Name: Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome (Video) | JoVE Journal
Uploaded: 2024-09-13T15:00:00.000+00:00
Duration: 5 min 22 s
Description: Learn 2-dimensional gel electrophoresis to analyze DNA replication through pathogenic repeats in human cells. This method visualizes replication fork stalling and secondary structures.

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5 minutes

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="Genomic mapping diagram, circular DNA plasmid, SV40 origin, probe sequence, fragment analysis." class="xfigimg" src="/files/ftp_upload/67229/67229fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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 °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 μg of repeat-containing plasmid DNA into the seeded cells using appropriate transfection reagents in accordance with the manufacturer'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 × g for 4 min.</li>
		<li>Aspirate the supernatant and wash the cell pellets with PBS. Spin again at 340 × g for 4 min and aspirate the supernatant. 
		NOTE: The experiment may be paused here by freezing cell pellets at -80 °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 μL of stock 2% SDS) and proteinase K to a final concentration of 100 μg/mL (approximately 10 μL of stock 20 mg/mL proteinase K) to remove nucleases.</li>
		<li>Mix gently by pipetting until homogeneous and incubate the mixture at 37 °C for at least 90 min.</li>
	</ol>
	</li>
	<li>Increase NaCl concentration to 1 M (approximately 540 μL of stock 5 M NaCl) and mix gently until homogeneous. Incubate overnight (18-24 h) at 4 °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 × g for 45 min at 4 °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 × 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 × g for 30 min at 4 °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 × g for 30 min at 4 °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 °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 °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 °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 × g for 30 min at 4 °C.</li>
		<li>Decant the supernatant and wash the sample with cold 70% ethanol. Spin again at 15,000 × g for 30 min at 4 °C.</li>
		<li>Decant the supernatant, air dry for 10 min, and resuspend the samples in 15 μL 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'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 μg/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 μg/mL ethidium bromide.
	<ol>
		<li>Upon cooling to approximately 55 °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 μg/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 °C to separate the intermediates with respect to their shape.</li>
	</ol>
	</li>
</ol>
<img alt="2D gel electrophoresis diagram, protein separation method with excision, rotation, second dimension pour." class="xfigimg" src="/files/ftp_upload/67229/67229fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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) 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 μm 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 μJ/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 &amp; Gilbert's hybridization buffer [1 mM EDTA, 1% bovine serum albumin (BSA), 0.5 M sodium phosphate, 7% SDS] at 65 °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 α-32P dATP or dCTP and a DNA labeling kit in accordance with the manufacturer'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/μL</li>
		<li>Combine 100 ng of the complementary PCR fragment with DNA Pol I, Klenow fragment (3' 5' exo-) buffer, and random decanucleotide oligos.</li>
		<li>Denature the fragment at 100 °C for 10 min.</li>
		<li>Add 5 units of DNA Pol I, Klenow fragment (3' 5' exo-), 50 μCi of α-32P dNTP, and 30-50 μmol of dNTP mix deficient in radiolabeled dNTP type to the sample.</li>
		<li>Incubate at 37 °C for 10 min to allow for polymerization and radiolabeled dNTP incorporation.</li>
		<li>Add 30-50 μmol of previously absent dNTP type to the sample and incubate at 37 °C for 10 min.</li>
		<li>Purify the radiolabeled fragment using a spin column at 3,000 × 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 °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 °C and 2x with wash buffer 2 (2x SSC, 0.1% SDS) at 65 °C. 
	​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="Capillary action diagram of blotting process; nylon membrane, agarose gel in NaOH for DNA transfer." class="xfigimg" src="/files/ftp_upload/67229/67229fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
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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<li>Giannattasio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+recombination-mediated+damage-bypass+by+template+switching%5BTitle%5D)+AND+%22Nat+Struct+Mol+Biol%22%5BJournal%5D)">Visualization of recombination-mediated damage-bypass by template switching.</a> Nat Struct Mol Biol. 21 (10), 884-892 (2014).</li>
<li>Hisey, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenic+CANVAS+%28AAGGG%29n+repeats+stall+DNA+replication+due+to+the+formation+of+alternative+DNA+structures%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.</a> Nucleic Acids Res. 52 (8), 4361-4374 (2024).</li>
<li>Kalejta, R. F., Lin, H. B., Dijkwel, P. A., Hamlin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+replication+intermediates+in+the+amplified+CHO+dihydrofolate+reductase+domain+by+two+novel+gel+electrophoretic+techniques%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques.</a> Mol Cell Biol. 16 (9), 4923-4931 (1996).</li>
