We thank Jorge Cebrian and Anastasia Rastokina who started developing this approach in our lab, Massimo Lopes for providing us with pML113 plasmid and invaluable advice, Ylli Doksani for insightful discussions, and members of the Mirkin lab for their support. The work in the Mirkin lab is supported by the National Institute of General Medical Sciences [R35GM130322] and NSF-BSF [2153071].

NOTE: The plasmid designed for our outlined 2DGE analysis in mammalian cells should contain an SV40 origin of replication several kb upstream of structure-prone repeats (Figure 2). Leading and lagging synthesis should be kept in mind when choosing what orientation relative to the origin the repeats should be cloned into the plasmid.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67229/67229fig02.jpg" /> 
Figure 2: Digestion of repeat-containing plasmid for 2DGE analysis. Structure-prone repeats are depicted several kb downstream from the right-moving replication fork. Digestion with unique cutters 1 and 2 will place the repeat sequence on the descending arm of the Y-arc, given the sequence beyond the halfway point of the digested fragment. Abbreviation: 2DGE = Two-dimensional neutral/neutral gel-electrophoresis. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Plasmid transfection into mammalian cells
<ol>
	<li>Seed 600,000 HEK293T cells in a 10 cm tissue culture plate prior to transfection. Allow the cells to recover at 37 &#176;C overnight. 
	NOTE: Many cell lines may be used for this experiment, although cells containing the SV40 Tag are recommended for optimal results. CAUTION: HEK293T cells are considered BSL-2 and all culturing work should be carried out in a biosafety cabinet using appropriate aseptic technique and proper PPE.</li>
	<li>When the cells reach 60% confluency, transfect 8 &#956;g of repeat-containing plasmid DNA into the seeded cells using appropriate transfection reagents in accordance with the manufacturer&#39;s protocol.</li>
	<li>If not isolating intermediates at this time point, aspirate old media and replace with 10 mL of fresh media after 24 h.</li>
	<li>Begin collecting cells 24-48 h following transfection.
	<ol>
		<li>Aspirate media and carefully wash with 10 mL of phosphate-buffered saline (PBS). Detach and collect cells using 0.5 mL of trypsin and spin down at 340 &#215; g for 4 min.</li>
		<li>Aspirate the supernatant and wash the cell pellets with PBS. Spin again at 340 &#215; g for 4 min and aspirate the supernatant. 
		NOTE: The experiment may be paused here by freezing cell pellets at -80 &#176;C. We have found the best resolution isolating replication intermediates 48 h following transfection; however, 24 h has yielded viable results.</li>
	</ol>
	</li>
</ol>
2. Isolation of replication intermediates
<ol>
	<li>Resuspend the cells in 1.5 mL of modified Hirt lysis buffer [10 mM tris-HCl (pH 7.5), 10 mM ethylenediaminetetraacetic acid (EDTA)] in 50 mL conical tubes and begin cell lysis.
	<ol>
		<li>Add sodium dodecyl sulfate (SDS) to a final concentration of 0.6% (approximately 650 &#956;L of stock 2% SDS) and proteinase K to a final concentration of 100 &#956;g/mL (approximately 10 &#956;L of stock 20 mg/mL proteinase K) to remove nucleases.</li>
		<li>Mix gently by pipetting until homogeneous and incubate the mixture at 37 &#176;C for at least 90 min.</li>
	</ol>
	</li>
	<li>Increase NaCl concentration to 1 M (approximately 540 &#956;L of stock 5 M NaCl) and mix gently until homogeneous. Incubate overnight (18-24 h) at 4 &#176;C to allow for precipitation of cell debris, RNA, and protein by salting out. 
	NOTE: The mixture will be highly viscous, so take care and be patient while mixing well.</li>
	<li>The following day, separate the DNA from cell debris, RNA, and protein.
	<ol>
		<li>Centrifuge the mixture at 29,500 &#215; g for 45 min at 4 &#176;C.</li>
		<li>Transfer the supernatant containing DNA, add one volume of phenol:chloroform:isoamyl alcohol 25:24:1 (v/v), and mix briefly until homogeneous. 
		CAUTION: Phenol:chloroform:isoamyl alcohol is a hazardous material and should be handled with appropriate PPE in a chemical fume hood.</li>
		<li>Centrifuge again at 15,000 &#215; g for 5 min at room temperature. Transfer the aqueous layer to a new conical tube.</li>
	</ol>
	</li>
	<li>Precipitate and wash the isolated DNA.
	<ol>
		<li>Add one volume of pure isopropanol and incubate at room temperature for at least 5 min. Spin down the DNA at 15,000 &#215; g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant and wash the pellet with cold 70% ethanol to remove excess salt.</li>
		<li>Spin once more at 15,000 &#215; g for 30 min at 4 &#176;C, air dry, and gently resuspend the pellet in Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA). 
