Name: continuous fluorescence-based endonuclease-coupled dna methylation assay to screen for dna methyltransferase inhibitors
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The authors thank Bucknell University and the Department of Chemistry for their support of this work.

1. Prepare assay solutions for the screen
NOTE: The concentrations of substrates used in this assay can be adapted. For RFTS-lacking DNMT1, the experimentally determined Km values for the hairpin DNA substrate and SAM are 1&#8211;2 nM and 2 &#181;M, respectively26,40.
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	<li>Prepare 600 &#181;L of each assay condition being tested in microcentrifuge tubes on ice. 
	NOTE: The total volume of assay s...

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M., Reisine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+compounds+that+modulate+epigenetic+regulation+of+the+transcriptome:+an+overview.">Screening for compounds that modulate epigenetic regulation of the transcriptome: an overview.</a> Journal of Biomolecular Screening. 16 (10), 1137-1152 (2011).</li><li>Holz-Schietinger, C., Matje, D. M., Reich, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+DNA+methyltransferase+(DNMT3A)+observed+in+acute+myeloid+leukemia+patients+disrupt+processive+methylation.">Mutations in DNA methyltransferase (DNMT3A) observed in acute myeloid leukemia patients disrupt processive methylation.</a> Journal of Biological Chemistry. 287 (37), 30941-30951 (2012).</li><li>Norvil, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dnmt3b+methylates+DNA+by+a+noncooperative+mechanism,+and+its+activity+is+unaffected+by+manipulations+at+the+predicted+dimer+interface.">Dnmt3b methylates DNA by a noncooperative mechanism, and its activity is unaffected by manipulations at the predicted dimer interface.</a> Biochemistry. 57 (29), 4312-4324 (2018).</li><li>Bashtrykov, P., Ragozin, S., Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+details+of+the+DNA+recognition+by+the+Dnmt1+DNA+methyltransferase.">Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase.</a> FEBS Letters. 586 (13), 1821-1823 (2012).</li><li>Bashtrykov, P., 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=Targeted+mutagenesis+results+in+an+activation+of+DNA+methyltransferase+1+and+confirms+an+autoinhibitory+role+of+its+RFTS+domain.">Targeted mutagenesis results in an activation of DNA methyltransferase 1 and confirms an autoinhibitory role of its RFTS domain.</a> Chembiochem. 15 (5), 743-748 (2014).</li><li>Berkyurek, A. C., 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+DNA+methyltransferase+Dnmt1+directly+interacts+with+the+SET+and+RING+finger-associated+(SRA)+domain+of+the+multifunctional+protein+Uhrf1+to+facilitate+accession+of+the+catalytic+center+to+hemi-methylated+DNA.">The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA.</a> Journal of Biological Chemistry. 289 (1), 379-386 (2014).</li><li>Kanada, K., Takeshita, K., Suetake, I., Tajima, S., Nakagawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+threonine+1505+in+the+catalytic+domain+stabilizes+mouse+DNA+methyltransferase+1.">Conserved threonine 1505 in the catalytic domain stabilizes mouse DNA methyltransferase 1.</a> Journal of Biochemistry. 162 (4), 271-278 (2017).</li><li>Bashtrykov, P., 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=Specificity+of+Dnmt1+for+methylation+of+hemimethylated+CpG+sites+resides+in+its+catalytic+domain.">Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain.</a> Chemistry &amp; Biology. 19 (5), 572-578 (2012).</li><li>Dolen, E. K., McGinnis, J. H., Tavory, R. N., Weiss, J. A., Switzer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-associated+mutations+G589A+and+V590F+relieve+replication+focus+targeting+sequence-mediated+autoinhibition+of+DNA+methyltransferase+1.">Disease-associated mutations G589A and V590F relieve replication focus targeting sequence-mediated autoinhibition of DNA methyltransferase 1.</a> Biochemistry. 58 (51), 5151-5159 (2019).</li><li>Kilgore, J. 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To identify and characterize inhibitors of DNA methyltransferases, the activity of the enzyme must be measured. Several methods for examining DNA methyltransferase activity exist. Activity is commonly monitored using radioactivity; transfer of the labeled methyl group of SAM can be quantified29,30,31,32,33,34. Gel-based assay...

