Name: deciphering molecular mechanism of histone assembly by dna curtain technique
Uploaded: 2022-03-09T14:30:00
Description: Watch this Scientific Journal Video about Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique at JoVE.com

The authors appreciate the kind support for Abo1 and Cy5-H3-H4 by Professor Ji-Joon Song, Carol Cho, Ph.D., and Juwon Jang, Ph.D., in KAIST, South Korea. This work is supported by the National Research Foundation Grant (NRF-2020R1A2B5B01001792), intramural research fund (1.210115.01) of Ulsan National Institute of Science and Technology, and the Institute for Basic Science (IBS-R022-D1).

1. Preparation of the flow cell
<ol>
	<li>Prepare a cleaned fused silica slide containing nano-trench patterns following previously published report25.
	<ol>
		<li>Drill two holes with 1 mm diameter in a cleaned fused silica slide (Figure 1A) using a diamond-coated drill bit (see Table of Materials).</li>
		<li>Deposit 250 nm of thick aluminum (Al) on the slide using DC sputter32 (see Table of Materia...

As a single-molecule imaging technique, DNA curtain has been used extensively to probe DNA metabolic reactions43. DNA curtain is a hybrid system that concatenates lipid fluidity, microfluidics, and TIRFM. Unlike other single-molecule techniques, DNA curtain enables high-throughput real-time visualization of protein-DNA interactions. Therefore, the DNA curtain technique is suitable for probing the mechanism behind molecular interactions, including sequence-specific association, protein movement alo...