		NOTE: The experiment can be paused here, and samples can be frozen at -20 &#176;C; however, freeze/thaw cycling should be avoided as this can decrease the quality of the DNA replication intermediates.</li>
	</ol>
	</li>
</ol>
3. Sample preparation and 2-dimensional gel electrophoresis
<ol>
	<li>Digest the isolated intermediates of plasmid replication.
	<ol>
		<li>Add 100 units of the appropriate restriction enzymes to the sample to digest the plasmid DNA, specifically placing the repeat-containing sequence on the origin-distal half of the linear fragment (Figure 2). In addition, add DpnI to cut the methylated DNA, thus removing any plasmid DNA that was not fully replicated in cultured human cells. 
		NOTE: For best results, restriction enzymes should be unique cutters that yield a 3-5 kb fragment that places the structure-prone sequence on the descending arm of the Y-arc.</li>
		<li>Incubate the samples at 37 &#176;C for 6-10 h to allow full plasmid digestion.</li>
		<li>Precipitate the DNA with either 2.5 volumes of cold pure ethanol and incubate at -20 &#176;C overnight, or add one volume of isopropanol and incubate at room temperature for 5 min.</li>
		<li>Centrifuge the digested and precipitated samples at 15,000 &#215; g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant and wash the sample with cold 70% ethanol. Spin again at 15,000 &#215; g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant, air dry for 10 min, and resuspend the samples in 15 &#956;L of TE buffer.</li>
	</ol>
	</li>
	<li>Prepare the first-dimension agarose gel at 0.4-0.5% in 1x Tris-borate-EDTA (TBE) (89 mM tris base, 89 mM boric acid, 2 mM EDTA). Allow the solution to solidify for at least 1 h.</li>
	<li>Begin loading the samples into the first dimension.
	<ol>
		<li>Load the ladder within the first 3 cm relative to the gel&#39;s leftmost edge. Then, load the entirety of the prepared samples, ensuring 3 cm between each pair.</li>
		<li>Run the gel in 1x TBE for 19-24 h at 0.85 V/cm to separate the intermediates with respect to their size. Ensure the chamber is covered to protect the samples from light, which may cause damage to the DNA.</li>
	</ol>
	</li>
	<li>The following day, remove the gel from the buffer and estimate the location of the digested linear fragment using a ruler.
	<ol>
		<li>Excise the first 3 cm of the gel containing the ladder and stain the gel segment in 1x TBE containing 0.3 &#956;g/mL ethidium bromide for 10-15 min. Visualize the ladder using a gel documentation system.</li>
		<li>Add 1.3 cm to the estimated location, yielding value a. Then, subtract 7.5 cm from value a, yielding value b. Align the ruler against the first-dimension gel and cut horizontally across at values a and b. Then, cut vertically down the 3 cm space reserved for each sample. Refer to Figure 3 for a visual schematic.</li>
		<li>In a new casting tray, rotate the segments clockwise and place them in the position of the sample wells (Figure 3).</li>
	</ol>
	</li>
	<li>Prepare the second-dimension agarose gel at a concentration of 1-1.3% in 1x TBE at 0.3 &#956;g/mL ethidium bromide.
	<ol>
		<li>Upon cooling to approximately 55 &#176;C, pour the second-dimension gel over the rotated first-dimension segments and allow it to solidify for at least 1 h.</li>
		<li>Transfer the second dimension to a chamber with 1x TBE at 0.3 &#956;g/mL ethidium bromide, and allow the gel to equilibrate for at least 30 min.</li>
		<li>Run the gel, again covered, for 9-10 h at 4.23 V/cm at 4 &#176;C to separate the intermediates with respect to their shape.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67229/67229fig03.jpg" /> 
Figure 3: Excision of first-dimension intermediates prior to second-dimension separation. Following visualization of the ladder, the mobility of unreplicated fragments can be estimated. (I)&#160;This value can then be used to determine appropriate cut sites (a and b) to excise it and its replicated counterparts (II). The section of the gel should then be rotated and placed in the position of wells for the second-dimension separation. Abbreviation: CW = clockwise. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Southern blotting and hybridization with radiolabeled probe
<ol>
	<li>Remove the second-dimension gel from the chamber and depurinate the DNA fragments for 10 min in a 0.24 M HCl solution with gentle rocking. Rinse the gel with deionized water and soak it in 0.4 M NaOH for 10-15 min. 
	CAUTION: HCl and NaOH are corrosive and should be handled with appropriate PPE in a chemical fume hood.</li>