DNA methylation is an important epigenetic mark that regulates gene expression and chromatin structure. Methylation occurs predominately in CpG dinucleotides &#8212; cytosine followed by guanosine; the methyl group is added to the 5-position of cytosine. Correct DNA methylation patterns, and thus proper gene expression, are needed for appropriate cellular development and function. Many disease states have been associated with changes to the normal methylation pattern1,2,3. For example, there is a link between cancer initiation and progression and alterations to the DNA methylation pattern. Typically, cancer cells exhibit lower overall levels of methylcytosine, which contributes to genome instability. At the same time, the methylcytosine that is present in the genome is concentrated in the promoter regions of tumor suppressor genes, which leads to gene silencing of these important proteins. Notably, epigenetic changes are dynamic and reversible, unlike the DNA mutations associated with tumorigenesis. This has made the proteins involved in epigenetic gene regulation interesting drug targets2,4.
DNA methyltransferases (DNMTs) are the proteins responsible for generating and maintaining DNA methylation patterns. Three catalytically active isozymes, DNMT1, DNMT3a, and DNMT3b, exist in humans. During development and differentiation, the de novo methyltransferases, DNMT3a and DNMT3b, establish methylation patterns. Both enzymes can bind the catalytically inactive DNMT3L protein to form complexes that exhibit increased activity1,5. Following cell division, daughter cells contain hemimethylated DNA &#8212; DNA containing methylcytosine in only one strand of the duplex &#8212; because the newly synthesized DNA is devoid of methylation marks. The major function of DNMT1 is to methylate this hemimethylated DNA, thus re-establishing the full methylation pattern1,5.
Links between DNMT activity and cancer are well established. Overexpression of DNMT1, either by transcriptional or post-translational mechanisms, is a consequence of several common oncogenic pathways6,7,8,9. Genetic approaches to lower DNMT1 activity using hypomorphic alleles result in decreased tumor formation in Apc(Min)mice10. Antisense oligonucleotides that knockdown DNMT1 inhibit neoplasia in cell culture and mouse tumor models11,12. Thus, inhibiting DNMT1 activity seems like a promising cancer therapy approach. However, the roles the DNMT3 isozymes play are not so straightforward. DNMT3a mutations are found in acute myeloid leukemia13 and myelodysplastic syndrome14. At least one of the identified mutations has been shown to decrease the DNA methylation activity of the enzyme15. However, DNMT3b is overexpressed in breast cancer16 and colorectal cancer17. With the various DNMT isozymes playing different roles in carcinogenesis, identifying isozyme-specific inhibitors will be critical. Not only will these compounds be useful for the development of therapeutics, but isozyme-specific inhibitors would also be an invaluable tool to dissect the role of each DNMT isozyme in cancer etiology.
Several DNMT inhibitors have been reported in the literature. Known DNMT inhibitors can be divided into two classes: nucleoside and non-nucleoside. Nucleoside inhibitors are typically cytidine analogs. These compounds are incorporated into DNA and covalently trap DNMTs. 