Eukaryotic DNA is packaged into a higher-order structure known as chromatin1,2. Nucleosome is the fundamental unit of chromatin, which consists of approximately 147 bp DNA wrapped around the octameric core histones3,4. Chromatin plays a critical role in eukaryotic cells; for example, the compact structure protects DNA from endogenous factors and exogenous threats5. Chromatin structure changes dynamically according to the cell cycle phase and environmental stimuli, and these changes control protein access during DNA transactions such as replication, transcription, and repair6. Chromatin dynamics are also important for genomic stability and epigenetic information.
Chromatin is dynamically regulated by various factors, including histone tail modifications and chromatin organizers such as chromatin remodelers, polycomb group proteins, and histone chaperones7. Histone chaperones coordinate the assembly and disassembly of nucleosomes via deposition or detachment of core histones8,9. Defects in histone chaperones induce genome instability and cause developmental disorders and cancer9,10. Various histone chaperones do not need chemical energy consumption like ATP hydrolysis to assemble or disassemble nucleosomes9,11,12,13. Recently, researchers reported that bromodomain-containing AAA+ (ATPase associated with diverse cellular activities) ATPases play a role in chromatin dynamics as histone chaperones14,15,16,17. Human ATAD2 (ATPase family AAA domain-containing protein 2) promotes chromatin accessibility to enhance gene expression18. As a transcriptional co-regulator, ATAD2 regulates the chromatin of oncogenic transcriptional factors14, and the overexpression of ATAD2 is related to poor prognosis in many types of cancer19. Yta7, the Saccharomyces cerevisiae (S. cerevisiae) homolog of ATAD2, decreases nucleosome density in chromatin15. In contrast, Abo1, the Schizosaccharomyces pombe (S. pombe) homolog of ATAD2, increases nucleosome density16. Using a unique single-molecule imaging technique, DNA curtain, whether Abo1 contributes to nucleosome assembly or disassembly is addressed17,20.
Traditionally, the biochemical properties of biomolecules have been examined by bulk experiments such as the electrophoretic mobility shift assay (EMSA) or co-immunoprecipitation (co-IP), in which a large number of molecules are probed, and their average properties are characterized21,22. In bulk experiments, molecular sub-states are veiled by the ensemble-average effect, and probing biomolecular interactions is restricted. In contrast, single-molecule techniques circumvent the limitations of bulk experiments and enable the detailed characterization of biomolecular interactions. In particular, single-molecule imaging techniques have been widely used to study DNA-protein and protein-protein interactions23. One such technique is DNA curtain, a unique single-molecule imaging technique based on microfluidics and total internal reflection fluorescence microscopy (TIRFM)24,25. In a DNA curtain, hundreds of individual DNA molecules are anchored to the lipid bilayer, which permits the two-dimensional motion of DNA molecules due to lipid fluidity. When hydrodynamic flow is applied, DNA molecules move along the flow on the bilayer and get stuck at a diffusion barrier, where they are aligned and stretched. While DNA is stained with intercalating agents, fluorescently labeled proteins are injected, and TIRFM is used to visualize protein-DNA interactions in real-time at a single-molecule level23. The DNA curtain platform facilitates the observation of protein movements such as diffusion, translocation, and collision26,27,28. Moreover, DNA curtain can be used for protein mapping on DNA with defined positions, orientations, and topologies or applied to the study of phase separation of protein and nucleic acids29,30,31.
In this work, the DNA curtain technique is used to provide evidence for the function of chaperones through direct visualization of specific proteins. Moreover, because DNA curtain is a high-throughput platform, it facilitates an extent of data collection sufficient for statistical reliability. Here, it is described how to conduct the DNA curtain assay in detail to investigate the molecular role of S. pombe bromodomain-containing AAA+ ATPase Abo1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL luer-lock syringe</td>
