Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures

Abstract

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A₂G₃)_n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the main pathogenic (A₂G₃)_n and the main nonpathogenic (A₄G)_n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in supercoiled DNA reveals triplex H-DNA formation by the pathogenic repeat. Consistently, bioinformatic analysis of the S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A₂G₃)_n motifs over (A₄G)_n motifs in vivo. Finally, the pathogenic, but not the non-pathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that CANVAS-causing (A₂G₃)_n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

Figure 1.. In vitro polymerization through pathogenic and nonpathogenic repeats.

(A) Polyacrylamide gel electrophoresis separation of ThermoSequenase sequencing reactions performed as described in Materials and Methods. Briefly, 5µg of each plasmid and 0.5pmol of primer pre-annealed and the USBio Thermo Sequenase Cycle Sequencing Kit’s 3’-dNTP internal label cycling sequencing instructions were followed with several modifications detailed in Materials and Methods. (B) Schematic of denatured double-stranded plasmid with primers annealed to allow for ThermoSequenase polymerization through the purine- or pyrimidine-rich strand of (A₂G₃)₁₀ or (A₄G)₁₀ in the template strand. Primers are 98 base pairs or 75 base pairs away from repeats for the purine-rich or pyrimidine-rich template, respectively. Created with BioRender. (C) Model for triplex formation as polymerase progresses through the repeats with the purine-rich strand as the template. Created with BioRender. (D) Polyacrylamide gel electrophoresis separation of in vitro T7 DNA polymerase primer extension reaction. 3.125µM single-stranded oligonucleotides bearing either (A₂G₃)₁₀ (JH267), (T₂C₃)₁₀ (JH268), or (A₄G)₁₀ (JH269) repeats were pre-annealed with a 2.5 µM 5’-³²P labeled primer (JH270) in 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA by incubation at 95°C for 5 minutes, followed by gradual return to room temperature. 50mM K⁺ and 10mM Mg²⁺ were added to the annealing buffer in the left panel. Primer extension reactions consisted of 10nM primer/template and 1µM T7 DNA polymerase in 40 mM Tris-HCl, pH 7.5, 5mM dithiothreitol (DTT), and 55mM K⁺ (left panel). Primer extension was initiated by addition of 10mM MgCl₂, followed by incubation at room temperature for 1 minute, and quenching with formamide/EDTA loading dye. Primer extension products were resolved by electrophoresis on a denaturing 10% polyacrylamide gel. NT=no template (primer added without oligonucleotide template). (E) The same general protocol was followed as in (D), with the following changes: 50mM LiCl, KCl, or NaCl were added to the annealing buffer and 55 mM of the indicated monovalent metal chloride salt was added to the primer extension buffer. Sequencing alongside primer extension reactions were conducted with the templates and primers used for the primer extension reactions and followed the protocol detailed in the Materials and Methods for the sequencing reactions in Figure 1A. NR=no reaction (no addition of MgCl₂). (F) Model for G-quadruplex formation as T7 DNA polymerase progresses along the purine-rich template in the presence of K⁺ in the annealing and primer extension buffer.

Figure 2.. Potassium permanganate probing of pathogenic and nonpathogenic repeats.

(A) Polyacrylamide gel electrophoresis separation of sequencing reactions and primer extension reactions on potassium permanganate or water treated repeat-containing plasmids using the pyrimidine-rich template. Repeat-containing supercoiled DNA was incubated with potassium permanganate or water, the DNA was precipitated, and used for a primer extension reaction as described in Materials and Methods. (B) H-r3 triplex predicted from chemical probing for (A₂G₃)₁₀ repeats. (C) DNA unwinding element (DUE) predicted from chemical probing for (A₄G)₁₀ repeats. Purple stars in (B) and (C) represent possible KMnO₄ modification sites.

Figure 3.. Bioinformatic analysis of S1-END-seq peaks to determine the triplex-forming potential of various repeats using data from .

For each graph: Top: Graph depicting the percentage of repeats found within 100 nucleotides of S1-END-seq peaks as the repeat length increases. Bottom: Graph depicting the number of repeats as the repeat length increases. (A) Comparison of pathogenic (A₂G₃)_n and (A₄G)_n motifs genome-wide. (B) Comparison of pentanucleotide motifs with increasing guanine:adenine ratio genome-wide.

Figure 4.. Analysis of yeast replication intermediates using two-dimensional gel electrophoresis.

(A) Representative gels of the no repeat control, (A₂G₃)₆₀, (C₃T₂)₆₀, and (A₄G)₆₀ in the lagging strand template of replication in yeast from the yeast 2µ origin of replication. The red arrow indicates replication fork stalling. (B) Densitometry profiles along the arc starting at the 1.5n spot to the 2n spot. These profiles were used for quantification, which was determined as described in ^,. (C) Quantification of replication fork slowing via area analysis with the fold change increased normalized to the no repeat control arc. Error bars represent standard error of the mean and non-overlapping error bars were used to determine significance. Created with BioRender and prism.

Figure 5.. Analysis of human cell replication intermediates using two-dimensional gel electrophoresis.

(A) Representative gels of the no repeat control, (A₂G₃)₆₀, (C₃T₂)₆₀, and (A₄G)₆₀ in the lagging strand template of replication from the SV40 origin of replication. The red arrow indicates replication fork stalling. (B) Densitometry profiles along the arc starting at the 1n spot to the 1.5n spot. These profiles were used for quantification, which was determined as described in ^,. (C) Quantification of replication fork slowing via area analysis with the fold change increased normalized to the no repeat control arc. Error bars represent standard error of the mean and non-overlapping error bars were used to determine significance. Created with BioRender and prism.