	<li>Begin assembling the Southern blot to facilitate the transfer of the separated intermediates from the gel to the membrane. Refer to Figure 4 for a comprehensive schematic.
	<ol>
		<li>Fill a sufficiently sized container with 1 L of 0.4 M NaOH.</li>
		<li>Align a long glass sheet across the container and fold (along the length) two long sheets of chromatography paper perpendicular across the glass sheet, extending into the container of NaOH.</li>
		<li>Wet the top of the paper with NaOH and carefully remove any air bubbles under its surface.</li>
		<li>Soak three sheets of chromatography paper with NaOH and place them over the folded paper, again removing any bubbles.</li>
		<li>Flip the second-dimension gel upside-down and transfer it over the papers.</li>
		<li>Wet a positively charged nylon membrane (0.45 &#956;m pore size) with DI water and place it over the gel.</li>
		<li>Finally, add three more sheets of paper, wet with DI water, over the membrane.</li>
		<li>Cover any exposed NaOH in the bottom container with plastic wrap to prevent evaporation. Place a stack of either napkins or paper towels over the blot, ensuring it is 0.3-0.5 m tall. Place a weight on the top, compressing the entire blot to facilitate tight capillary action. Allow at least 2 days for DNA to transfer to the membrane. 
		NOTE: The length and width of the chromatography paper and membrane depend on the size of the agarose gel used for the second dimension. For the most efficient transfer, use paper and membrane with the same dimensions as those of the gel.</li>
	</ol>
	</li>
	<li>Following transfer, crosslink the DNA to the membrane using a UV crosslinker at 120 &#956;J/cm2 for 1 min. 
	NOTE: The experiment may be paused here by placing the membrane in an airtight, dry, and clean sheet protector at room temperature.</li>
	<li>Wash the membrane 2x for 5 min with 2x saline sodium citrate (SSC buffer) (0.3 M NaCl, 0.03 M sodium citrate).</li>
	<li>Prehybridize the membrane with 0.18 mL/cm2 of Church &#38; Gilbert&#39;s hybridization buffer [1 mM EDTA, 1% bovine serum albumin (BSA), 0.5 M sodium phosphate, 7% SDS] at 65 &#176;C, rotating in a hybridization incubator for at least 2 h. 
	NOTE: The membrane may prehybridize for several days.</li>
	<li>Prepare the radiolabeled probe using &#945;-32P dATP or dCTP and a DNA labeling kit in accordance with the manufacturer&#39;s protocol. 
	CAUTION: Radiolabeled dNTPs are hazardous, and appropriate PPE should be worn when handling them. All radioactive work should be performed behind shielding, and trained individuals should be monitored for radiation uptake using dosimeters.
	<ol>
		<li>Design a 400-900 bp linear DNA fragment complementary to the restriction digested sequence (Figure 2) and amplify the fragment using polymerase chain reaction (PCR). 
		NOTE: We recommend having a stock PCR fragment of 50-100 ng/&#956;L</li>
		<li>Combine 100 ng of the complementary PCR fragment with DNA Pol I, Klenow fragment (3&#39; 5&#39; exo-) buffer, and random decanucleotide oligos.</li>
		<li>Denature the fragment at 100 &#176;C for 10 min.</li>
		<li>Add 5 units of DNA Pol I, Klenow fragment (3&#39; 5&#39; exo-), 50 &#956;Ci of &#945;-32P dNTP, and 30-50 &#956;mol of dNTP mix deficient in radiolabeled dNTP type to the sample.</li>
		<li>Incubate at 37 &#176;C for 10 min to allow for polymerization and radiolabeled dNTP incorporation.</li>
		<li>Add 30-50 &#956;mol of previously absent dNTP type to the sample and incubate at 37 &#176;C for 10 min.</li>
		<li>Purify the radiolabeled fragment using a spin column at 3,000 &#215; g for 2 min.</li>
	</ol>
	</li>
	<li>Add radiolabeled probe to 50 mL of hybridization buffer and incubate with the membrane overnight at 65 &#176;C rotating in a hybridization incubator.</li>
	<li>The following day, remove the probe and wash the membrane 2x with wash buffer 1 (0.1x SSC, 0.1% SDS) at 42 &#176;C and 2x with wash buffer 2 (2x SSC, 0.1% SDS) at 65 &#176;C. 
	&#8203;NOTE: All washes should be performed with fast rotations for 15 min in the incubator.</li>
	<li>Dry the membrane for 10 min and place it in a thin, transparent sheet protector. Store the sealed membrane in a controlled, radiation-resistant cassette with a phosphor-sensitive screen. Allow the membrane to be exposed to the screen for 1-10 days.</li>
	<li>Visualize the results using a biomolecular imager set to phosphor imaging. Wash and re-expose if necessary.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67229/67229fig04.jpg" /> 
Figure 4: Assembly of Southern blot. Comprehensive schematic of a typical apparatus used for Southern blot transfer of intermediates from the second dimension onto a nylon membrane. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

