RESUMEN
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.
Asunto(s)
Ataxia Cerebelosa , Expansión de las Repeticiones de ADN , Replicación del ADN , Enfermedades del Sistema Nervioso Periférico , Enfermedades Vestibulares , Humanos , ADN/metabolismo , ADN/química , ADN/genética , Expansión de las Repeticiones de ADN/genética , Replicación del ADN/genética , Conformación de Ácido Nucleico , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Ataxia Cerebelosa/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades Vestibulares/genéticaRESUMEN
Friedreich's ataxia (FRDA) is caused by expansions of GAAâ¢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 GAAâ¢TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAAâ¢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 GAAâ¢TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAAâ¢TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.
Asunto(s)
Ataxia de Friedreich , Oligonucleótidos , Ácidos Nucleicos de Péptidos , Expansión de Repetición de Trinucleótido , Humanos , ADN , Replicación del ADN/efectos de los fármacos , Ataxia de Friedreich/genética , Proteínas de Unión a Hierro/genética , Oligonucleótidos/farmacología , Ácidos Nucleicos de Péptidos/farmacología , Saccharomyces cerevisiae/genéticaRESUMEN
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.