Biology subjects

Rastokina, A.

Publications and source records attributed to Rastokina, A..

3 recordsLinked to original sources

bioRxiv2023

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

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.

genetics↗

bioRxiv2022

Large-scale expansions and replication stalling of Friedreich's ataxia GAA repeats in an experimental mammalian system

1Human disease Friedreichs ataxia (FRDA) is caused by large-scale expansions of (GAA)n repeats in the first intron of the FXN gene. While repeat expansions during intergenerational transmissions are causative for the disease development, somatic expansions additionally contribute to the disease progression. We and others have previously shown that (GAA)n repeats transiently pause the replication fork progression in cultured human cells. However, whether and by which mechanisms fork stalling underlies repeat expansions remained unclear. Here we developed a new genetically tractable experimental system to simultaneously analyze repeat-mediated fork stalling and large-scale repeat expansions in cultured human cells. It is based on a mammalian/yeast shuttle vector that can transiently replicate from the SV40 replication origin in human HEK-293T cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6; it also contains a cassette for selecting repeat expansions in yeast. Repeat expansions accumulate in mammalian cells and are then detected upon plasmid transformation into yeast. We found that large-scale expansions of (GAA)n repeats do occur in this experimental mammalian system. Further, we observed that repeat expansions frequency depends on several previously implicated proteins in replication fork stalling, reversal, and restart. These proteins include SHPRH, RAD52, ZRANB3, DDX11, SMARCAL1, HLTF, RECQ1 and WRN. Therefore, we propose that GAA repeat expansions might occur as a consequence of deregulated replication fork regression and restoration process.

genetics↗

bioRxiv2022

Large-scale expansions of Friedreich's ataxia GAA·TTC repeats in human cells are prevented by LNA-DNA oligonucleotides and PNA oligomers

The human disease Friedreichs ataxia (FRDA) is caused by expansions of GAA*TTC repeats in the first intron intron of the frataxin (FXN) gene, and both intergenerational and somatic expansions are crucial for disease development. We and others have shown earlier that expanded GAA*TTC repeats can form an intramolecular triplex structure (H-DNA). Here we studied the effects of locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers on the expansion of GAA*TTC repeats in cultured human cells. Our experimental system employes a mammalian/yeast shuttle plasmid containing a selectable cassette to detect repeat expansions. Using our in-house in vitro triplex-specific DNA cleavage assay, we first confirmed H-DNA formation by the (GAA)100*(TTC)100 repeat in the selectable cassette and demonstrated that the designed LNA-DNA oligonucleotides as well as PNA oligomers are able to disrupt this structure. We then found that both LNA-DNA mixmers and PNA oligomers prevent repeat expansions in human cells. In the accompanying paper, we show that expansions of GAA*TTC repeats in this experimental system occur during replication fork stalling, regression and restart at the repetitive run. We hypothesize, therefore, that triplex DNA formation by the GAA*TTC repeats is a key to their instability, while LNA-DNA oligonucleotides and PNA oligomers counteract repeat expansions by disrupting the triplex at the fork or preventing triplex formation upon fork reversal.

genetics↗