Name: atomic force microscopy investigations of dna lesion recognition in nucleotide excision repair
Uploaded: 2017-05-24T14:00:00
Description: Watch this Scientific Journal Video about Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair at JoVE.com

PUC19N，含CPD的寡核苷酸和p44分别由Samuel Wilson，Korbinian Heil和Thomas Carell以及Gudrun Michels和Caroline Kisker提供。这项工作得到德意志民主共和国（DFG）FZ82和TE-671/4向IT部门的资助。

样品制备<ol><li> DNA底物的制备11 <ol><li>在质粒中产生ssDNA间隙<ol><li>在具有合适的切口酶（这里为Nt.BstNBI）的反应管中完全消化质粒样品（这里：修饰的pUC19，pUC19N），然后使用根据制造商的方案的条件进行酶热灭活（参见图1的示意图介绍）。以〜50μL和〜500nM质粒开始，得到足够的产率。 </li><li>通过琼脂糖凝胶电泳验证稀释样品（〜20nM） 15质粒切...

<ol>
	<li>Janicijevic, A., Ristic, D., Wyman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+machines+of+DNA+repair:+scanning+force+microscopy+analysis+of+their+architecture.">The molecular machines of DNA repair: scanning force microscopy analysis of their architecture.</a> J. Microsc. 212 (3), 264-272 (2003).</li><li>Wang, H., 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=DNA+bending+and+unbending+by+MutS+govern+mismatch+recognition+and+specificity.">DNA bending and unbending by MutS govern mismatch recognition and specificity.</a> Proc. Natl. Acad. Sci. USA. 100 (25), 14822-14827 (2003).</li><li>Tessmer, I., 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+of+MutS+searching+for+DNA+mismatches+and+signaling+repair.">Mechanism of MutS searching for DNA mismatches and signaling repair.</a> J. Biol. Chem. 283 (52), 36646-36654 (2008).</li><li>Wagner, K., Moolenaar, G., van Noort, J., Goosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+reveals+two+separate+DNA-binding+domains+in+the+Escherichia+coli+UvrA+dimer.">Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer.</a> Nucleic Acids Res. 37 (6), 1962-1972 (2009).</li><li>Buechner, C. N., 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=Strand-specific+recognition+of+DNA+damages+by+XPD+provides+insights+into+nucleotide+excision+repair+substrate+versatility.">Strand-specific recognition of DNA damages by XPD provides insights into nucleotide excision repair substrate versatility.</a> J Biol. Chem. 289 (6), 3613-3624 (2014).</li><li>Van der Linden, E., Sanchez, H., Kinoshita, E., Kanaar, R., Wyman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAD50+and+NBS1+form+a+stable+complex+functional+in+DNA+binding+and+tethering.">RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.</a> Nucleic Acids Res. 37 (5), 1580-1588 (2009).</li><li>Fuentes-Perez, M. E., Dillingham, M., Moreno-Herrero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM+volumetric+methods+for+the+characterization+of+proteins+and+nucleic+acids.">AFM volumetric methods for the characterization of proteins and nucleic acids.</a> Methods. 60, 113-121 (2013).</li><li>Shlyakhtenko, L. S., Lushnikov, A. Y., Miyagi, A., Lyubchenko, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+binding+of+single-stranded+DNA-binding+protein+to+its+target.">Specificity of binding of single-stranded DNA-binding protein to its target.</a> Biochemistry. 51, 1500-1509 (2012).</li><li>Wirth, N., 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=Conservation+and+Divergence+in+Nucleotide+Excision+Repair+Lesion+Recognition.">Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition.</a> J. Biol. Chem. 291 (36), 18932-18946 (2016).</li><li>Kuper, J., Kisker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+recognition+in+nucleotide+excision+DNA+repair.">Damage recognition in nucleotide excision DNA repair.</a> Curr. Opin. Struct. Biol. 22, 88-93 (2012).</li><li>Buechner, C. N., Tessmer, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+substrate+preparation+for+atomic+force+microscopy+studies+of+protein-DNA+interactions.">DNA substrate preparation for atomic force microscopy studies of protein-DNA interactions.</a> J. Mol. Recognit. 26 (12), 605-617 (2013).</li><li>Sun, Z., Tan, H. Y., Bianco, P. R., Lyubchenko, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+RecG+Helicase+at+the+DNA+Replication+Fork+by+SSB+Protein.">Remodeling of RecG Helicase at the DNA Replication Fork by SSB Protein.</a> Sci. Rep. 5, 9625 (2015).</li><li>Billingsley, D. J., Bonass, W. A., Crampton, N., Kirkham, J., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+studies+of+DNA+transcription+using+atomic+force+microscopy.">Single-molecule studies of DNA transcription using atomic force microscopy.</a> Phys. Biol. 9 (2), 021001 (2012).