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Updated: May 20, 2026
Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
Published on: June 19, 2018
DNA mismatch repair complex MutSβ promotes GAA·TTC repeat expansion in human cells
Anasheh Halabi1, Scott Ditch, Jeffrey Wang
1Department of Genetics, Health Sciences Center, Louisiana State University, New Orleans, LA 70112, USA.
Abstract:
While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future.
Insights
DNA repair mechanisms, specifically MutSβ (MSH2/MSH3), influence Friedreich ataxia (FRDA) GAA·TTC repeat expansion. Targeting MSH3, not MSH2, shows therapeutic promise for slowing FRDA repeat instability.
Area of Science:
- Genetics
- Molecular Biology
- Neuroscience
Background:
- Friedreich ataxia (FRDA) is linked to GAA·TTC repeat expansions in the FXN gene.
- The precise role of DNA repair in GAA·TTC repeat expansion, unlike CAG·CTG repeats, remains unclear.
- Previous work linked GAA·TTC expansion rates to transcription within the repeats.
Purpose of the Study:
- To investigate the role of DNA repair enzymes, particularly MutSβ (MSH2/MSH3), in GAA·TTC repeat expansion in FRDA.
- To develop and utilize a human cellular model to study FRDA repeat instability.
- To assess the therapeutic potential of targeting specific DNA repair proteins for FRDA.
Main Methods:
- Development of a human cellular model recapitulating FRDA DNA repeat expansion.
- Utilizing shRNA knockdown to assess the impact of MSH2 and MSH3 on GAA·TTC expansion.
- Employing functional assays in FRDA patient-derived fibroblasts with ectopic MSH2/MSH3 expression.
Main Results:
- Knockdown of either MSH2 or MSH3 significantly slowed GAA·TTC repeat expansion in the cellular model.
- Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in native FXN genes in FRDA fibroblasts.
- MSH2's critical role in mismatch repair and cancer predisposition makes it an unattractive therapeutic target.
- MSH3 absence/reduction is not strongly linked to cancer predisposition, suggesting its potential as a therapeutic target.
Conclusions:
- MutSβ (MSH2/MSH3) plays a crucial role in the expansion of GAA·TTC repeats in the context of FRDA.
- MSH3 inhibition represents a potential therapeutic strategy to mitigate GAA·TTC repeat expansion and treat FRDA.
- Further research into MSH3's mechanism in GAA·TTC repeat expansion could lead to novel FRDA therapies.
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