			<td>BecktonDickinson</td>
			<td>301321</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#39; x 3&#39; fused-silca slide glass</td>
			<td>G. Finkenbeiner</td>
			<td></td>
			<td>1 inch x 3 inch rectangular and 1 mm thickness</td>
		</tr>
		<tr>
			<td>10 mL luer-lock syringe</td>
			<td>BecktonDickinson</td>
			<td>302149</td>
			<td></td>
		</tr>
		<tr>
			<td>18:1 (&#916;9-Cis) PC (DOPC)</td>
			<td>Avanti</td>
			<td>850375</td>
			<td>This is a component of biotinylated lipid stock</td>
		</tr>
		<tr>
			<td>18:1 Biotinyl cap PE</td>
			<td>Avanti</td>
			<td>870273</td>
			<td>This is a component of biotinylated lipid stock</td>
		</tr>
		<tr>
			<td>18:1 PEG2000 PE</td>
			<td>Avanti</td>
			<td>880130</td>
			<td>This is a component of biotinylated lipid stock</td>
		</tr>
		<tr>
			<td>3 mL luer-lock syringe</td>
			<td>BecktonDickinson</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>6-way sample injection valve</td>
			<td>IDEX</td>
			<td>MX series II</td>
			<td></td>
		</tr>
		<tr>
			<td>950K PMMA</td>
			<td>All-resist</td>
			<td>671.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SAMCHUN</td>
			<td>A1759</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum (Al)</td>
			<td>TASCO, South Korea</td>
			<td>LT50AI414</td>
			<td>Diameter 4 inch, thickness 1/4 inch</td>
		</tr>
		<tr>
			<td>Amicon Ultra centrifugal filter, MWCO 10 kDa</td>
			<td>Millipore</td>
			<td>Z648027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Mbcell</td>
			<td>MB-A4128</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>AZ 300 MIF developer</td>
			<td>Merck</td>
			<td>10454110521</td>
			<td>Used for removing aluminum</td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>DORCO</td>
			<td>DN52</td>
			<td>12 mm x 6 m</td>
		</tr>
		<tr>
			<td>Boron trichloride (BCl3)</td>
			<td>UNIONGAS</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C40-1g</td>
			<td>This is a component of 100x gloxy stock</td>
		</tr>
		<tr>
			<td>Chlorine (Cl2)</td>
			<td>UNIONGAS</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>Clear double-sided tape</td>
			<td>3M</td>
			<td>313770</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>DC sputter</td>
			<td>Sorona</td>
			<td>SRM-120</td>
			<td>Used for deposition aluminum on a slide</td>
		</tr>
		<tr>
			<td>Diamond-coated drill bit</td>
			<td>Eurotool</td>
			<td>DIB-211.00</td>
			<td>Used for making holes in a fusced silica slide</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Dove-prism</td>
			<td>Korea Electro-Optics Co. Ltd.</td>
			<td>1906-106</td>
			<td>Custom-made fused-silica dove prism with anti-reflection coating</td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>Dremel 3000</td>
			<td>Used for making holes in a fusced silica slide</td>
		</tr>
		<tr>
			<td>Electron Bean Lithography</td>
			<td>Nanobeam Ltd.</td>
			<td>NB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene-diamine-tetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fingertight fittings</td>
			<td>IDEX</td>
			<td>F-300</td>
			<td>It is connected with &#34;PFA Tubing Natural&#34; to form luer-lock tubing</td>
		</tr>
		<tr>
			<td>Flangeless male nut</td>
			<td>IDEX</td>
			<td>P-235</td>
			<td>It is connected with &#34;PFA Tubing Natural&#34; to form luer-lock tubing</td>
		</tr>
		<tr>
			<td>Freeze Dryer, HyperCOOL</td>
			<td>Labogene</td>
			<td>HC3110</td>
			<td>Used for lyophilizing liquid proteins</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133-50KU</td>
			<td>This is a component of 100x gloxy stock</td>
		</tr>
		<tr>
			<td>Guanidinium hydrochloride</td>
			<td>Acros Organics</td>
			<td>364790025</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton Company</td>
			<td>80065</td>
			<td>This syringe is used for sample injection</td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 26/600 SuperdexTM 200 pg</td>