5-azacytidine and 5-aza-2&#39;-deoxycytidine have been approved for the treatment of myelodysplastic syndrome and acute myeloid leukemia4,18. The high toxicity, low bioavailability, and chemical instability of these compounds present problems. Ongoing work is examining the efficacy of the next generation of nucleoside inhibitors; SGI-110, derived from 5-aza-2&#39;-deoxycytidine, is one example19,20. Nucleoside inhibitors are not isozyme-specific and will inactivate any DNMT isozyme encountered. Therefore, treatment with a nucleoside-demethylating agent results in the depletion of all DNMT isozymes4,18. Non-nucleoside inhibitors do not need to be incorporated into DNA to exert their inhibitory effects. Instead, these molecules bind directly to DNMTs, introducing the possibility for isozyme-specific inhibition. Several non-nucleoside inhibitors have been discovered to date, including SGI-102721, hydralazine22, procainamide23, RG108 and derivatives24, and natural products, (&#8722;)-epigallocatechin 3-gallate (EGCG)25 and laccaic acid A26,27. Most of the non-nucleoside inhibitors discovered to date are not isozyme-selective or display weak preferences for one DNMT isozyme. In addition, the potency of these molecules needs to be improved, especially in cells4,18. Thus, there is a need to discover or develop more potent, isozyme-selective DNMT inhibitors.
A hurdle to discovering new small molecule inhibitors of DNMTs is the laborious assays traditionally used to examine DNMT activity28. Assays are usually discontinuous with multiple steps. The enzymatic activity of DNMTs is still routinely assayed using radioactive S-adenosyl methionine (SAM)29,30,31,32,33,34. Non-radioactive assays for DNA methylation have been developed as well. For example, assays utilizing methyl-sensitive restriction endonucleases and electrophoresis to separate the digestion products have been described35,36. These types of discontinuous, multistep assays are not readily amenable to drug discovery. Since the mid-2000s, several DNA methylation assays with a higher throughput have been developed28. A scintillation proximity assay was used to screen for DNMT1 inhibitors37. Another assay utilizing a methyl-sensitive restriction endonuclease was used to screen for DNMT3a inhibitors25,38. While both assays allowed for higher throughput than traditional DNA methylation assays, the assays require multiple steps and do not allow the observation of methylation activity in real time. More recently, a continuous kinetics assay has been described that couples the formation of S-adenosylhomocysteine (SAH), one product of the methylation reaction, to the spectroscopic change at 340 nm associated with NADPH oxidation39. This assay utilizes three coupling enzymes to generate a spectroscopic signal.
We developed a fluorescence-based endonuclease-coupled DNA methylation assay that utilizes a single commercially available coupling enzyme and can generate data in real time (Figure 1). A hairpin oligonucleotide containing three methylcytosines is used as a substrate. The substrate DNA contains a fluorophore on the 5&#39; end and a quencher on the 3&#39; end. Methylation of the hemimethylated CpG site generates the cleavage site for the endonuclease Gla I &#8212; fully methylated GCGC. Gla I cleavage of the product oligonucleotide releases the fluorophore from the quencher and generates fluorescence in real time. The assay can be used to examine the activity of any isoform of DNMT; however, higher activity is observed with DNMT1 as this isozyme preferentially methylates hemimethylated DNA1,5. Even more robust activity is observed if the autoinhibitory Replication Foci Targeting Sequence (RFTS) domain is removed from DNMT1. This domain, found in the N-terminal regulatory region, binds to the catalytic site and prevents DNA binding. Removal of the first ~600 amino acids results in a truncated enzyme that is significantly more active than the full-length enzyme (~640-fold increase in kcat/Km)40. This activated form of the enzyme, referred to as RFTS-lacking DNMT1 (amino acids 621&#8211;1616), allows for the easier identification of inhibitors due to its increased catalytic power. This paper presents a protocol to utilize RFTS-lacking DNMT1 in assays to screen for potential small molecule inhibitors. Using the endonuclease-coupled continuous assay, the initial velocity is determined in the presence and absence of a few small molecules. Each potential inhibitor is examined at two concentrations to look for concentration-dependent DNMT1 inhibition. The percent activity observed in the presence of the small molecules was calculated in each case.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62949/62949fig01.jpg" /> 
