Name: Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis (Video) | JoVE Journal
Uploaded: 2010-12-21T16:00:00.000+00:00
Duration: 10 min 36 s
Description: Learn to visualize UV-induced DNA replication intermediates in E. coli using two-dimensional agarose-gel analysis. This method reveals structural changes and repair dynamics in bacterial cells.

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5 minutes

Work in our lab is supported by CAREER award MCB0551798 from the National Science Foundation and AREA grant R15GM86839 from the NIGMS-NIH.

1. Growth and UV Irradiation.
<ol>
	<li>200μl of a fresh overnight culture containing the plasmid pBR322 grown in Davis medium1 supplemented with 0.4% glucose, 0.2% casamino acids, and 10 μg/ml thymine (DGCthy medium) and 100 μg/ml ampicillin is pelleted. The cell pellet is then resuspended in 200μl DGCthy medium lacking ampicillin and used to inoculate 20 ml of DGCthy medium.</li>
	<li>Cultures are grown without ampicillin selection in a shaking incubator at 37° C to an OD600 of 0.5 (~ 5 x 108 cells/ml). Growth without ampicillin avoids selection against abnormal or unproductive replication intermediates that may arise in some mutants. In addition, if using UV light to induce damage, the removal of the ampicillin from the media is necessary because it absorbs strongly at these wavelengths and shields the cells, reducing the effective dose of UV.</li>
	<li>Working under yellow lights, the culture is placed in a 15cm diameter Petri dish on a rotating platform for agitation. Our cultures are placed at a distance from a 15-watt germicidal lamp that produces an exposure rate of ~1 J/m2/sec, which is measured using a UVC photometer. Cultures are irradiated with 50 J/m2 and then placed immediately back into the shaking 37° C incubator for the duration of the experiment. This dose produces, on average, 1 cyclobutane pyrimidine dimer every 4.5 kb of ssDNA . The yellow lighting prevents photoreactivation-reversal of cyclobutane pyrimidine dimers by photolyase.</li>
</ol>
2. DNA Isolation.
<ol>
	<li>At times when replication intermediates are to be examined, a 0.75 ml aliquot of the culture is placed into 0.75 ml ice cold NET30 Buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 30 mM EDTA) and placed on ice until the end of the time course. We typically run a 90 minute time course, with samples examined at 0, 15, 30, 45, 60, and 90 minutes. The EDTA and cold temperature serve to effectively stop replication and nucleotide excision repair.</li>
	<li>Each sample is then pelleted, resuspended in 150 μl of 1.5 mg/ml lysozyme and 0.2 mg/ml RNaseA in TE (10 mM Tris, pH 8.0, 1 mM EDTA), and lysed at 37°C for 20 min. At this time, 10μl of proteinase K (10mg/ml) and 10μl of 20% sarkosyl are added and the incubation is allowed to continue for 1 hr. Proteinase K and sarkosyl help to release DNA fragments that may be associated with the membrane or proteins before phenol extraction occurs. Since actively replicating DNA is often bound to protein or membrane complexes, this helps increase the yield of replication fragments that are recovered.</li>
	<li>Samples are then extracted by adding 2 volumes of phenol to each sample and the tubes are gently inverted for 5 minutes. Then 2 volumes of chloroform/isoamyl alcohol (24/1) are added and the tubes are gently inverted again for 5 minutes.</li>
	<li>The samples are centrifuged at 14,000 rpm in a microcentrifuge for 5 minutes and the top aqueous phase of each sample is removed, and placed into a fresh tube. Then 4 volumes of chloroform/isoamyl alcohol (24/1) are added, the tubes are gently inverted for 5 minutes, and centrifuged again at 14,000 rpm for 5 minutes.</li>
	<li>The top, aqueous phase in each sample is then dialyzed for 1 hour by spotting 100μl of each sample on a 47mm Whatman 0.025 μm pore disk which floats on 250 ml of 0.1X TE in a beaker. Because replicating structures with single strand regions or branch points are more susceptible to shearing, we typically cut our pipette tips off with a razor blade to make the mouth wider and minimize shearing forces. In general, pipetting should be kept to a minimum until after the DNA has been digested with restriction enzymes.</li>
	<li>Each sample is then digested with PvuII (New England Biolabs) which linearizes the plasmid just downstream from the origin of replication.</li>
	<li>Prior to loading in the agarose gel, 100μl chloroform and 20μl of 6X loading dye that contains Bromophenol Blue and Xylene Cyanol are added to each sample and mixed. Then, 40μl of the aqueous phase containing the loading dye is loaded into the gel. To minimize structural biases, structural artifacts, and DNA shearing, this isolation procedure does not include any steps to enrich for single strand regions, precipitate, or concentrate the DNA samples following cell lysis.</li>
</ol>
3. 2D Gel and Southern Analysis.
<ol>
	<li>The 2-dimensional agarose-gel analysis is modified from 3. For the 1ST dimension, the restricted DNA samples are run through a 0.4% agarose gel in 1X TBE at 1V/cm. One Liter of 10X TBE stock solution contains:
 <ul>
		<li>108 g Tris Base</li>
		<li>55 g Boric Acid</li>
		<li>40 ml 0.5M EDTA (pH 8.0)</li>
 </ul>
</li>
	<li>A lambda Hind III size marker is loaded in the first lane, then samples are loaded in every other lane. We typically run the first dimension ~12-15 hrs. Skipping lanes makes it easier to slice the lanes out to cast in the second dimension. The low voltage and low percent agarose gel serves to separate the DNA fragments primarily based upon size.</li>
	<li>For the second dimension, gel lanes are sliced out of the first dimension gel using a large butcher's knife. The first lane containing the lambda Hind III marker can be stained with ethidium bromide. Using the lambda Hind III marker as a guide, crop and discard the region of the gel that is below where the linearized plasmid is expected to run. Under these conditions, we find that linearized pBR322 runs slightly above the xylene cyanol stain.</li>
	<li>To cast the second dimension, each lane is placed horizontally across the top of an empty gel caster. A solution of 1.0% agarose in 1X TBE is prepared and cooled to 55°C. At this time, the gel solution is poured in to cast the second dimension, making sure to completely cover the gel slices. Once the gel has set, it is run at 6.5V/cm in an electrophoresis unit that allows the buffer to recirculate. We typically run the second dimension ~5.5-7 hrs. The high voltage and high percent agarose gel effectively separates the DNA fragments based on their shape, as well as size. Nonlinear shapes run more slowly through the second dimension.</li>
	<li>Following electrophoresis, the gel is then rinsed in H2O, washed twice in 400ml of 0.25M hydrochloric acid for 15 minutes, rinsed with H2O;, and then washed twice in 400 ml 0.4M NaOH for 30 minutes. The acid washes serve to partially nick the DNA molecules into smaller fragments that transfer more efficiently. This step is necessary due to the large size and unusual shapes of the DNA replication intermediates.</li>
	<li>The DNA in the gels is then transferred to a Hybond N+ nylon membrane 4. We use a downward alkali transfer system with 0.4M NaOH. Briefly, two sheets of blotting paper soaked in 0.4M NaOH are placed on a large stack of paper towels. The nylon membrane is also soaked in 0.4M NaOH and placed on top of the blotting paper. The gel is then carefully layered on top of this, followed by another piece of blotting paper, wetted in the NaOH solution. Finally, a long piece of wetted blotting paper is layered across the top and its two ends are placed into a dishes containing 1 L of 0.4M NaOH solution to serve as a wick. We typically let the DNA transfer for 6-12 hrs.</li>
	<li>The nylon membrane is removed, washed 20-30 sec in 5X SSC Buffer, and placed in a hybridization roller bottle containing 10.5ml Prehybridization Solution. Then, the membrane is incubated at 42°C with rotation for more than 6hrs. Prehybridization solution:
	<ul>
		<li>5.0 ml formamide</li>
		<li>0.5 ml 20% SDS</li>
		<li>2.5 ml 20X SSC*</li>
		<li>2.0 ml 50X Denhardt's*</li>
		<li>0.5 ml Salmon Sperm DNA (10mg/ml) *recipes for SSC and Denhardt's can be found in 5</li>
 </ul>
 </li>
	<li>During the prehybridization period, 1 μg of pBR322 plasmid is labeled with 32P by nick translation according to the protocol supplied by Roche using alpha labeled [32-P]-dCTP. The radiolabeled probe (100μl) is denatured by incubating at 98° C for 10 minutes in a screw cap microcentrifuge tube, and then placed immediately on ice.</li>
	<li>The denatured probe is added to 5.9 ml hybridization solution, which contains:
 	<ul>
		<li>3.0 ml formamide</li>
		<li>1.5 ml SSC</li>
		<li>1.0 ml H2O</li>
		<li>0.15 ml 50X Denhardt's</li> 
		<li>0.10 ml 20% SDS</li>
		<li>0.15 ml 10mg/ml Salmon Sperm DNA</li>
	</ul>
</li>
	<li>The prehybridization solution is poured out of the roller bottle and the hybridization solution is poured in. The roller bottle is then returned to the 42° C incubator and rotated for more than 12hrs.</li>
	<li>The hybridization solution is poured out of the roller bottle. Then, the blot is washed 4 times for 20 minutes in the roller bottle with ~150 ml of a wash solution containing 0.5X SSC, 0.1% SDS at 42° C with rotation.</li>
	<li>Following the last wash, the membrane is placed on a paper towel until the liquid has visibly disappeared. The membrane is then wrapped in polyvinyl-chloride plastic wrap and exposed to a phosphorimager screen. We visualize and quantify the radioactivity using a Storm 840 and its associated ImageQuant Software.</li>
</ol>
4. Representative Results:
<img src="/files/ftp_upload/2220/2220fig1.jpg" alt="DNA damage repair process diagram with UV-induced intermediates; UV irradiation effects shown." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Plasmid replication intermediates observed in the presence and absence of UV-induced DNA damage. The migration pattern of PvuII digested pBR322 plasmid observed by 2D-agarose-gel analysis is diagrammed. Non-replicating linear plasmids run as a linear 4.4-kb fragment. Replicating plasmids form Y-shaped structures that migrate slower than the non-replicating fragments due to their larger size and nonlinear shape. This migration pattern forms an arc that extends out from the linear region towards the well. Following UV-irradiation, double-Y or X-shaped molecules are observed in the cone region that migrates more slowly than the arc of Y-shaped molecules. An example of the Southern analysis of the 2D gel probed with 32P-labeled pBR322 is shown for cells immediately after UV irradiation and 15 minutes after UV irradiation (reproduced with permission from the publisher).