			<td>Cytiva</td>
			<td>28-9893-36</td>
			<td>Used for FPLC (size exclusion)</td>
		</tr>
		<tr>
			<td>Hot plate stirrer</td>
			<td>Corning</td>
			<td>PC-420D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>H1759</td>
			<td>Used for Tris-HCl</td>
		</tr>
		<tr>
			<td>Index matching oil</td>
			<td>ZEISS</td>
			<td>444970-9000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma-reactive ion etching</td>
			<td>Top Technology Ltd.</td>
			<td>FabStar</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Glentham Life Sciences</td>
			<td>GC6586-100g</td>
			<td>Used for induction of &#946;-galactosidase activity</td>
		</tr>
		<tr>
			<td>Lambda phage DNA</td>
			<td>NEB</td>
			<td>N0311</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>BD difco</td>
			<td>244610</td>
			<td>Media for E.coli cell growth</td>
		</tr>
		<tr>
			<td>Luer adapter 10-32</td>
			<td>IDEX</td>
			<td>P-659</td>
			<td>This connects luer-lock syringe and tubing</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>fisher bioreagents</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl isobutyl ketone (MIBK)</td>
			<td>KAYAKU ADVANCED MATERIALS</td>
			<td></td>
			<td>Used for developing solution</td>
		</tr>
		<tr>
			<td>Microscope (Eclipse Ti2)</td>
			<td>Nikon</td>
			<td>Eclipse Ti2</td>
			<td>Inverted fluorescence microscope</td>
		</tr>
		<tr>
			<td>Microscope glass coverslip</td>
			<td>MARIENFELD</td>
			<td>101142</td>
			<td>22 x 50 mm (No. 1)</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>DURAN GROUP</td>
			<td>DU.2355013</td>
			<td>Slide glass ground edge 45&#176;, plain 26 x 76 mm</td>
		</tr>
		<tr>
			<td>Nanoport</td>
			<td>IDEX</td>
			<td>N-333-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td>CFI Plan Apochromat VC 60XC WI</td>
			<td>Immersion type: water, magnification: 60x, correction: 18, working distance: 0.29 (0.31-0.28)</td>
		</tr>
		<tr>
			<td>One Shot BL21 (DE3)pLysS Chemically Competent E. coli</td>
			<td>Thermo Fisher Scientific</td>
			<td>C6060-03</td>
			<td>Competent cell for overexpressing proteins</td>
		</tr>
		<tr>
			<td>Oxygen (O2)</td>
			<td>NOBLEGAS, South Korea</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>PFA tubing natural</td>
			<td>IDEX</td>
			<td>1512L</td>
			<td>It is connected with &#34;Fingertight Fittings&#34; to form luer-lock tubing</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche</td>
			<td>11359061001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Sephacryl S-200 High Resolution</td>
			<td>Cytiva</td>
			<td>17-0584-01</td>
			<td>Used for FPLC (size exclusion)</td>
		</tr>
		<tr>
			<td>Shut-off valve</td>
			<td>IDEX</td>
			<td>P-732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>791741</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>s5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra/Por molecularporous membrane tubing, MWCO 6-8 kDa</td>
			<td>Spectrum laboratories</td>
			<td>132660</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>S888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfur tetralfluoride (SF4)</td>
			<td>NOBLEGAS, South Korea</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>KD Scientific</td>
			<td>78-8210</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrafluoromethane (CF4)</td>
			<td>NOBLEGAS, South Korea</td>
			<td></td>
			<td>Purity: &#62;99.99%</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Used for Tris-HCl</td>
		</tr>
		<tr>
			<td>TSKgel SP-5PW</td>
			<td>TOSOH</td>
			<td>14715</td>
			<td>Used for FPLC (ion exchange)</td>
		</tr>
		<tr>
			<td>Union assembly</td>
			<td>IDEX</td>
			<td>P-760</td>
			<td>This connects tubings</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Jeio Tech</td>
			<td>OV-11</td>
			<td></td>
		</tr>
		<tr>
			<td>YOYO-1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Y3601</td>
			<td>This intercalation dye is diluted in DMSO</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol (BME)</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
	</tbody>
</table>

deciphering molecular mechanism of histone assembly by dna curtain technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

This work describes the procedure for flow cell preparation for the DNA curtain assay (Figure 1A). The DNA curtain assay facilitated the study of histone H3-H4 dimer assembly on DNA by Abo1. First, DNA curtain formation was checked by staining DNA molecules with YOYO-1, an intercalating dye. Green lines were shown in parallel arrays, indicating that YOYO-1 intercalated into DNA molecules, which were well-aligned and stretched at a diffusion barrier under hydrodynamic flow (<strong class="xfi...

Watch this Scientific Journal Video about Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique at JoVE.com

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Ce protocole montre comment la technique du rideau d’ADN est utilisée pour étudier la dynamique de la chromatine, en particulier pour étudier la fonction moléculaire des chaperons d’histones. La technique d’imagerie à haut débit d’une seule molécule d’ADN permet de surmonter les limites des expériences en vrac et facilite l’imagerie en temps réel, permettant la caractérisation détaillée des interactions entre les biomolécules. Mettez un papier propre de dimension 5 par 35 millimètres au centre du ruban adhésif double face.Fixez le ruban adhésif sur la diapositive pour couvrir les deux trous et les nano-motifs avec le papier. Retirez ensuite le papier à l’aide d’une lame propre. Placez une lamelle de verre sur le ruban adhésif double face et frottez la lamelle de protection à l’aide d’une pointe de pipette pour former une chambre microfluidique.Placez la cellule d’écoulement assemblée entre deux lames de microscope et clipsez-les. Faites cuire la cellule d’écoulement dans un four sous vide à 120 degrés Celsius pendant 45 minutes. Fixez le connecteur de fluide pour l’analyse à base de puce à chaque trou ouvert à l’aide d’un pistolet à colle chaude.Et connectez les deux lignes de tubes de verrouillage du leurre. Branchez une seringue contenant trois millilitres d’eau déminéralisée et lavez la chambre. Lavez à nouveau la chambre avec trois millilitres de tampon lipidique via une connexion goutte à goutte.Injectez un millilitre de lipide biotinylé 0,04x dans le tampon lipidique, dans la chambre, en deux fois, avec une incubation de cinq minutes par injection. Injectez 0,025 milligramme par millilitre de streptavidine dans un millilitre de BSA, avec deux injections et une incubation de 10 minutes par injection. Injecter environ 30 picomolaires d’ADN de phage lambda biotinylé dans un millilitre de tampon BSA, dans la chambre avec deux injections et des incubations de 10 minutes par injection.Connectez une seringue contenant 10 millilitres de tampon d’imagerie à un pousse-seringue et éliminez les bulles dans toutes les conduites de tubulure du système microfluidique. Couplez la cellule d’écoulement préparée avec le système microfluidique via une connexion goutte à goutte pour éviter l’injection de bulles dans la cellule d’écoulement. Assemblez la cellule d’écoulement et les supports de cellule d’écoulement, et montez l’ensemble sur un microscope à fluorescence à réflexion interne totale construit sur mesure.Injecter 200 microlitres de colorant YOYO-1 dans le tampon d’imagerie à travers 100 microlitres de boucle d’échantillon pour colorer les molécules d’ADN Lambda. Exécutez un logiciel d’imagerie et allumez le laser de 488 nanomètres pour vérifier si les rideaux d’ADN sont bien formés. Une fois que le colorant YOYO-1 a été retiré, injectez l’échantillon de protéine pré-incubé.Lorsque les protéines atteignent le rideau d’ADN, éteignez le pousse-seringue, fermez la vanne d’arrêt et incubez pendant 15 minutes pour la charge d’histones par Abo1. Lavez les protéines Abo1 et histones non liées pendant cinq minutes. Arrêtez temporairement le flux tampon pour vérifier si les protéines histones sont chargées sur l’ADN.La formation d’un rideau d’ADN a été vérifiée en colorant les molécules d’ADN avec un colorant interpolant. Les lignes vertes montrées dans des réseaux parallèles indiquaient que YOYO1 intercalé en molécules d’ADN étaient bien alignés et étirés au niveau d’une barrière de diffusion sous écoulement hydrodynamique. Lorsque des dimères Cy5-H3-H4 ont été injectés dans le rideau d’ADN en l’absence d’Abo1, Cy5-H3-H4 ne s’est pas lié à l’ADN, ce qui indique une absence de liaison spontanée des dimères H3-H4 à l’ADN.Lorsque Cy5-H3-H4 a été injecté avec Abo1, des particules fluorescentes rouges ont été observées sur les molécules d’ADN, ce qui suggère qu’Abo1 charge des dimères H3-H4 sur l’ADN. Les signaux fluorescents ont disparu en l’absence d’écoulement tampon et sont réapparus lorsque le flux a été repris, ce qui suggère que les dimères H3-H4 se lient à l’ADN. La liaison des dimères H3-H4 à l’ADN a également été confirmée par le kymographe, dans lequel les signaux de fluorescence disparaissaient chaque fois que le flux était coupé.Peu de particules fluorescentes sont apparues dans le rideau d’ADN, soit en l’absence de nucléotide, soit en présence d’ADP, ce qui indique que les dimères H3-H4 se lient rarement à l’ADN, que ce soit à l’état Apo ou en présence d’ADP. L’analyse quantitative montre que le nombre de dimères H3-H4 liés à l’ADN augmente en présence d’ATP. Les résultats suggèrent que l’hydrolyse de l’ATP est essentielle pour le chargement de H3-H4 sur l’ADN par Abo1.Éviter l’injection de bulles dans la cellule d’écoulement est le plus critique pour toutes les étapes. Ainsi, dans ce protocole, nous mettons l’accent sur les connexions goutte à goutte. Le test de rideau d’ADN peut être étendu aux rideaux d’ADN à double attache avec deux nano-motifs.De plus, nous pouvons fabriquer un autre type de rideau d’ADN avec des filaments d’ADN simple brin, d’ARN ou d’actine. Le rideau d’ADN permet de visualiser le mouvement des protéines sur l’ADN et facilite l’étude du mécanisme de recherche de cibles. Le rideau d’ADN peut être étendu pour étudier le métabolisme de l’ADN, y compris la réplication et la transcription.

Research

JoVE Journal

Biochemistry

Analysis of Histone Antibody Specificity with Peptide Microarrays

Analyse de la spécificité d&#39;anticorps d&#39;histone avec des microarrays de peptides

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Recherche

Biochimie

Real-time Tracking of DNA Fragment Separation by Smartphone

Suivi en temps réel de la séparation des fragments d&#39;ADN par téléphone intelligent

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Analyse d’activité de l’ADN polymérase utilisant l’ADN marqué Fluorescent proche infrarouge visualisée par électrophorèse sur Gel d’Acrylamide

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

ADN coloration méthode basée sur la précipitation de Formazan induite par l’exposition à la lumière bleue

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

Observation en temps réel de la réaction d’échange de brin ADN médiée par Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Enrichissement en une seule étape d’une déacétylase d’histone marquée par un robinet du champignon filamenteux Aspergillus nidulans pour le dosage enzymatique d’activité

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Reconnaissance de séquence d'ADN par primase d'ADN utilisant le profilage primase à haut débit

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Les deux règles d'ADN étudieront le mécanisme de la translocation de Ribosome avec la résolution d'un seul nucléotide

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation

Décryptage des effets structurels de l’activation des mutations somatiques EGFR avec simulation de dynamique moléculaire

Numerous somatic mutations occurring in the epidermal growth factor receptor (EGFR) family (ErbB) of receptor tyrosine kinases (RTK) have been reported ...