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

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 instability^3,4. 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 stalling^5,6,7, or in severe cases, fork collapse and DNA breakage^8,9. Both the restart of stalled forks and DNA repair pathways have been shown to lead to repeat instability, such as repeat expansions^10,11 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 others^14,15 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 yeast^19,20,21 and triplex-forming repeats in human cells^22,23,24. 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 reversal^20,24,26. 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.

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. Please click here to view a larger version of this figure.

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 infection^30,31. 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.