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1.
Nucleic Acids Res ; 52(8): 4361-4374, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38381906

RESUMO

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 pathogenic (A2G3)n and 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 vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.


Assuntos
Ataxia Cerebelar , Expansão das Repetições de DNA , Replicação do DNA , Doenças do Sistema Nervoso Periférico , Doenças Vestibulares , Humanos , DNA/metabolismo , DNA/química , DNA/genética , Expansão das Repetições de DNA/genética , Replicação do DNA/genética , Conformação de Ácido Nucleico , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Ataxia Cerebelar/genética , Doenças do Sistema Nervoso Periférico/genética , Doenças Vestibulares/genética
2.
Nucleic Acids Res ; 51(16): 8532-8549, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37216608

RESUMO

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.


Assuntos
Ataxia de Friedreich , Oligonucleotídeos , Ácidos Nucleicos Peptídicos , Expansão das Repetições de Trinucleotídeos , Humanos , DNA , Replicação do DNA/efeitos dos fármacos , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Oligonucleotídeos/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Saccharomyces cerevisiae/genética
3.
J Vis Exp ; (211)2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39345159

RESUMO

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.


Assuntos
Replicação do DNA , Plasmídeos , Vírus 40 dos Símios , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/química , Humanos , Plasmídeos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Transfecção/métodos , DNA Viral/genética , DNA Viral/química , Southern Blotting/métodos
4.
bioRxiv ; 2023 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-37546920

RESUMO

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.

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