setting up two-dimensional gel electrophoresis and southern blotting for dna fragment analysis within the sv40-based human episome

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

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.

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

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

The scope of our research is to characterize DNA replication of unstable pathogenic repeats in human cells. We hope to answer questions such as, what specific repeats cause replication fork stalling? What unique secondary structures are these repeats forming?And how do these repeats expand during DNA replication? While we know that many of these structure forming repeats can impair DNA replication, it's still unclear what exact steps happen next. We're hoping that this protocol will help answer this question and shed light on the mechanisms of how these pathogenic repeats expand.2D gels provide the unique opportunity to actually visualize the structures in relative populations of molecules that can arise during DNA replication. Within one single experiment, you essentially have the complete map of what replication progression looks like through a specific DNA sequence.

Setting Up Two-Dimensional Gel Electrophoresis and Southern Blotting for DNA Fragment Analysis Within the SV40-Based Human Episome

To begin, place the reagents required for the preparation of gel electrophoresis on a working platform. Add Tris/Borate/EDTA, or TBE, to agarose and heat the solution in a microwave. Then, pour the 0.4-0.5%agarose into a casting tray to prepare a first-dimension agarose gel and let it solidify for one hour.After solidification, load the ladder within the first 3 centimeters relative to the gel's leftmost edge. Then load the DNA samples digested with restriction enzymes, ensuring a 3 centimeter distance between each pair. Run the gel in TBE for 19-24 hours at 0.85 volts per centimeter to separate the intermediates by size.The following day, remove the gel from the buffer. Excise the first 3 centimeters of the gel containing the ladder. Stain the gel segment in TBE containing 0.3 micrograms per milliliter of ethidium bromide for 10-15 minutes.Then visualize the ladder using a gel documentation system. On the agarose gel, add 1.3 centimeters to the estimated location, yielding a value of A.Then subtract 7.5 centimeters 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 centimeter space reserved for each sample. In a new casting tray, rotate the segments clockwise and place them in the position of the sample wells.Prepare the second-dimension agarose gel at a concentration of 1-1.3%in TBE with 0.3 micrograms per milliliter of ethidium bromide. Heat the gel, and upon cooling to approximately 55 degrees Celsius, pour it over the rotated first-dimension segments. Allow the gel to solidify for one hour.After solidification, transfer the gel to a chamber containing TBE at 0.3 micrograms per milliliter of ethidium bromide, and allow it to equilibrate for 30 minutes. Cover the gel and run for 9-10 hours at 4.23 volts per centimeter at 4 degrees Celsius. Remove the second-dimension gel from the chamber and depurinate the DNA fragments for 10 minutes in a 0.24 molar hydrochloric acid solution with gentle rocking.Rinse the gel with deionized water and soak it in 0.4 molar sodium hydroxide for 10-15 minutes. Then fold two long sheets of chromatography paper perpendicular across the glass sheet, extending into the container. To assemble the southern blot, fill a container with 1 liter of 0.4 molar sodium hydroxide.Align a long glass sheet across the container. Wet the top of the paper with sodium hydroxide and carefully remove any air bubbles under its surface. Soak three sheets of chromatography paper with sodium hydroxide and place them over the folder paper.Remove any air bubbles. Flip the second-dimension gel upside down and place it over the chromatography papers. Wet a positively charged nylon membrane with deionized water and place it over the gel.Then place three chromatography sheets wet with deionized water over the membrane. Cover any exposed sodium hydroxide in the container with plastic wrap to prevent evaporation. Place a 0.3-0.5 meter tall stack of napkins or paper towels over the blot.Compress the entire blot with a weight to facilitate tight capillary action, and leave it for two days for DNA transfer to the membrane.

setting-up-two-dimensional-gel-electrophoresis-southern-blotting-for

Research

JoVE Journal

Genetics

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.

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 ...