PUC19N, CPD-haltige Oligonukleotide und p44 wurden freundlicherweise von Samuel Wilson, Korbinian Heil und Thomas Carell und Gudrun Michels bzw. Caroline Kisker bereitgestellt. Diese Arbeit wurde durch Stipendien der Deutschen Forschungsgemeinschaft (DFG) FZ82 und TE-671/4 an IT unterstützt.

<ol>
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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. 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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. 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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>

Die Autoren haben nichts zu offenbaren.

AFM statistische Analysen von Bindungspositionen von Proteinen auf langen DNA-Fragmenten, die spezifische Zielstellen enthalten, können interessante Details über die speziellen Strategien des Proteins zeigen, um diese Stellen 2 , 3 , 4 , 5 , 6 zu erkennen. Um die daraus resultierenden Positionsverteilungen zu interpretieren, müssen die Positionen der Targe...

Atomkraftmikroskopie (AFM) ist eine leistungsfähige Technik für die Analyse der Protein-DNA-Wechselwirkungen 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Es erfordert nur geringe Mengen an Probenmaterial, um heterogene Proben direkt mit einer Auflösung auf Einzelmolekülebene zu visualisieren. Heterogenität kann aus verschiedenen konformationellen oder oligomeren Zuständen eines Proteins resultieren. Insbesondere können im Rahmen von Protein-DNA-Proben Proteinkomplexe unterschiedliche Stöchiometrien und / oder Konformationen aufweisen, die durch DNA-Bindung im Allgemeinen induziert werden oder an eine spezifische Zielstelle innerhalb der DNA binden. Heterogene Proben können auch zwei (oder mehrere) verschiedene Arten von Proteinen und verschiedene Proteinkomplexe enthaltenFormen ( zB bestehend aus nur einer Art von Protein gegenüber heteromeren Komplexen) können unterschiedlich mit DNA interagieren. Die hier diskutierten Studien nutzten die AFM-Bildgebung in der Luft auf statischen, getrockneten Proben von DNA-Reparaturproteinen, die an lange (~ 900 Basenpaare, bp) DNA-Fragmente gebunden sind, die eine Läsion enthalten, die ein Ziel dieser Proteine ​​darstellt. Die hohe molekulare Auflösung von AFM ermöglicht die Unterscheidung zwischen verschiedenen Arten von Proteinkomplexen und die Bestimmung der Bindungspositionen der Proteine ​​an den DNA-Fragmenten. Wichtig ist, dass die Läsionen in gut definierten Positionen in die DNA-Substrate eingebracht werden. Da die Position der Läsionsstelle in der DNA bekannt ist, liefern die Verteilungen von Proteinen, die an DNA gebunden sind, einen Einblick in (verschiedene) Läsionserkennungseigenschaften der (verschiedenen) Proteinkomplexe, zB wie gut sie eine bestimmte Art von Läsion erkennen (verglichen Zu nicht beschädigter DNA) 2 , 3 , <supClass = &quot;xref&quot;&gt; 4 , 5 , 6 . Ihre Positionen auf der DNA erlauben auch die Unterscheidung zwischen Proteinkomplexen, die spezifisch an den Läsionen und Komplexen gebunden sind, die unspezifisch anderswo an der DNA gebunden sind. Eine getrennte Charakterisierung dieser verschiedenen komplexen Typen (Komplexe, die spezifisch an der Läsion gegenüber unspezifischen Komplexen gebunden sind) können potentielle Konformationsänderungen in den Komplexen zeigen, die bei der Zielortidentifizierung induziert werden. Die DNA-Reparaturproteine, die hier konzentriert sind, sind Helicasen, die für die Läsionserkennung in der Nucleotide Exzision Reparatur (NER) Weg verantwortlich sind. In Bakterien wird NER durch die Proteine ​​UvrA, UvrB und UvrC erreicht. UvrA ist verantwortlich für die initiale Läsionserfassung in einem UvaA 2 / UvrB 2 DNA-Scan-Komplex. Nach der Läsionsüberprüfung durch UvrB wandelt sich dieser Komplex in monomeres UvrB um, das an der Läsionsstelle gebunden ist, und dieser spezifische Komplex kann dann das p rekrutierenRokaryotische NER Endonuklease UvrC. UvrC verbreitet eine kurze (12-13 nt) Strecke einzelsträngiger DNA (ssDNA), die die Läsion enthält. Die fehlende Dehnung wird dann durch DNA-Polymerase nachgefüllt. Schließlich versiegelt die DNA-Ligase die neu synthetisierte Dehnung mit der ursprünglichen DNA 9 , 10 . Bei Eukaryonten sind die meisten Proteine ​​der NER-Kaskade Teil des großen, multimeren Transkriptionsfaktors II H (TFIIH) -Komplexes. Nach der ersten Läsionserfassung über den trimeren CEN2-XPC-HR23B-Komplex wird TFIIH an die DNA-Zielstelle rekrutiert. Wenn XPD innerhalb des Komplexes die Anwesenheit einer NER-Zielläsion verifiziert, werden die eukaryotischen NER-Endonukleasen XPG und XPF rekrutiert, um eine kurze (24-32 nt) Strecke von ssDNA, die die Läsion 9 , 10 enthält , zu exzimieren. Hier wurden insbesondere die Helicasen UvrB und XPD aus prokaryotischem und eukaryotischem NER untersucht. Diese Helicases erfordern eine ungepaarte Region in derDNA (eine DNA-Blase), um auf einen der beiden DNA-Einzelstränge zu fädeln und anschließend entlang diesem durch ATP-Hydrolyse angefeuerten Strang zu translozieren. Zusätzlich zu den DNA-Läsionen wurde daher eine DNA-Blase in die Substrate eingeführt, die als Ladestelle für die Proteine ​​fungieren. Das Verfahren zur Herstellung von spezifischen Läsions-DNA-Substraten wurde zuvor beschrieben 11 . Es erfordert ein kreisförmiges DNA-Konstrukt (Plasmid) mit zwei eng beabstandeten Restriktionsstellen für eine Nickase. Im Rahmen dieser Studie wurde das Plasmid pUC19N (2729 bp) verwendet (erstellt von S. Wilsons Laboratorium, NIEHS). Dieses Plasmid enthält drei eng beabstandete Restriktionsstellen für die Nickel Nt.BstNBI, die eine 48 Nukleotid (nt) Streckung bilden. Nach der Inkubation mit der Nickel kann die Strecke der ssDNA zwischen diesen Stellen entfernt und durch ein Oligonukleotid ersetzt werden, das ein beliebiges Zielmerkmal enthält. Nach jedem Schritt wird die vollständige enzymatische Verdauung über Agarosegel getestetElektrophorese Die vernetzte zirkuläre DNA kann aufgrund ihrer geringeren elektrophoretischen Beweglichkeit gegenüber dem ursprünglichen supercoiled Plasmid unterschieden werden. Das Anhängen der DNA und das Ersetzen der entfernten Dehnung durch das spezifische Substrat-Oligonukleotid können durch Verdauung mit einem Restriktionsenzym ausgewertet werden, das das Substrat ausschließlich innerhalb des Bereichs zwischen den Nicks aufnimmt. Die Linearisierung des zirkulären Plasmids durch das Enzym wird daher für die gapped DNA unterdrückt und nach der Insertion des spezifischen Oligonukleotids wiederhergestellt. Schließlich erlauben zwei Endonuklease-Restriktionsstellen (idealerweise Einzelschneider) die Erzeugung eines linearen DNA-Substrats mit der gewünschten Länge und mit der spezifischen Zielstelle an einer definierten Position sowie einer DNA-Blase in einem Abstand von der Läsion entweder in 5 &#39;Oder 3&#39; Richtung. Die Erkennung der Läsionen durch die NER-Helicasen kann mittels AFM-Bildgebung untersucht werden . Stabile DNA-Translokation der Helicasen bei tEr Läsionsstelle ist als Peak in der Proteinpositionsverteilung auf der DNA sichtbar und zeigt eine Läsionserkennung an. Da die DNA-Translokation dieser Helicinen weiterhin mit einer 5&#39;-bis-3&#39;-Polarität gerichtet ist, zeigt die Abhängigkeit der Läsionserkennung an der Position der Ladestelle (DNA-Blase stromaufwärts oder stromabwärts der Läsion) auch an, ob die Läsion bevorzugt erkannt wird Auf dem translozierten oder auf dem entgegengesetzten, nicht translozierten ssDNA-Strang 5 , 9 . In den folgenden Abschnitten werden die angewandten Methoden eingeführt und wichtige Erkenntnisse aus diesen Experimenten werden kurz diskutiert. Wichtig ist, dass analog zur beispielhaften Arbeit zur hier beschriebenen DNA-Reparatur die AFM-Bildgebung auf die Untersuchung verschiedener DNA-interagierender Systeme wie der DNA-Replikation oder der Transkription 8 , 12 , 13 , 14 angewendet werden kann </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.