2D Gel Electrophoresis for Analyzing DNA Replication and Fork Stalling Events

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+and+termination+of+DNA+replication+in+human+rRNA+genes.">Initiation and termination of DNA replication in human rRNA genes.</a> Mol Cell Biol. 13 (10), 6600-6613 (1993).</li><li>Fanning, E., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV40+DNA+replication:+From+the+A+gene+to+a+nanomachine.">SV40 DNA replication: From the A gene to a nanomachine.</a> Virology. 384 (2), 352-359 (2009).</li><li>Sogo, J. M., Stahl, H., Koller, T., Knippers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+replicating+simian+virus+40+minichromosomes:+The+replication+fork,+core+histone+segregation+and+terminal+structures.">Structure of replicating simian virus 40 minichromosomes: The replication fork, core histone segregation and terminal structures.</a> J Mol Biol. 189 (1), 189-204 (1986).</li><li>Weisshart, K., Taneja, P., Fanning, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+replication+protein+A+binding+site+in+simian+virus+40+(SV40)+T+antigen+and+its+role+in+the+initial+steps+of+SV40+DNA+replication.">The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication.</a> J Virol. 72 (12), 9771-9781 (1998).</li><li>Sowd, G. A., Fanning, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wolf+in+sheep's+clothing:+SV40+co-opts+host+genome+maintenance+proteins+to+replicate+viral+DNA.">A Wolf in sheep's clothing: SV40 co-opts host genome maintenance proteins to replicate viral DNA.</a> PLoS Pathog. 8 (11), e1002994 (2012).</li><li>. BLAST: Basic Local Alignment Search Tool Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/Blast.cgi">https://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (2024)</li><li>Hirt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures.">Selective extraction of polyoma DNA from infected mouse cell cultures.</a> J Mol Biol. 26 (2), 365-369 (1967).</li><li>Lopes, M., Foiani, M., Sogo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mechanisms+control+chromosome+integrity+after+replication+fork+uncoupling+and+restart+at+irreparable+UV+lesions.">Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.</a> Mol Cell. 21 (1), 15-27 (2006).</li></ol>

The authors have no conflicts of interest to disclose.

2DGE provides a semi-quantitative and comprehensive image of the relative populations of intermediates that arise during the replication of a particular sequence. Given that the fragile molecular structures of replication forks must be maintained throughout this procedure, great care should be implemented to prevent physical shearing and chemical denaturation. Therefore, it is highly recommended that any alkaline treatment be avoided during plasmid isolation. To avoid this, we and others have implemented a modified form ...

