是, 低效的蛋白质交联到质粒将导致较高的背景值。在表 1B中提供的示例数据中描述了这一点, 其中低效的蛋白质-DNA 交联导致了时间0的增量 CT 值较高。因此, 在启动 transfections 之前, 控制实验总是执行的, 而在0.8 阈值水平以上升高的三角洲 CT 值的基底会被丢弃。如 qPCR 协议所述, 每个样本分为3个井, 以确保吹打的一致性。这些复制然后平均获得该样本的 CT 值。从数据集中消除了偏离0.1 的复制。如果所有的三复制偏离了 0.1, 从彼此之间, SSPE-qPCR 化验是重做, 直到一致的价值得到。经验表明, 至少3个独立 transfections 必须进行, 以获得可靠的平均值, 然后可以进行统计分析, 以确定各种参数对 DPC 维修效率的影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57413/57413fig1.jpg"> 图1。生成含 DPC 的质粒.(A) 含有 8-氧鸟嘌呤残留物 (红色) 的寡核苷酸被退火为单链 DNA (粗黑色圆), 并扩展以创建双绞线质粒 (由虚线表示的底漆延伸产品)。溴溴化酶电泳后, 超螺旋 DNA (红盒) 的波段从低熔琼脂糖凝胶中切除, 并经β-agarase 消化。车道: (1) 分子量标记, (2) 单链 dna, (3) 双链 dna, (4) 底漆扩展样本。(B) Oxoguanine glycosylase (简称为橙色圆圈的缩写 hOGG1) 通过钠 cyanoborohydride 交联到 8-羰基-鸟嘌呤残留物, 由此产生的 DPC 基底被消化生成2800基对、游离 DNA 片段和4400附着在蛋白质上的基对片段。将 SDS 添加到样品中, 然后将其分为两部分, 其中之一是用氯化钾和离心处理含有含 DPC DNA 的沉积物 (如橙色颗粒)。后一种样品中的上清 (如离心管中的蓝液) 和不接触氯化钾的样品均受凝胶电泳。车道: (1) 分子量标记, (2)-氯化钾, (3) + 氯化钾.<a href="//cloudflare.jove.com/files/ftp_upload/57413/57413fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57413/57413fig2.jpg"> 图 2: 通过 SSPE-qPCR 对受损质粒进行量化.质粒 DNA 变性, 对受损链 (R) 的底漆进行退火和扩展。这一过程总共重复了8个周期。由于损坏的基底 (顶部), Taq 聚合酶被 DPC 的损害阻止, 并不会产生全长的产品链。相比之下, 用修复后的基板 (底部), Taq 聚合酶将产生全长的产品链包含结合部位的底漆 L。8循环后, 添加底漆 L, 并使用 qPCR 确定周期阈值。例子放大结果为损坏和未损坏的底物在右边被说明在红色线代表接受了底漆引伸之前在 qPCR 和蓝色代表脱氧核糖核酸之前在 qPCR 之前未被延伸。<a href="//cloudflare.jove.com/files/ftp_upload/57413/57413fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">表1A</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td rowspan="2">时间</td>
			<td rowspan="2">-底漆延伸</td>
			<td rowspan="2">+ 底漆延伸</td>
			<td rowspan="2">Δ CT</td>
			<td>修复百分比</td>
		</tr>
<tr>
<td>(2ΔCT/23x 100)</td>
		</tr>
<tr>
<td>3小时</td>
			<td>23。5</td>
			<td>21。1</td>
			<td>2。4</td>
			<td>66</td>
		</tr>
<tr>
<td>8小时</td>
			<td>29。4</td>
			<td>26。5</td>
			<td>2。9</td>
			<td>93</td>
		</tr>
<tr>
<td colspan="2">表1B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td rowspan="2">时间</td>
			<td rowspan="2">-底漆延伸</td>
			<td rowspan="2">+ 底漆延伸</td>
			<td rowspan="2">Δ CT</td>
			<td>百分比背景</td>
		</tr>
<tr>
<td>(2ΔCT/23x 100)</td>
		</tr>
<tr>
<td>0小时</td>
			<td>15。4</td>
			<td>14。6</td>
			<td>0。8</td>
			<td>22</td>
		</tr>
<tr>
<td>0小时</td>
			<td>15。4</td>
			<td>12。9</td>
			<td>2。5</td>
			<td>71</td>
		</tr>
</tbody></table>表 1: 使用从 SSPE qPCR 生成的 CT 值计算修复百分比.(A) 修复 V79 中国仓鼠肺成纤维细胞3小时和8小时转染后的含 DPC 基底。(B) SSPE-qPCR 的0个 h 样本未转染到细胞中。在一个样本中, DNA 与 oxoguanine glycosylase 交联反应的效率很高 (这个样本产生了较低的三角洲 CT 值), 而在另一个样品中, 蛋白质交联的效率较低 (这个样本产生了相对较高的三角洲CT 值)。有关详细信息, 请参见代表结果的文本。

Watch this Scientific Journal Video about A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells at JoVE.com

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

本协议的目的是量化在质粒 dna 上对定义的 dna 蛋白 crosslinks 的修复。损伤质粒被转染成受体哺乳动物细胞系和低分子量, 在多时间点后再染。DNA 修复动力学的量化使用链特定引伸后, qPCR。

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

a-simple-rapid-quantitative-assay-to-measure-repair-dna-protein

Universidad San Sebastian

Research

JoVE Journal

Genetics

8.2K 次观看.  University of Minnesota. 本协议的目的是量化在质粒 dna 上对定义的 dna 蛋白 crosslinks 的修复。损伤质粒被转染成受体哺乳动物细胞系和低分子量, 在多时间点后再染。DNA 修复动力学的量化使用链特定引伸后, qPCR。

视频: A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

科研

遗传学

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

多拷贝产质粒在抗生素耐药性演变中的作用试验

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...