Unterscheidung zwischen verschiedenen komplexen Typen auf der Basis von Protein komplexen Volumina Die Helicaseaktivität der prokaryotischen NER-Helicase UvrB wird durch DNA-Bindung 22 , 23 stimuliert. UvrB benötigt eine ungepaarte Region in der DNA (eine DNA-Blase), um korrekt auf einen der beiden einzelnen ssDNA-Stränge zu laden. In vivo wird diese DNA-Struk...

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

Rasterkraftmikroskopische Untersuchungen zur Erkennung von DNA-Läsionen bei der Reparatur von Nukleotid-Exzisionen | Genetik | Video

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.

Atommikroskopie Untersuchungen zur DNA-Läsionserkennung bei der 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 ...

atomic-force-microscopy-investigations-dna-lesion-recognition

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Unit 1

DNA Structure and Function

DNA Repair and Recombination

SCIENTISTS IN ACTION

9.3K Aufrufe.  University of Würzburg. 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.

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

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

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

Der Visual kolorimetrischen Nachweis von Multi-Nukleotid-Polymorphismen auf einem pneumatischen Droplet Manipulation Platform

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

Forschung

Genetik

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

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

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

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ Labeling der mitochondrialen DNA - Replikation in Drosophila Adult Ovarien von EdU Anfärben

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

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

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

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Kinetik der Verzögerungs-DNA-Synthese<em&gt; In Vitro</em&gt; Von den Bakteriophagen T7 Replikationsproteine

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

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

Bewertung der Kontamination der DNA in RNA-Proben anhand der ribosomalen DNA

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 is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

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

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genom-weite Abbildung von Protein-DNA-Wechselwirkungen mit ChEC-seq in<em&gt; 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

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

Sequenz-spezifische und gezielte Anerkennung der Doppelstrang-RNA über Single-Stranded RNA durch chemisch modifiziert Peptid-Nukleinsäuren

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

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

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

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

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA-Replikation Timing mit Zebrafisch als ein In Vivo Modell-System-Profiling

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

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 mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

Ein einfacher, rasches und quantitativen Assay zur Maßnahme Reparatur von DNA-Protein-Querverbindungen auf Plasmide transfiziert in Säugerzellen

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

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Methodik für die präzise Erkennung der mitochondrialen DNA-Methylierung

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

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

Verwendung von gefrorenem Gewebe im Kometentest zur Bewertung von DNA-Schäden

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Atommikroskopie Untersuchungen zur DNA-Läsionserkennung bei der Nucleotide Excision Repair

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