Short tandem repeats (STR) are small, typically 2-9 base pairs (bp), repetitive sequences of DNA that constitute around 3% of the human genome1. STR play an important role in gene regulation2; however, their repetitive composition leaves them prone to non-canonical DNA secondary structure formation and subsequent genetic instability3,4. From left-handed helices to hairpins/cruciforms, to three and four-stranded helices, these alternative DNA structures cause intrinsic challenges for the replisome. A natural prerequisite for secondary structure formation is DNA unwinding, which is a prerequisite for DNA replication. This presents a unique conundrum for genome functioning as many of these structures can form during replication, hindering replisome progression and ultimately causing replication fork stalling5,6,7, or in severe cases, fork collapse and DNA breakage8,9. Both the restart of stalled forks and DNA repair pathways have been shown to lead to repeat instability, such as repeat expansions10,11 and complex genome rearrangements (CGR)12,13. These events can result in the development of roughly 60 human diseases known as repeat expansion disorders, including Fragile X syndrome, Huntington&#39;s disease, Friedreich&#39;s ataxia, and others14,15 as well as CGR diseases, such as Emmanuel syndrome16. Therefore, to better understand the mechanisms of human disease driven by repeat instability, it is imperative to study the details of replication fork progression through those repeats.
A technique for studying replication progression emerged in the mid-1980s when Brewer and Fangman sought to provide direct evidence that replication initiation in Saccharomyces cerevisiae occurs at autonomous replication sequence (commonly known as ARS) elements17. In doing so, they separated structures of yeast replication intermediates in agarose, adapting an earlier method from Bell and Byers known as 2-dimensional Neutral/Neutral gel-electrophoresis (2DGE)18. This technique utilized the fact that nonlinear DNA travels differently in agarose gel than its linear equivalent of the same mass. More specifically, in 2DGE, isolated DNA is separated in two perpendicular dimensions, first primarily by size and then primarily by shape, to create a comprehensive map of replication at a particular region of interest. In their original paper, Brewer and Fangman demonstrated this as an arc composed of &#34;simple Y&#34; structures or replication forks bridging unreplicated DNA to their replicated counterparts. They further describe other observed intermediates as &#34;bubbles&#34; and &#34;double Ys,&#34; representing replication origins and converging forks, respectively.
2DGE can be used to study the relative populations of DNA replication intermediates at a given time. Therefore, if one population of intermediates is more prevalent than another, this would be evident upon visualization. This makes 2DGE an especially useful tool for studying replication progression through challenging sequences, like structure-forming repeats. For example, if the region analyzed contains a sequence capable of inducing replication fork stalling, this would present as a bulge on the arc (Figure 1A), indicating an accumulation of replication forks at that locus. This can be seen with the replication of both hairpin-forming repeat sequences in yeast19,20,21 and triplex-forming repeats in human cells22,23,24. In addition to stalling, 2DGE can be used to observe DNA structures that do not conform to the standard simple Ys formed during replication, as in the case of recombinant intermediates25. These intermediates have a heavier and more branched X-shaped structure and, therefore, travel slower in both the first- and second dimensions than standard replication forks. Similar results can also be observed with regards to replication fork reversal20,24,26. In response to strong replication stress, eukaryotic cells have been shown to utilize replication fork reversal to rescue stalled forks. These reversed forks have similar molecular weight to stalled forks; yet their chicken-foot structure results in slower electrophoretic mobility in the second dimension relative to their Y-shaped complements, resulting in an extension up and out from the arc.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67229/67229fig01.jpg" /> 
Figure 1: 2D Gel electrophoresis analysis of DNA replication. (A)&#160;Schematic of a typical 2DGE depicting replication through a structure-forming repeat capable of inducing fork stalling. Intermediate size and structure will influence electrophoretic mobility. (B)&#160;Sample Y-arc with ascending and descending arms respectively labeled. Abbreviation: 2DGE = Two-dimensional neutral/neutral gel-electrophoresis. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Naturally, one of the most important aspects of 2DGE concerns the quality and quantity of replication intermediates. However, the resolution of 2DGE analysis of replication through endogenous loci in mammalian cells is insufficient for a single copy target sequence within the 6 &#215; 109 bp diploid human genome, though it has been done for multi-copy genes, such as heavily amplified DHFR locus27 or ribosomal RNA28. SV40-based replication is an efficient and well-characterized means of studying replication in eukaryotic cells29. It provides a reliable model of eukaryotic replication that utilizes most of the host replisome machinery to replicate the viral genome, which is parceled into nucleosomes upon infection30,31. Two notable exceptions from the mammalian replisome are that the T-antigen (Tag), instead of the host CMG complex, serves as replicative DNA helicase, and DNA polymerase delta synthesizes both leading and lagging DNA strands32. We have taken advantage of this system by placing pathogenic stretches of structure-forming repeats downstream from an SV40 origin of replication within a plasmid that was originally created in the Massimo Lopes lab22. Importantly, this plasmid also contains the gene encoding for Tag itself, thus resulting in its constitutive and extremely potent replication upon transfection into a variety of cultured human cells. This feature gives rise to a large quantity of products, ideal for 2DGE analysis of the intermediates formed during and in response to the replication of pathogenic repeats in human cells. Here, we describe a detailed method of visualizing the replication of structure-forming repeats within the SV40-based human episome using 2-dimensional gel electrophoresis.