Figure 1: DNA methylation assay. A hemimethylated hairpin DNA with a fluorophore on the 5&#39; end and a quencher on the 3&#39; end is used as a substrate. DNMT1 catalyzes the transfer of the methyl group from S-adenosylmethionine to the nonmethylated CpG site, generating S-adenosylhomocysteine and fully methylated DNA. The DNA product contains the cleavage site for the endonuclease Gla I, which cleaves fully methylated GCGC sites. Cleavage of the product DNA releases the 5&#39; fluorophore from the 3&#39; quencher, generating fluorescence. Abbreviations: Fl = fluorophore; Q =quencher; DNMT1 = DNA methyltransferase 1; SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine. <a href="https://www.jove.com/files/ftp_upload/62949/62949fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:791px;" width="791">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">96-well Half Area Black Flat Bottom Polystyrene Not Treated Microplate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:119px;">3694</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">96-Well Polystyrene Conical Bottom Plates</td>
			<td style="width:100px;">ThermoFisher</td>
			<td style="width:119px;">249570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Bovine Serum Albumin</td>
			<td style="width:100px;">NEB</td>
			<td style="width:119px;">B9000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">compound 1</td>
			<td style="width:100px;">ChemBridge</td>
			<td>5812086</td>
			<td>screening compound; resuspended in DMSO to 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">compound 2</td>
			<td style="width:100px;">ChemBridge</td>
			<td>6722175</td>
			<td>screening compound; resuspended in DMSO to 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">compound 3</td>
			<td style="width:100px;">ChemBridge</td>
			<td>5249376</td>
			<td>screening compound; resuspended in DMSO to 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dithiothreitol</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Gla I</td>
			<td style="width:100px;">SibEnzyme</td>
			<td style="width:119px;">E494</td>
			<td>methyl-sensitive endonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glycerol</td>
			<td style="width:100px;">RPI</td>
			<td style="width:119px;">G22025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Magnesium Chloride</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">M0250</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:251px;">Oligonucleotide (5&#39;-FAM-CCTATGCGmCATCAGTTTTCTGATGmCGmCATAGG-3&#39;-Iowa Black Quencher)</td>
			<td style="width:100px;">IDT</td>
			<td style="width:119px;">custom synthesized</td>
			<td>internally quenched hairpin DNA (substrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium Glutamate</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">G1501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">S-adenosylmethionine</td>
			<td style="width:100px;">Sigma</td>
			<td style="width:119px;">A4377</td>
			<td>methyl-donating co-factor (substrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tris Base</td>
			<td style="width:100px;">RPI</td>
			<td style="width:119px;">T60040</td>
			<td></td>
		</tr>
	</tbody>
</table>

continuous fluorescence-based endonuclease-coupled dna methylation assay to screen for dna methyltransferase inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

Active DNMT1 is a prerequisite for this analysis. RFTS-lacking DNMT1 was expressed in E.coli and purified to homogeneity following previously published procedures41. To ensure the purified enzyme was active, a discontinuous endonuclease-coupled assay was used to examine DNA methylation activity36. This assay utilizes a 32 base pair duplex DNA with a single hemimethylated CpG positioned in a Sau3A1 cleavage site. Sau3A1 can cleave the hemimethylated substrate DNA; h...

Watch this Scientific Journal Video about Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors at JoVE.com

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

A robust plate reader assay like ours is very useful for the initial screening of potential methyltransferase inhibitors. The ability to collect data in real time is a big advantage of the continuous endonuclease-coupled assay. Start by preparing 600 microliters of the assay conditions containing 20 micromolar and 50 micromolar compounds separately in the microcentrifuge tubes on ice.To do so, add double distilled water and 5X methylation buffer to each tube to achieve a final concentration of 1X methylation buffer. Next, add 3.15 microliters of 20 milligrams per milliliter bovine serum albumen, or BSA, to each sample. Then, add two microliters of 3.15 micromolar hairpin DNA substrate and 1.33 microliters of 4.75 micromolar S-adenosylmethionine, or SAM, to each sample.Add inhibitor to the appropriate samples to achieve a final concentration of 21 micromolar or 52.5 micromolar and an equivalent amount of dimethyl sulfoxide, or DMSO, to a control sample before mixing the solutions by vortexing at 3, 000 rotations per minute for three seconds. Then, spin the samples for a few seconds in a table table mini centrifuge at 1, 200 times G to ensure the solution is collected at the bottom of the tube. Aliquot 95 microliters of each assay solution into six consecutive wells in a black half area 96 well plate.Prepare 75 microliters of Gla I or DNMT1 plus Gla I enzyme solution in the microcentrifuge tubes as described before to a final concentration of one-time buffer. Next, add 1.2 microliters of 10 units per microliter Gla I to each solution to achieve a final concentration of 0.16 units per microliter. Add 0.6 microliters of five micromolar RFTS-lacking DNMT1 to the DNMT1 plus Gla I solution for a final concentration of 40 nanomolar.After gentle mixing, spin the samples for a few seconds in a tabletop mini centrifuge at 1, 200 G to ensure that the solution is collected at the bottom of the tube. Then, aliquot 12 microliters of each enzyme solution into six wells in a conical-bottomed 96 well plate. For the methylation assay of the DNA sample, preheat the plate reader to 37 degrees Celsius.Then, insert the black plate containing the assay solutions in the plate reader. After shaking the plate at 425 CPM, measure the fluorescence with an excitation wavelength of 485 nanometers and an emission wavelength of 528 nanometers. Then, incubate the plate at 37 degrees Celsius for five minutes.Remove the assay plate from the plate reader and add five microliters of the enzyme solution to each well, followed by mixing the solution by pipetting up and down. Insert the plate into the plate reader and shake the plate before recording the fluorescence every 53 seconds for 30 minutes. Obtain the average fluorescence of the triplicate Gla I control assays for each condition and subtract the average Gla I containing control reaction trays from the triplicate traces obtained in the presence of RFTS-lacking DNMT1.Then, determine the average and standard error of the mean for the corrected replicates. Plot the average corrected reaction trace and fit the initial linear portion to a line to determine the initial velocity. Determine the percent activity by dividing the velocity observed in the presence of a compound by the velocity observed in the DMSO containing control reaction and multiplying by 100.The results of a discontinuous endonuclease-coupled assay showed that fully methylated product DNA is protected from cleavage, confirming that RFTS-lacking DNMT1 is active and able to methylate the hemimethylated substrate DNA. In the fluorescence-based assay, in the absence of Gla I, the addition of DNMT1 or buffer alone did not affect the background fluorescence. Subsequent addition of Gla I to all assays resulted in fluorescence generation only in assays that contained DNMT1.The concurrent addition of RFTS-lacking DNMT1 and Gla I to three assays resulted in robust fluorescence generation. The addition of Gla I alone produced no fluorescence. To screen for potential inhibitors, the DNA methylation activity of RFTS-lacking DNMT1 was examined in the presence of DMSO, compound one, compound two, or compound three at 20 and 50 micromolar concentrations.The compounds that reduce fluorescence generation in this coupled assay must be further validated. Ensuring the compounds are not inhibiting the coupling enzyme is an important next step.

continuous-fluorescence-based-endonuclease-coupled-dna-methylation

Research

JoVE Journal

Biochemistry

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified 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 ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

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. ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is dependent on its ability to hydrolyze adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and free phosphate. The ATPase activity of Hsp90 is linked to its chaperoning function; ATP binds to the N-terminal domain of the Hsp90, and disrupting its binding was found to be the most successful strategy in suppressing Hsp90 function. The ATPase activity can be measured by a colorimetric malachite green assay, which determines the amount of free phosphate formed by ATP hydrolysis. Here, a procedure for determining the ATPase activity of yeast Hsp90 by using the malachite green phosphate assay kit is described. Further, detailed instructions for the discovery of Hsp90 inhibitors by taking geldanamycin as an authentic inhibitor is provided. Finally, the application of this assay protocol through the high-throughput screening (HTS) of inhibitor molecules against yeast Hsp90 is discussed.

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...