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 ...

This protocol shows how DNA curtain technique is utilized for studying chromatin dynamics, in particular investigating the molecular function of histone chaperones. High-throughput single-molecule imaging technique DNA curtain overcomes limitations of bulk experiments and facilitates Real-Time imaging, enabling the detailed characterization of Biomolecule interactions. Put a clean paper of dimension 5 by 35 millimeters on the center of the double-sided tape.Attach the tape over the slide to cover the two holes and nano patterns with the paper. Then excise the paper using a clean blade. Put a glass cover slip on top of the double-sided tape, and rub the cover slip using a pipet tip to form a microfluidic chamber.Place the assembled flow cell between two microscope slides, and clip them. Bake the flow cell in a 120 degrees Celsius vacuum oven for 45 minutes. Attach the fluid connector for the chip-based analysis to each open hole using a hot glue gun.And connect the two lines of lure lock tubing. Connect a lure lock syringe containing three milliliters of deionized water, and wash the chamber. Wash the chamber again with three milliliters of lipid buffer via drop-by-drop connection.Inject one milliliter of 0.04x biotinylated lipid in lipid buffer, into the chamber, in two shots, with a five minute incubation per shot. Inject 0.025 milligrams per milliliter of streptavidin in one milliliter of BSA, with two shots, and a 10-minute incubation per shot. Inject approximately 30 picomolar of biotinylated lambda phage DNA in one milliliter of BSA buffer, into the chamber with two shots, and 10-minute incubations per shot.Connect a syringe containing 10 milliliters of imaging buffer to a syringe pump, and remove bubbles in all tubing lines in the microfluidic system. Couple the prepared flow cell with the microfluidic system via drop-to-drop connection to avoid bubble injection into the flow cell. Assemble the flow cell and flow cell holders, and mount the assembly on a custom built, total internal reflection fluorescence microscope.Inject 200 microliters of YOYO-1 dye in imaging buffer through 100 microliters of sample loop to stain Lambda DNA molecules. Run imaging software, and turn on the 488-nanometer laser to check whether DNA curtains are well-formed. After YOYO-1 dye has been removed, inject the pre-incubated protein sample.When the proteins reach the DNA curtain, turn off the syringe pump, switch off the shutoff valve, and incubate for 15 minutes for histone loading by Abo1. Wash out the unbound Abo1 and histone proteins for five minutes. Transiently turn off the buffer flow to check if histone proteins are loaded onto DNA.DNA curtain formation was checked by staining DNA molecules with an interpolating dye. Green lines shown in parallel arrays indicated that YOYO1 intercalated into DNA molecules were well aligned and stretched at a diffusion barrier under hydrodynamic flow. When Cy5-H3-H4 dimers were injected into the DNA curtain in the absence of Abo1, Cy5-H3-H4 did not bind to DNA, indicating a lack of spontaneous binding of H3-H4 dimers to DNA.When Cy5-H3-H4 was injected with Abo1, red fluorescent puncta were seen on DNA molecules, suggesting that Abo1 loads H3-H4 dimers onto DNA. The fluorescent signals disappeared in the absence of buffer flow, and reappeared when the flow was resumed, suggesting that H3-H4 dimers bind to DNA. The binding of H3-H4 dimers to DNA was also confirmed by the kymograph, in which the fluorescence signals disappeared whenever the flow was turned off.Few fluorescent puncta appeared in the DNA curtain, either in the absence of nucleotide or in the presence of ADP, indicating that H3-H4 dimers rarely bind to DNA, either in the Apo state or in the presence of ADP. The quantitative analysis shows that the number of H3-H4 dimers bound to DNA increases in the presence of ATP. The results suggest that ATP hydrolysis is essential for H3-H4 loading onto DNA by Abo1.Avoiding bubble injection into flow cell is the most critical for all steps. So in this protocol, we emphasize drop-by-drop connections. DNA curtain assay can be extended to double-tether DNA curtains with dual nano-patterns.In addition, we can make another type of DNA curtain with single stranded DNA, RNA, or actin filaments. DNA curtain enables visualization of protein motion on DNA and facilitates studying on target search mechanism. DNA curtain can be extended for studying DNA metabolism including replication and transcription.

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Biochemistry

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

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

Real-time Tracking of DNA Fragment Separation by Smartphone

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 others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

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

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

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

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

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

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

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

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

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

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

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

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

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

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

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

Numerous somatic mutations occurring in the epidermal growth factor receptor (EGFR) family (ErbB) of receptor tyrosine kinases (RTK) have been reported from cancer patients, although relatively few have been tested and shown to cause functional changes in ErbBs. The ErbB receptors are dimerized and activated upon ligand binding, and dynamic conformational changes of the receptors are inherent for induction of downstream signaling. For two mutations shown experimentally to alter EGFR function, A702V and the &#916;746ELREA750 deletion mutation, we illustrate in the following protocol how molecular dynamics (MD) simulations can probe the (1) conformational stability of the mutant tyrosine kinase structure in comparison with wild-type EGFR; (2) structural consequences and conformational transitions and their relationship to observed functional changes; (3) effects of mutations on the strength of binding ATP as well as for binding between the kinase domains in the activated asymmetric dimer; and (4) effects of the mutations on key interactions within the EGFR binding site associated with the activated enzyme. The protocol provides a detailed step-by-step procedure as well as guidance that can be more generally useful for investigation of protein structures using MD simulations as a means to probe structural dynamics and the relationship to biological function.

The objective of this protocol is to use molecular dynamics simulations to examine the dynamic structural changes that occur due to activating mutations of the EGFR kinase protein.

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

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.