</li><li>Maurer, S., Fritz, J., Muskhelishvili, G., Travers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+and+an+activator+form+discrete+subcomplexes+in+a+transcription+initiation+complex.">RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex.</a> EMBO J. 25 (16), 3784-3790 (2006).</li><li>Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. 1, (2012).</li><li>Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., Kisker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+UvrB,+a+DNA+helicase+adapted+for+nucleotide+excision+repair.">Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair.</a> EMBO J. 18 (24), 6899-6907 (1999).</li><li>Kuper, 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=In+TFIIH,+XPD+helicase+is+exclusively+devoted+to+DNA+repair.">In TFIIH, XPD helicase is exclusively devoted to DNA repair.</a> PLoS Biol. 12 (9), e1001954 (2014).</li><li>Shlyakhtenko, L. 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=Silatrane-based+surface+chemistry+for+immobilization+of+DNA,+protein-DNA+complexes+and+other+biological+materials.">Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials.</a> Ultramicroscopy. 97, 279-287 (2003).</li><li>Roth, H. M., 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=XPB+helicase+regulates+DNA+incision+by+the+Thermoplasma+acidophilum+endonuclease+Bax1.">XPB helicase regulates DNA incision by the Thermoplasma acidophilum endonuclease Bax1.</a> DNA Repair. 11 (3), 286-293 (2012).</li><li>Ratcliff, G. C., Erie, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Single-Molecule+Study+to+Determine+Protein-Protein+Association+Constants.">A Novel Single-Molecule Study to Determine Protein-Protein Association Constants.</a> J. Am. Chem. Soc. 123 (24), 5632-5635 (2001).</li><li>Yang, Y., Sass, L. E., Du, C., Hsieh, P., Erie, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+protein-DNA+binding+constants+and+specificities+from+statistical+analyses+of+single+molecules:+MutS-DNA+interactions.">Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.</a> Nucleic Acids Res. 33 (13), 4322-4334 (2005).</li><li>Caron, P. R., Grossman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+a+cryptic+ATPase+activity+of+UvrB+and+its+proteolysis+product,+UvrB*+in+DNA+repair.">Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair.</a> Nucleic Acids Res. 16 (22), 10891-10902 (1988).</li><li>Wang, H., 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=UvrB+domain+4,+an+autoinhibitory+gate+for+regulation+of+DNA+binding+and+ATPase+activity.">UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.</a> J. Biol. Chem. 281 (22), 15227-15237 (2006).</li><li>Chammas, O., Billingsley, D. J., Bonass, W. A., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stranded+DNA+loops+as+fiducial+markers+for+exploring+DNA-protein+interactions+in+single+molecule+imaging.">Single-stranded DNA loops as fiducial markers for exploring DNA-protein interactions in single molecule imaging.</a> Methods. 60 (2), 122-130 (2013).</li><li>Truglio, J. 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=Structural+basis+for+DNA+recognition+and+processing+by+UvrB.">Structural basis for DNA recognition and processing by UvrB.</a> Nat. Struct. Mol. Biol. 13 (4), 360-364 (2006).</li><li>Kuper, J., Wolski, S. C., Michels, G., Kisker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+studies+of+the+nucleotide+excision+repair+helicase+XPD+suggest+a+polarity+for+DNA+translocation.">Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation.</a> EMBO J. 31 (2), 494-502 (2012).</li><li>Verhoeven, E. E., Wyman, C., Moolenaar, G. F., Goosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+two+UvrB+subunits+in+the+UvrAB+complex+ensures+damage+detection+in+both+DNA+strands.">The presence of two UvrB subunits in the UvrAB complex ensures damage detection in both DNA strands.</a> EMBO J. 21 (15), 4196-4205 (2002).</li><li>Verhoeven, E. E., Wyman, C., Moolenaar, G. F., Hoeijmakers, J. H., Goosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+nucleotide+excision+repair+complexes:+DNA+is+wrapped+by+UvrB+before+and+after+damage+recognition.">Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition.</a> EMBO J. 20 (3), 601-611 (2001).</li><li>Moolenaar, G. F., 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=The+Role+of+ATP+Binding+and+Hydrolysis+by+UvrB+during+Nucleotide+Excision+Repair.">The Role of ATP Binding and Hydrolysis by UvrB during Nucleotide Excision Repair.</a> J. Biol. Chem. 275, 8044-8050 (2000).</li></ol>

蛋白质在含有特定靶位点的长DNA片段上的结合位置的AFM统计分析可以揭示蛋白质识别这些位点2,3,4,5,6的特定策略的有趣细节。为了解释所得到的位置分布，DNA中靶标的位置需要精确地知道。这是通过将定义明确的序列位置的特定位点引入循环质粒DNA并使用限制酶技术切割包含该特异位点的DNA片段来实现的。在蛋白质结合位置的AFM图像分析中，只有具有正确长度的DNA片段（与切出的片段的全长一致?...

原子力显微镜（AFM）是分析蛋白质 - DNA相互作用1,2,3,4,5,6,7,8,9的有力技术。它只需要少量的样品材料，以单分子水平的分辨率直接观察异质样品。蛋白质的不同构象或寡聚状态可能产生异质性。特别地，在蛋白质-DNA样品的上下文中，蛋白质复合物可以显示通常的DNA结合诱导的不同化学计量学和/或构象，或者结合DNA中的特定靶位点。不均匀的样品也可能含有两种（或多种）不同种类的蛋白质和不同的蛋白质复合物形式（ 例如 ，仅由一种类型的蛋白质与异源复合物组成）可能与DNA不同的相互作用。这里讨论的研究利用空气中的AFM成像技术，将DNA修复蛋白的静态，干燥样品结合到长（〜900个碱基对，bp）的含有病变的DNA片段，其代表这些蛋白质的靶标。 AFM的高分子分辨率允许区分不同类型的蛋白质复合物并确定蛋白质在DNA片段上的结合位置。重要的是，将损伤引入到确定位置的DNA底物中。因为DNA中的病变部位的位置是已知的，所以与DNA结合的蛋白质的分布提供了（不同的）蛋白质复合物的（不同的）病变识别特性的洞察， 例如 ，它们如何识别特定类型的病变（比较对未损伤的DNA） 2，3 ， <supclass =“xref”&gt; 4，5，6 。他们在DNA上的位置也可以区分特异性结合病变的蛋白质复合物和DNA上非特异性结合的复合物。这些不同复杂类型（复合物特异性结合病变与非特异性复合物）的分离表征可以揭示在靶位点鉴定中诱导的复合物中潜在的构象变化。 在这里聚集的DNA修复蛋白是解旋酶，其负责核苷酸切除修复（NER）途径中的病变识别。在细菌中，NER通过蛋白质UvrA，UvrB和UvrC实现。 UvrA负责UvaA 2 / UvrB 2 DNA扫描复合物中的初始病变感测。通过UvrB的病变验证，该复合物转化为在病变部位结合的单体UvrB，然后该特异性复合物可以招募p核型NER核酸内切酶UvrC。 UvrC切除含有病变的短（12-13nt）单链DNA（ssDNA）。然后通过DNA聚合酶重新填充缺失的片段。最后，DNA连接酶用原始DNA 9,10密封新合成的片段。在真核生物中，NER级联的大多数蛋白质是大的多聚体转录因子II H（TFIIH）复合物的一部分。 通过三聚体CEN2-XPC-HR23B复合物进行初始病变感染后，TFIIH被引入DNA靶位点。当复合物中的XPD验证NER靶病变的存在时，招募真核NER核酸内切酶XPG和XPF以消除含有病变9,10的短（24-32nt）片段的ssDNA。具体来说，研究了分离自原核和真核NER的解旋酶UvrB和XPD。这些解剖器需要不配对的区域DNA（DNA气泡）连接到两条DNA单链之一上，随后沿着ATP水解促进的该链转移。除DNA损伤之外，DNA泡也被引入作为蛋白质负载位点的底物中。 以前已经描述了用于制备特定病变DNA底物的方法11 。它需要一个循环DNA构建体（质粒），其具有用于切口酶的两个紧密间隔的限制性位点。在本研究的上下文中，使用质粒pUC19N（2729bp）（由S.Wilson的实验室，NIEHS创建）。该质粒含有三个紧密间隔的限制性位点，用于构建48个核苷酸（nt）延伸的切口酶Nt.BstNBI。与切口酶孵育后，可以除去这些位点之间的ssDNA片段，并用含有任何靶标特征的寡核苷酸代替。在每个步骤之后，通过琼脂糖凝胶测试完全酶消化电泳。由于与原来的超螺旋质粒相比，镍环状DNA的电泳迁移率较低，因此可以区别。通过用特异性底物寡核苷酸替代除去的拉伸的替代物可以通过限制酶进行消化来进行评估，所述限制酶将底物专门在切口之间的区域内切割。因此，通过酶对圆形质粒的线性化将被抑制，并且在插入特异性寡核苷酸后恢复。最后，两个核酸内切酶限制性位点（理想的是单个切割器）允许产生线性DNA底物，其长度根据需要和特定的靶位点在确定的位置以及与病变相距一定距离的DNA气泡&#39;或3&#39;方向。 可以通过 AFM成像来检查NER解旋酶对病变的识别。在t处的解旋酶失调的DNA易位他的病变部位作为DNA上的蛋白质位置分布的峰值可见，表明病变识别。因为这些解旋酶的DNA易位进一步是定向的，具有5&#39;至3&#39;极性，病变识别对加载位点的位置（病毒上游或下游的DNA气泡）的依赖性还指示是否优先识别病变在易位或相对的非易位的ssDNA链5,9上 。在以下部分中，将介绍所使用的方法，并简要讨论这些实验的主要发现。重要的是，类似于本文所示的DNA修复的示范性工作，AFM成像可以应用于不同DNA相互作用系统的研究，例如DNA复制或转录8,12,13,14 </SUP&gt;。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Molecular Force Probe (MFP) 3D</td>
			<td>Asylum Research</td>
			<td>N/A</td>
			<td>atomic force microscope (AFM)</td>
		</tr>
		<tr>
			<td>Precision 390</td>
			<td>DELL</td>
			<td>N/A</td>
			<td>computer</td>
		</tr>
		<tr>
			<td>ThermoMixer and 1.5 ml block</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>heat block for DNA preparation</td>