<li>Little, R. D., Platt, T. H., Schildkraut, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+and+termination+of+DNA+replication+in+human+rRNA+genes%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((SV40+DNA+replication%3A+From+the+A+gene+to+a+nanomachine%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">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/pubmed?term=((Structure+of+replicating+simian+virus+40+minichromosomes%3A+The+replication+fork%2C+core+histone+segregation+and+terminal+structures%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((The+replication+protein+A+binding+site+in+simian+virus+40+%28SV40%29+T+antigen+and+its+role+in+the+initial+steps+of+SV40+DNA+replication%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">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/pubmed?term=((A+Wolf+in+sheep%27s+clothing%3A+SV40+co-opts+host+genome+maintenance+proteins+to+replicate+viral+DNA%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">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>National Library of Medicine: National Center for Biotechnology Information. BLAST: Basic Local Alignment Search Tool. , 
 <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/pubmed?term=((Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Multiple+mechanisms+control+chromosome+integrity+after+replication+fork+uncoupling+and+restart+at+irreparable+UV+lesions%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">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 of the Hirt isolation of DNA, established by Hirt in 196733. Here, DNA is separated from proteins, RNA, and other cellular debris by treating cell lysate with 1 M NaCl at 4 &#176;C overnight. While this method has proven exceptional for preserving the quality of replication intermediates, it does not appear to fully separate plasmid DNA from genomic DNA. This should be kept in mind when designing the sequence for radiolabeled probing, as the designed probe should not have high sequence similarity with any genomic DNA to best avoid off-target binding. Further, the integrity of replication intermediates may be greatly increased through UV-psoralen crosslinking. Here, cells can be incubated with psoralen for several minutes in the dark before being UV irradiated at 366 nm34, thus creating covalent bonds between the DNA molecules. In addition to reinforcing the cohesiveness of intermediate structures, this may also alleviate any concern of structures forming during isolation rather than in vivo.
One aspect that should be considered when designing this experiment is repeat placement on the resolved final arc. The first half of the arc extending out from the 1n spot is denoted as the ascending arm, while the second half is known as the descending arm (Figure 1B). To observe replication fork stalling, repeat placement on either arm of the arc should be sufficient. However, to observe more structured intermediates that arise at repeat sequences, such as postreplicative junctions, we recommend placing the repeat sequence on the descending arm. This is largely due to the slower electrophoretic mobility of these intermediates in both dimensions, which may result in co-migration with simple Ys structures farther along in their progression if the repeat-containing sequence were placed on the ascending arm, thus hindering any detailed analysis. For the best resolution, we recommend placing the repeat sequence somewhere between 60% and 90% within the fragment of interest relative to the origin of replication. 
Attention to detail should be utilized during the DNA transfer step and subsequent treatment of the membrane. It is extremely important that the Southern blot is assembled in a manner that facilitates an even and tight transfer of DNA to the membrane. This means that all components on the transfer are free of air bubbles and that the apparatus is uniform across its surface. To assist in this, it is standard to flip the second-dimension gel over prior to adding it to the Southern blot. The bottom of the gel is assumed to be flatter than the top; therefore, flipping the gel over ensures the smoothest possible transfer of DNA to the membrane. 
If a strong background is present in the final resolution of the blot, this may be indicative of the non-specific binding of the radiolabeled probe. This can be alleviated in a number of ways. First, it should be checked that the probe does not contain high sequence similarity to any genomic DNA. This can be easily verified using the Nucleotide Basic Local Alignment Search Tool (BLAST), which is readily available through the NIH website35. Otherwise, nonspecifically bound probes can be removed through additional stringent washes. We recommend beginning with two additional washes of buffer 1 (0.1x SSC, 0.1% SDS) at 42 &#176;C in 15 min intervals each; however, more washes may be needed. Finally, the temperature at which hybridization occurs may be altered as well. For most probes containing a G/C content of 40-60%, 65 &#176;C tends to be sufficient, though this is not a static value. Efficient binding of the probe is dependent on its melting temperature and, therefore, may require alterations to the hybridization temperature.
Given that 2DGE provides an overview of the relative populations of replication intermediates at a given time, one of its greatest drawbacks is that it does not provide a strong measure of replication progression timing. For this purpose, we recommend using single-molecule analysis like DNA combing. Further, while 2DGE allows for analysis of the replication intermediates that arise in response to stalling, genetic controls must be established to uncover their exact identity, which can be timely. Although costly, electron microscopy remains superior in identifying the exact structure of DNA molecules.

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's disease, Friedreich'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 "simple Y" structures or replication forks bridging unreplicated DNA to their replicated counterparts. They further describe other observed intermediates as "bubbles" and "double Ys," 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="DNA replication fork stalling diagram, Y-arc structure, size vs. branched, molecular biology." class="xfigimg" src="/files/ftp_upload/67229/67229fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: 2D Gel electrophoresis analysis of DNA replication. (A) 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) 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 × 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

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'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 replicated molecule with significant junctional variation from conventional replication forks. In the case of lagging-strand synthesis of the (GAA)100 repeat, fork stalling is indicated by a darkened defined spot on the arc (I), representing an accretion of replication forks upon encounter of the repeat (Figure 5B), likely due to triplex formation. This is accompanied by heavier, more branched intermediates (II and III) above the stall site. We have described the nature of these intermediates in more depth in Rastokina et al. 202324. In short, these likely represent a combination of forks reversing in response to the stall (II), in addition to the advent of the converging fork (Figure 5B) (III).
One observation that may be made upon visualization is a less-than ideal, broad arc. At worst, this may present as a complete doubling of the arc. Typically, this represents poor separation of intermediates in the first dimension. This may be attributed to several factors, the most likely of which is either salt or protein contamination in the replication intermediate sample. A phenol:chloroform purification after digesting intermediates, followed by extensive washing with 70% ethanol, can minimize this risk. However, it should be noted that additional phenol exposure can increase the chance of denaturing intermediates, which may present as a darkened intensity of linear DNA under the arc, representing collapsed replication intermediates (Figure 5C).
In the worst-case scenario, visualization may result in a complete absence of replication intermediates, evidenced by the lack of arc (Figure 5D). It is unlikely that the absence of an arc is attributed to denatured replication intermediates, as there is no darkened spot under the expected area of the arc. Given the presence of a particularly small 1n spot (IV), the overall quantity of DNA present in this depicted example is minimal, and, therefore, this issue may be caused by under-replication of the plasmid or an overall poor transfection or isolation of DNA.
<img alt="DNA replication diagram; SV40 origin with (GAA) repeat. Gel electrophoresis analysis of DNA fragments." class="xfigimg" src="/files/ftp_upload/67229/67229fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Potential outcomes of 2DGE analysis. (A) Successful 2DGE analysis of replication through the structure-forming (GAA)100 repeat in HEK293T cells. (I) refers to forks stalled at the repeat, whereas (II) and (III) refer to more structured intermediates that arise in response to the stall. An illustration of the 2.7 kb fragment is depicted above. (B) Representative intermediates that arise during replication of the (GAA)100 repeat. Using siRNA knockdowns of replication fork reversal and restart genes, genetic controls were established to identify the nature of these intermediates. (C) Depiction of unresolved replication intermediates that may appear if forks are denatured during isolation. (D) An unsuccessful 2DGE experiment as shown by a complete absence of replication intermediates. IV represents linear, unreplicated fragments of the digested plasmid. Abbreviation: 2DGE = Two-dimensional neutral/neutral gel-electrophoresis. <a href="https://www.jove.com/files/ftp_upload/67229/67229fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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Research

JoVE Journal

Genetics

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Learn 2-dimensional gel electrophoresis to analyze DNA replication through pathogenic repeats in human cells. This method visualizes replication fork stalling and secondary structures.

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

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