<ol><li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li>
<li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+Damage-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li>
<li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li>
<li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+damage+and+repair+in+specific+DNA+sequences%5BTitle%5D)+AND+%22METHODS%3A+A+Companion+to+Methods+in+Enzymology%22%5BJournal%5D)">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li>
<li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+Cloning+-+A+laboratory+manual%22">Molecular Cloning - A laboratory manual</a>. , CSHL Press. Cold Spring Harbor. (2001).</li>
<li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li>
<li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li>
<li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li>
<li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork%3A+a+comparison+to+UV-induced+arrest%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li>
<li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RecBCD+and+RecJ%2FRecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli%5BTitle%5D)+AND+%22Radiat+Res%22%5BJournal%5D)">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li>
<li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li>
</ol>

No conflicts of interest declared.

Typical results obtained from wild type cells in the presence and absence of UV-induced damage are shown in Figure 1. In the absence of damage, ~1% of the total plasmid DNA can be found in the Y arc when cells are rapidly growing in exponential phase. Following irradiation, a transient increase in Y shaped molecules is observed as blocked replication forks accumulate at damaged sites. The X-shaped replication intermediates also transiently accumulate and persist until a time that correlates with when the lesions are rep...

<table><tbody><tr><td>An example of the Southern analysis of the 2D gel probed</td><td>GE Healthcare</td><td>G15T8</td><td>Yellow Lighting</td></tr><tr><td>15 &amp;mu;watt germicidal lamp</td><td>Sylvania</td><td>F20T12/GO</td><td>UV Lamp</td></tr><tr><td>Blak-Ray UV Intensity Meter 254nm</td><td>Daigger</td><td>EF28195T</td><td>UVC photometer</td></tr><tr><td>0.025 &amp;mu;m pore disks</td><td>Whatman, GE Healthcare</td><td>VSWP04700</td><td>Floating dialysis disks</td></tr><tr><td>PvuII</td><td>Fermentas</td><td>ER0632</td><td>Restriction Endonuclease</td></tr><tr><td>Nick-translation kit</td><td>Roche Group</td><td>976776</td><td>To make 32P-labeled probe</td></tr><tr><td>Blotting Paper</td><td>Whatman, GE Healthcare</td><td>3030-704</td><td>For Southern transfer</td></tr><tr><td>Nylon membrane</td><td>GE Healthcare</td><td>RPN203S</td><td>For Southern transfer</td></tr></tbody></table>

visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

The overall goal of this procedure is to visualize the DNA structural intermediates that occur when replication encounters DNA lesions using two dimensional gel analysis. This is accomplished by first isolating the DNA from actively dividing cells in which replication blocking lesions have been induced. Then gel electrophoresis is used to separate the recover DNA based on size.Next, the lanes are cut out and recast horizontally in a second gel that further separates DNA based on both size and shape. Finally, southern analysis is used to visualize the DNA structures that are associated with replicating DNA fragments at times before and at various times after the introduction of UV induced damage. Ultimately, this procedure can be used to show that the inability to process DNA lesions in certain mutants results in accumulation of DNA repair intermediates.Hi, my name is Arthur Ian. I'm from the Corella Lab at Portland State University. Hi, my name is Brand she, I'm also from the Corella Lab.Today we're gonna show you a procedure for two dimensional gel electrophoresis. We use this procedure in our laboratory to study DNA replication and repair intermediates. So let's get started.To begin this procedure, grow a 200 microliter overnight culture of e coli containing the plasmid PBR 3 22 in DGC thigh medium with ampicillin at 37 degrees Celsius. The use of DGC thigh medium minimizes UV shielding. Following overnight incubation pellet the cells at 14, 000 RRP M for 30 seconds.Then resuspend the cell pellet in 200 microliters of DG C th medium lacking ampicillin and inoculate. 20 milliliters of DG C thigh medium grow cultures without ampicillin selection. In a shaking incubator at 37 degrees Celsius to an OD 600 of 0.5, which corresponds to about five times 10 to the eighth cells Per milliliter growth without ampicillin avoids selection against abnormal or unproductive replication intermediates that may arise in some mutants.In addition, if using UV light to induce damage, the removal of the ampicillin from the media is necessary because it absorbs strongly at these wavelengths and shields. The cells reducing the effective dose of UV while the cells are growing. Use a UVC photometer to determine the distance from a 15 watt germicidal lamp that produces an exposure rate of approximately one JUUL per meter meter squared per second.Subsequent steps should be performed under yellow light as this will prevent photo reactivation reversal of cyclo butane perine dimers by photo liase after UV irradiation. Once the desired cell density is reached, use a pipette to transfer the culture to a 15 centimeter diameter Petri dish on a rotating platform to ensure even irradiation of the entire cell population. Next, irradiate the cultures with 50 joules per meter squared and immediately transfer them into a sterile prewarm flask and place them back into the shaking 37 degrees Celsius incubator.For the duration of the experiment, this dose produces on average one cyclo butane perine dimer, every 4.5 kilobases of single stranded DNA for each time point that is to be examined. Pipette a 0.75 milliliter aliquot of the culture into 0.75 milliliters of ice cold net 30. Buffer net 30 serves as a stop buffer that prevents replication and nucleotide excision repair from proceeding.Once the stop buffer has been added, the sample can be kept on ice until the end of the time course. After the time course pellet each sample and resuspend the pellets. In 150 microliters of lysis buffer lyce the cells at 37 degrees Celsius for 20 minutes without agitation.Because replicating structures with single strand regions or branch points are more susceptible to shearing cut pipette tips off with a razor blade to make the mouth wider and minimize shearing forces. In general, pipetting and agitation should be kept to a minimum until after the DNA has been digested with restriction enzymes, following lysis, add proteinase K and sarcas cell and allow the incubation to continue for one hour. This serves to release DNA fragments associated with protein.Since actively replicating DNA is often bound to protein or membrane complexes, this helps increase the yield of replication fragments. After one hour, extract the samples by adding two volumes of phenol to each sample and gently inverting the tubes for five minutes. Then add two volumes of chloroform, isoamyl, alcohol, and gently invert the tubes for an additional five minutes.Next, centrifuge the samples at 14, 000 RPM in a micro centrifuge for five minutes and transfer the top aqueous phase of each sample into a fresh tube. Then add four volumes of chloroform ISO alcohol, and again, gently invert the tubes for five minutes. Centrifuge the samples again at 14, 000 RPM for five minutes.Place a dialysis membrane atop 250 milliliters of 0.1 xte in a beaker and spot 100 microliters of each sample onto the membrane. Let the samples dialyze for one hour after dialysis. Place 80 microliters of each sample into a fresh 1.5 milliliter tube containing 20 microliters of enzyme mix.Digest the samples overnight at 37 degrees Celsius. PV two linearizes the plasmid just downstream from the origin of replication. Prior to loading the agros gel, add 100 microliters of chloroform and 20 microliters of six x loading dye containing bromo.Phenol blue and xylene cyan to each sample and mix chloroform will strip any remaining PV two bound to DNA ends. Begin two dimensional aros gel analysis by running the restricted DNA samples through the first dimension. First load a Lambda hind three size marker into the first lane of a previously prepared 0.4%agros gel in one XTBE.Then load 40 microliters of the aqueous phase containing the loading dye for each sample, skipping lanes to make gel slicing easier. Following electrophoresis, run the first dimension at one volt per centimeter for approximately 12 to 15 hours. The low voltage and low percent AGROS gel serves to separate the DNA fragments primarily based upon size.After the first dimension has run, use a large butcher's knife to cut out the first lane containing the Lambda hind three marker and stain with atherium bromide. For the second dimension, slice out the gel lanes using the large butcher's knife using the Lambda Hind three marker as a guide crop and discard the region below where the linearized plasmid is expected to run on each lane. To cast the second dimension, place each slice horizontally across the top of an empty gel caster.Prepare a solution of 1%AROS in one XTBE and cool to 55 degrees Celsius. Once cooled, pour the gel solution in to cast the second dimension, making sure to completely cover the gel slices. It is important to ensure the slices are evenly oriented and the castor's level before pouring the gel.Once the gel has set, run the second dimension, five and a half to seven hours at 6.5 vols per centimeter in an electrophoresis unit that allows the buffer to recirculate the high voltage and high percent Agros gel effectively separates the DNA fragments based on their shape as well as their size. Non-linear shapes run more slowly through the second dimension following electrophoresis. Rinse the gel in water once and then wash it twice in 400 milliliters of 0.25 molar hydrochloric acid for 15 minutes with gentle agitation.The acid washes serve to partially nick the DNA molecules into smaller fragments that transfer more efficiently during the southern analysis and are necessary due to the large size and unusual shape of the DNA replication intermediates. To prepare the gel for alkali transfer, rinse the gel again in water, then wash it twice in 400 milliliters of 0.4 molar sodium hydroxide for 30 minutes. The DNA in the gels is then transferred to a high bond n plus nylon membrane using a downward alkali transfer system with 0.4 molar sodium hydroxide as described in the written portion of this procedure, let the DNA transfer for six to 12 hours following DNA transfer continue with probe hybridization as outlined in the written protocol.Once the membrane has been probed and washed, place the membrane on a paper towel until the liquid has visibly disappeared. Then wrap the membrane in polyvinyl chloride plastic wrap and expose it to a phospho imager screen. Finally, the radioactivity can be visualized and quantified using a storm eight 40 and its associated image quant.The migration pattern of PV two digested PBR 3 22 plasmid observed by 2D aros gel analysis is diagrammed. Non replicating linear plasmids run as linear 4.4 KB fragment replicating plasmid form Y shaped structures that migrate slower than the non replicating fragments due to their larger size and non-linear shape. This migration pattern forms an arc that extends out from the linear region towards the well following UV irradiation.Double Y or X shaped molecules are observed in the cone region and migrate more slowly than the arc of Y shaped molecules. An example of the southern analysis of the 2D gel probed with P 32 labeled PBR 3 22 is shown for cells immediately after UV irradiation and 15 minutes after UV irradiation immediately after UV irradiation. Approximately 1%of the total plasma DNA can be found in the Y arc when cells are rapidly growing in exponential phase following a radiation.A transient increase in YS shaped molecules is observed as blocked replication forks accumulate at damaged sites. The L-shaped replication intermediates also transiently, accumulate and persist until a time that correlates with when the lesions are repaired. We've just shown you how to visualize DNA repair intermediates using two dimensional gel electrophoresis.When doing this procedure, it's important to be aware of shearing forces that can reduce yield and to be gentle with all samples and gels. So that's it. Thanks for watching and good luck with your experiments.