		</tr>
		<tr>
			<td>Rotilabo Block-Heater H 250 &#38; blocks for 0.5 ml tubes</td>
			<td>Carl Roth GmbH</td>
			<td>Y264.1 &#38; Y267.1</td>
			<td>heat block for protein-DNA incubations</td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT</td>
			<td>Bio-Rad Laboratories GmbH</td>
			<td>1704467</td>
			<td>electrophoresis chamber with gel caster and power supply</td>
		</tr>
		<tr>
			<td>Power Pac Basic</td>
			<td>Bio-Rad Laboratories GmbH</td>
			<td>1645050</td>
			<td>electrophoresis power supply</td>
		</tr>
		<tr>
			<td>Centifuge 5415 D with rotor</td>
			<td>Eppendorf</td>
			<td>2262120-3</td>
			<td>table centrifuge</td>
		</tr>
		<tr>
			<td>Ultra-Lum electronic UV transillumonator MEB-15</td>
			<td>Ultralum</td>
			<td>900-1322-02</td>
			<td>UV irradiation table</td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000</td>
			<td>VWR International / PEQLAB Biotechnologie GmbH</td>
			<td>N/A</td>
			<td>UV spectrophotometer</td>
		</tr>
		<tr>
			<td>TKAX-CAD with 0.2 &#956;m capsule filter</td>
			<td>Unity Lab Services</td>
			<td>N/A</td>
			<td>water deionization and filter unit</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MFP software on Igor Pro</td>
			<td>Asylum Research</td>
			<td>N/A</td>
			<td>AFM software</td>
		</tr>
		<tr>
			<td>ImageJ (open source Java image processing)</td>
			<td>NIH Image</td>
			<td>N/A</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Excel (Microsoft Office)</td>
			<td>Microsoft Corporation</td>
			<td>N/A</td>
			<td>data analysis software</td>
		</tr>
		<tr>
			<td>Origin9 / Origin2016</td>
			<td>OriginLab Corporation</td>
			<td>N/A</td>
			<td>statistical data analysis and graphing software</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OMCL-AC240TS</td>
			<td>Olympus</td>
			<td>OMCL-AC240TS</td>
			<td>AFM cantilevers</td>
		</tr>
		<tr>
			<td>grade V-5 muscovite</td>
			<td>SPI Supplies</td>
			<td>1805</td>
			<td>mica sheets</td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5ml 50k Ultracell</td>
			<td>Millipore Ireland Ltd.</td>
			<td>UFC505096</td>
			<td>centrifuge filters</td>
		</tr>
		<tr>
			<td>NucleoSpin Extract II&#160;</td>
			<td>&#160;Macherey-Nagel GmbH</td>
			<td>740 609.250</td>
			<td>Agarose gel extraction kit</td>
		</tr>
		<tr>
			<td>Rotilabo cellulose paper type 111A</td>
			<td>Carl Roth GmbH</td>
			<td>AP59.1</td>
			<td>AFM deposition blotting paper</td>
		</tr>
		<tr>
			<td>Anatop 25 (0.02 &#956;m)</td>
			<td>Whatman GmbH</td>
			<td>6809-2102</td>
			<td>syringe filter</td>
		</tr>
		<tr>
			<td>SSpI, BspQI</td>
			<td>New England Biolabs (NEB)</td>
			<td>R0132, R0712</td>
			<td>restriction enzymes for DNA substrate preparation</td>
		</tr>
		<tr>
			<td>XhoI, BglII</td>
			<td></td>
			<td>R0146, R0144</td>
			<td>restriction enzymes for DNA preparation controls</td>
		</tr>
		<tr>
			<td>nicking restriction enzyme Nt.BstNBI</td>
			<td>New England Biolabs (NEB)</td>
			<td>R0607</td>
			<td>nickase</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs (NEB)</td>
			<td>M0202S</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>Tris, HEPES</td>
			<td>Carl Roth GmbH</td>
			<td>4855, 9105</td>
			<td>buffer chemicals</td>
		</tr>
		<tr>
			<td>NaCl, MgCl2, KCl, MgAcetate</td>
			<td>Carl Roth GmbH</td>
			<td>3957, HN03, HN02, P026</td>
			<td>salt chemicals</td>
		</tr>
		<tr>
			<td>NaAc</td>
			<td>Sigma-Aldrich Chemie GmbH</td>
			<td>32318</td>
			<td>salt chemicals</td>
		</tr>
		<tr>
			<td>DTT, TCEP, EDTA</td>
			<td></td>
			<td>6908, HN95, 8040</td>
			<td>chemicals/reagents</td>
		</tr>
		<tr>
			<td>agarose, acetic acid, HCl</td>
			<td>Carl Roth GmbH</td>
			<td>2267, 3738, K025</td>
			<td>reagents</td>
		</tr>
		<tr>
			<td>ATP&#404;S</td>
			<td>Jena Bioscience</td>
			<td>NU-406</td>
			<td>nucleotides</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Carl Roth GmbH</td>
			<td>K054</td>
			<td>nucleotides</td>
		</tr>
		<tr>
			<td>oligonucleotide #1 in Table 1</td>
			<td>Biomers</td>
			<td>custom</td>
			<td>complementary DNA oligonucleotide</td>
		</tr>
		<tr>
			<td>oligonucleotides #2, #3, and #6 in Table 1</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>custom</td>
			<td>fluorescein containing oligonucleotides</td>
		</tr>
		<tr>
			<td>oligonucleotides #4&#160; and #5 in Table 1</td>
			<td>private (available from e.g. TriLink or GlenResearch)</td>
			<td></td>
			<td>CPD containing oligonucleotides</td>
		</tr>
		<tr>
			<td>SafeSeal reaction tube 0.5 ml and 1.5 ml</td>
			<td>Sarstedt</td>
			<td>72.704 and 72.706</td>
			<td>incubation tubes</td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb</td>
			<td>Thermo Scientific</td>
			<td>SM0311</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>6x concentrate gel loading dye purple</td>
			<td>New England Biolabs (NEB)</td>
			<td>51406</td>
			<td>DNA loading dye</td>
		</tr>
		<tr>
			<td>Midori Green&#160;</td>
			<td>Nippon Genetics Europe GmbH</td>
			<td>999MG28055</td>
			<td>DNA stain</td>
		</tr>
	</tbody>
</table>

atomic force microscopy investigations of dna lesion recognition in nucleotide excision repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

基于蛋白质复合体积区分不同复杂类型原核NER解旋酶UvrB的解旋酶活性受DNA结合22,23刺激。 UvrB需要DNA中的未配对区域（DNA气泡）才能正确地加载到两条单链DNA链之一上。 在体内 ，该DNA结构通过NER级联的另一种蛋白质的DNA相互作用提供;在体外实验中，气泡可以人造地引入到与目标病变附近的dsDNA片?...

Watch this Scientific Journal Video about Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair at JoVE.com

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

原子力显微镜检查DNA损伤识别核苷酸切除修复

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

The overall goal of this atomic force microscopy experiment is to resolve interactions of DNA repair proteins with damage sites in DNA and to distinguish between different DNA interactions by various protein complexes. This method can help answer key questions in the DNA repair field, such as how a particular DNA repair system achieves the recognition of its target lesions in the DNA. The main advantage of this technique is that we can directly see and analyze the different types of complexes that are involved in the DNA lesion recognition process.Demonstrating the procedure will be Nicolas Wirth, who used this approach during his bachelor research. To begin, prepare a suitable DNA substrate by generating a single-stranded DNA gap in a plasmid. Refill the gap with modified, single-stranded DNA oligonucleotides, and finally, linearize the plasma DNA using appropriate restriction enzymes.For these experiments, the double-stranded DNA substrates are about 1, 000 base pairs long and contain a DNA lesion as well as an unpaired region called a DNA bubble for protein loading onto the DNA. Next, prepare a protein DNA sample for AFM analysis by first pipetting 10x Reaction Buffer and water into a microreaction tube. Then, add the DNA and proteins under investigation.For example, here, 100 nanomolar of the DNA sample and one micromolar or each of the eukaryotic nucleotide excision repair proteins XPD and p44. Then, incubate the reaction for 30 minutes at 37 degrees Celsius in a heat block. Following the incubation, briefly spin down the sample.Next, prepare a mica substrate to place the sample onto. Use a scalpel to cut a one-centimeter by one-centimeter piece of mica from a larger strip. Then, use a piece of adhesive tape to remove the top layers of the mica to reveal a clean, flat, anatomically smooth substrate surface.Dilate the protein DNA sample fifty to a hundredfold in AFM deposition buffer, and immediately deposit 20 microliters of the diluted sample onto the mica surface. The dilution factor depends on the sample concentration. Immediately after depositing the sample on the mica surface, rinse the sample three to four times with a few milliliters of filtered, deionized water and blot off excess liquid.Use a gentle stream of nitrogen to blow dry the sample. Finally, use adhesive tape to fix the sample at its edges onto a microscope slide. Place the sample in the middle of the AFM stage and use magnetic pads to fix the microscope slide on the stage.Then, insert the AFM tip into the tip holder. Fix the tip in the holder under a clamp by tightening the screw so that it is finger tight. Once secured, enter the tip holder into the AFM measurement head.Use the positioning screws to move the sample in a central position. Place the AFM measurement head on top of the sample, making sure the head stands stably with its legs inside the stage indentations. Next, align the AFM laser on the back of the cantilever for optimal signal strength.Watch the reflection signal in the AFM video window to crudely position the laser. For this AFM model, turn the wheels of the right side and back of the AFM measurement head to adjust the X and Y positions of the AFM laser in order to direct it centrally onto the end of the cantilever. Then, optimize the detector sum signal by fine-tuning the laser position with the two wheels.Direct the AFM laser reflection onto the detector center by zeroing the different signal from the top and bottom diodes of the detector array. For this step, turn the screw on the left-side of the measurement head for this AFM model. Next, determine the cantilever residence frequency by going into the master panel tune window in the software.Choose an amplitude corresponding to a one-volt input for the Piezo that drives the cantilever oscillation and then set the oscillation frequency to negative 5%slightly lower than the residence frequency. Now perform an auto frequency tune and zero the phase of the oscillation. Leave the system to reach thermal equilibrium for about one hour before you start imaging, in order to avoid drift.When you are ready to start imaging, in the software main panel, set a protecting