<table><tbody><tr><td>10x TBE Buffer</td><td>Bio Rad</td><td>1610733</td><td></td></tr><tr><td>20x SSC Buffer</td><td>Fisher Scientific</td><td>BP1325-1</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>a-&lt;sup&gt;32&lt;/sup&gt;P dATP, 3000 Ci/mmol&amp;nbsp;</td><td>Revvity</td><td>BLU512H250UC</td><td></td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP160-500</td><td></td></tr><tr><td>Amersham Hybond-N+</td><td>Fisher Scientific</td><td>RPN303B</td><td></td></tr><tr><td>BAS Storage Phosphor Screens</td><td>Fisher Scientific</td><td>28956482</td><td></td></tr><tr><td>Church and Gibert&#039;s hybriddization buffer</td><td>Fisher Scientific</td><td>50-103-5408</td><td></td></tr><tr><td>DecaLabel DNA labeling kit</td><td>ThermoFisher Scientific</td><td>K0622</td><td></td></tr><tr><td>DMEM, high gluctose, GltaMAX Supplement, pyruvate</td><td>ThermoFisher Scientific</td><td>10569010</td><td></td></tr><tr><td>DpnI</td><td>New England Biolabs</td><td>R0176S</td><td>Additional restriction enzymes will need to be purchased as well</td></tr><tr><td>EDTA 0.5 M, pH 8</td><td>Fisher Scientific</td><td>BP2482500</td><td></td></tr><tr><td>Ethanol, 70%</td><td>Fisher Scientific</td><td>BP82031GAL</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>Hydrochloric acid solution, 12 M</td><td>Millipore Sigma</td><td>13-1683</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP26184</td><td></td></tr><tr><td>jetPRIME DNA and siRNA Transfection Reagent with Buffer</td><td>VWR</td><td>101000027</td><td></td></tr><tr><td>MycoZap Plus-CL</td><td>VWR</td><td>75870-448</td><td></td></tr><tr><td>NaCl</td><td>Millipore Sigma</td><td>746398-500G</td><td></td></tr><tr><td>Nalgene Oak Ridge High-Speed Centrifuge Tubes</td><td>ThermoFisher Scientific</td><td>3139-0050</td><td></td></tr><tr><td>Phosphate Buffer Saline, pH 7.4</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Phosphate Buffer Saline, pH 7.5</td><td>ThermoFisher Scientific</td><td>10010024</td><td></td></tr><tr><td>Proteinase K</td><td>ThermoFisher Scientific</td><td>EO0491</td><td></td></tr><tr><td>Proteinase K</td><td>ThermoFisher Scientific</td><td>EO0492</td><td></td></tr><tr><td>Pure Cellulose Chromatography Paper</td><td>Fisher Scientific</td><td>05-714-4</td><td></td></tr><tr><td>Pure Cellulose Chromatography Paper</td><td>Fisher Scientific</td><td>05-714-5</td><td></td></tr><tr><td>Ruler</td><td>Fisher Scientific</td><td>09-016</td><td></td></tr><tr><td>Scalpel</td><td>Fisher Scientific</td><td>12-460-451</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Millipore Sigma</td><td>436143-25G</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S25548</td><td></td></tr><tr><td>Sorval LYNX 4000 Superspeed Centrifuge</td><td>ThermoFisher Scientific</td><td>75006580</td><td></td></tr><tr><td>Sub-cell Horizontal Electrophoresis System</td><td>Bio Rad</td><td>1704401</td><td></td></tr><tr><td>TH13-6 x 50 Swinging Bucket Rotor</td><td>ThermoFisher Scientific</td><td>75003010</td><td></td></tr><tr><td>Tris-HCl 1 M, pH 7.5</td><td>Fisher Scientific</td><td>BP1757-500</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr></tbody></table>

electrophoretic analysis of replication through structure-prone dna repeats within the sv40-based human episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

If successful, upon visualization, a sharp arc of replication forks can be observed extending up and out from the massive 1n spot (Figure 5A). The size of a fragment, or percentage that is replicated, determines the fragment&#39;s mobility in the first dimension. As the intermediates develop a more jointed structure, they will begin to travel more slowly in the second dimension. Therefore, if an intermediate has traveled slowly in both dimensions, it can be asserted that it is a highly repli...

Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

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

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

electrophoretic-analysis-replication-through-structure-prone-dna

Research

JoVE Journal

Genetics

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

Video: Author Spotlight: Characterizing DNA Replication of Pathogenic Repeats to Uncover Mechanisms of Replication Fork Stalling and Expansion

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Immunology and Infection

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

Biology

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

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

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.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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

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

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites