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Published on: March 21, 2022
MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination
Tim Arlow1, Junwon Kim2, Joanna E Haye-Bertolozzi3
1Ophthalmic Associates, Johnstown, PA.
Abstract:
In eukaryotes, DNA mismatch recognition is accomplished by the highly conserved MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3) complexes. Previously, in the yeast Saccharomyces cerevisiae, we determined that deleting MSH6 caused wild-type Msh2 levels to drop by ∼50%. In this work, we determined that Msh6 steady-state levels are coupled to increasing or decreasing levels of Msh2. Although Msh6 and Msh2 are reciprocally regulated, Msh3 and Msh2 are not. Msh2 missense variants that are able to interact with Msh6 were destabilized when Msh6 was deleted; in contrast, variants that fail to dimerize were not further destabilized in cells lacking Msh6. In the absence of Msh6, Msh2 is turned over at a faster rate and degradation is mediated by the ubiquitin-proteasome pathway. Mutagenesis of certain conserved lysines near the dimer interface restored the levels of Msh2 in the absence of Msh6, further supporting a dimer stabilization mechanism. We identified two alternative forms of regulation both with the potential to act via lysine residues, including acetylation by Gcn5 and ubiquitination by the Not4 ligase. In the absence of Gcn5, Msh2 levels were significantly decreased; in contrast, deleting Not4 stabilized Msh2 and Msh2 missense variants with partial function. The stabilizing effect on Msh2 by either the presence of Msh6 or the absence of Not4 are dependent on Gcn5. Taken together, the results suggest that the wild-type MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.
Insights
DNA mismatch repair protein Msh2 stability is regulated by its partner Msh6, acetylation by Gcn5, and ubiquitination by Not4. This ensures proper function of the MutSα complex.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Protein Regulation
Background:
- Eukaryotic DNA mismatch recognition relies on MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3) complexes.
- Previous studies in yeast showed MSH6 deletion reduces wild-type Msh2 levels by approximately 50%.
- The interplay between Msh2, Msh6, and Msh3 in complex stability and regulation requires further elucidation.
Purpose of the Study:
- To investigate the reciprocal regulation between Msh2 and Msh6 steady-state levels.
- To determine the role of subunit interaction, acetylation, and ubiquitination in Msh2 stability.
- To understand the impact of Msh6 absence on Msh2 turnover and degradation pathways.
Main Methods:
- Analysis of Msh2 and Msh6 protein levels in wild-type and mutant yeast strains (e.g., ΔMSH6, ΔGcn5, ΔNot4).
- Characterization of Msh2 missense variants with altered dimerization capabilities.
- Investigation of Msh2 degradation via the ubiquitin-proteasome pathway.
- Mutagenesis of conserved lysine residues in Msh2 to assess their role in stability.
- Assessing the impact of Gcn5 (acetylation) and Not4 (ubiquitination) on Msh2 levels.
Main Results:
- Msh6 and Msh2 exhibit reciprocal regulation of steady-state levels, unlike Msh3 and Msh2.
- Msh2 variants that dimerize with Msh6 are destabilized upon Msh6 deletion, indicating a dimer stabilization mechanism.
- In the absence of Msh6, Msh2 undergoes faster turnover mediated by the ubiquitin-proteasome pathway.
- Mutagenesis of specific Msh2 lysines near the dimer interface partially restored Msh2 levels without Msh6.
- Gcn5-mediated acetylation and Not4-mediated ubiquitination are key regulators of Msh2 stability.
- Absence of Gcn5 decreased Msh2 levels, while deletion of Not4 stabilized Msh2.
- The stabilizing effects of Msh6 or absence of Not4 on Msh2 are dependent on Gcn5 activity.
Conclusions:
- Wild-type MutSα protein stability is governed by a complex interplay of subunit interaction, acetylation, and ubiquitination.
- Msh6 binding stabilizes Msh2, protecting it from ubiquitin-proteasome mediated degradation.
- Gcn5 and Not4 act as critical regulators of Msh2 stability through acetylation and ubiquitination, respectively, influencing MutSα function.
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