set point and prepare to approach the tip to the sample surface by selecting engage. Lower the front wheel of the AFM head manually until the protective setting, or set point, is reached. Then, carefully lower the front wheel further until the Z sensor signal is fully extended to the left.Activate the vibration oscillation table and close the acoustic isolation enclosure. Next, lower the set point in the AFM software using the hamster wheel to fine engage the AFM tip with the sample. To perform tapping mode imaging in the repulsive regime, lower the set point until the phase signal becomes slightly lower than the free level phase.Then, begin scanning the sample. Use the scan speed of 2.5 micrometers per second in image surface areas of four micrometer by four micrometer or eight micrometer by eight micrometer squares with pixel resolutions of 2, 048 or 4, 096, respectively;and press the Do Scan button. Preselect relevant protein DNA complexes by direct visual inspection of the images in the AFM software.For XPD samples, possible complexes include XPD p44 DNA, as well as XPD DNA and p44 DNA with distinctly different sizes due to the different molecular masses of XPD and p44. Measure volumes of individual protein peaks on the DNA by using the section tool in the analysis window to measure the height and diameter of the protein peak sections with the section window cursors. Use these values to determine the volume of the peaks using simple mathematical models to verify the complex type.Finally, calibrate your AFM system with a range of proteins with known molecular weights to translate these volumes into approximate molecular weights of proteins and protein complexes. In order to determine additional features, such as DNA fragment lengths, the positions of protein complexes on the DNA and their target-site specificities or DNA band angles, see the accompanying text protocol. Shown here is an AFM image with three different types of DNA-bound complexes that can be distinguished based on their different sizes.Class one complexes are consistent with only the helicase activating co-factor p44, class two complexes consist of only the XPD protein, and class three complexes consist of both XPD and p44 as legion recognition by XPD requires the activation of its helicase activity by p44, and only the class three complexes show lesion recognition. In order to identify different lesion recognition strategies, the locations of proteins on the DNA substrates were measured for protein loading at a DNA bubble in either the five prime or three prime directions from a DNA lesion shown in red for prokaryotic and eukaryotic NER helicases. In the protein position distributions, DNA lesion recognition can be seen as a peak due to enhanced binding at the DNA lesion position.The prokaryotic NER helicase UVRB and its eukaryotic counterpart, XPD, show different strategies of CPD lesion recognition that indicate different DNA strand preferences. Specifically, CPD lesions are recognized on the translocated strand by UVRB, but preferentially on the opposite, non-translocated strand by XPD. Once mastered, this method can be done in a few hours or days including analysis.It's important to work with highly pure protein and DNA samples to be able to interpret the features in the AFM images and to carefully design the DNA substrate depending on the biological system and on the question you want to address. In addition, ensemble methods, such as gel electrophoresis space, electromobility shift, or DNA incision assays are, of course, highly complementary to these AFM experiments;and these methods can help address additional questions, such as on DNA binding affinities or DNA lesion excision activities. This technique is a relatively fast and simple approach to directly visualize protein DNA complexes, and these complexes give us a good picture of the processes that occur in a biological system.Of course, this is not only useful in understanding DNA repair processes, but it can also be applied to other protein DNA systems. After watching this video, you should have a pretty good idea of how to prepare a sample on your favorite protein DNA system, how to image it by AFM, and of how to extract useful parameters for an interpretation of your data.

Education

Research

JoVE Core

JoVE Journal

Molecular Biology

Biology

Genetics

Unit 1

DNA Structure and Function

DNA Repair and Recombination

SCIENTISTS IN ACTION

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

科研

遗传学

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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

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 ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...