visualization-uv-induced-replication-intermediates-e-coli-using-two

Research

JoVE Journal

Biology

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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Source: Cayron, J., and Lesterlin, C. Multi-scale Analysis of Bacterial Growth Under Stress Treatments. J. Vis. Exp. (2019)This video demonstrates the procedure for assessing E. coli filamentation and DNA replication under cell division-inhibiting antibiotic treatment using plating to measure viability and flow cytometry to analyze cell size and DNA content.

Assessing Antibiotic-Induced Filamentation in E. coli Using Plating Assay and Flow Cytometry

Source: Mettrick, K. A., Lawrence, et al., Inducing a Site-Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System. J. Vis. Exp. (2016)This video demonstrates site-specific replication fork blockage in Escherichia coli using arabinose-induced expression of yellow fluorescent protein-tagged TetR that binds chromosomal tetO arrays.

Fluorescently Tagged Repressor Protein-Mediated Replication Blockage in Escherichia coli

Source: Bratton, B. P., et al., Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization. J. Vis. Exp. (2019)This video demonstrates the method for acquiring and analyzing three-dimensional fluorescence images of bacterial cells to study protein organization at the single-cell level.

Visualizing Bacterial Protein Distribution in Three Dimensions by Fluorescence Imaging

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Isolation and Visualization of Rotavirus Genome via Gel Electrophoresis

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Learn to visualize UV-induced DNA replication intermediates in E. coli using two-dimensional agarose-gel analysis. This method reveals structural changes and repair dynamics in bacterial cells.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

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