Triplex formation of Friedreich ataxia (FA) GAA•TTC repeats in an experimental human system may slow down the replication fork progression and cause repeat expansions during replication fork restart, according to an article recently published in Nucleic Acids Research.
As part of the study, the researchers used an experimental system to analyze large-scale repeat expansions in cultured human cells. The system is composed of a shuttle plasmid that can replicate from the SV40 origin in human cells or remain preserved in Saccharomyces cerevisiae with ARS4-CEN6, as well as a selectable cassette with the ability to identify repeat expansions accumulated in human cells upon plasmid transformation into yeast.
According to the results, there were massive expansions of GAA•TTC repeats and their frequency was dependent on proteins implicated in replication fork stalling, reversal, and restart. The repeats slowed down the replication fork progression significantly.
Moreover, the expansion of GAA•TTC repeats was prevented in human cells by locked nucleic acid-DNA mixmer oligonucleotides and peptide nucleic acid oligomers that normally interfere with the triplex formation of these repeats in vitro.
Read more about FA etiology
“The only known instance where massive repeat expansions were observed in a mammalian experimental system was a specific transgenic DM1 mouse, yet the reasons for big jumps in the number of CTG repeats in these mice remain unclear. Thus, our experimental system is unique in allowing the study of mechanisms and genetic controls of large-scale repeat expansions in human cells,” Rastokina and colleagues wrote.
FA is a genetic condition caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene. This system holds great potential for analyzing large-scale expansions in human cells as well as for evaluating potential FA drugs.
Reference
Rastokina A, Cebrián J, Mozafari N, et al. Large-scale expansions of Friedreich’s ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers. Nucleic Acids Res. Published online May 22, 2023. doi:10.1093/nar/gkad441
