MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.

In eukaryotes, DNA mismatch recognition is accomplished by the highly conserved MutSα (Msh2/Msh6) and MutSß (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.

Morning glory disc anomaly with Chiari type I malformation.

Morning glory disc anomaly is a rare optic nerve dysplasia associated with various neovascular abnormalities. Due to these associations, children with morning glory disc anomaly have brain imaging and angiography to detect other congenital defects. The authors report the case of an infant with morning glory disc anomaly and coexisting Chiari type I malformation.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Cell-cycle and DNA damage regulation of the DNA mismatch repair protein Msh2 occurs at the transcriptional and post-transcriptional level.

DNA mismatch repair during replication is a conserved process essential for maintaining genomic stability. Mismatch repair is also implicated in cell-cycle arrest and apoptosis after DNA damage. Because yeast and human mismatch repair systems are well conserved, we have employed the budding yeast Saccharomyces cerevisiae to understand the regulation and function of the mismatch repair gene MSH2. Using a luciferase-based transcriptional reporter, we defined a 218-bp region upstream of MSH2 that contains cell-cycle and DNA damage responsive elements. The 5' end of the MSH2 transcript was mapped by primer extension and was found to encode a small upstream open reading frame (uORF). Mutagenesis of the uORF start codon or of the uORF stop codon, which creates a continuous reading frame with MSH2, increased Msh2 steady-state protein levels ∼2-fold. Furthermore, we found that the cell-cycle transcription factors Swi6, Swi4, and Mbp1-along with SCB/MCB cell-cycle binding sites upstream of MSH2-are all required for full basal expression of MSH2. Mutagenesis of the cell-cycle boxes resulted in a minor reduction in basal Msh2 levels and a 3-fold defect in mismatch repair. Disruption of the cell-cycle boxes also affected growth in a DNA polymerase-defective strain background where mismatch repair is essential, particularly in the presence of the DNA damaging agent methyl methane sulfonate (MMS). Promoter replacements conferring constitutive expression of MSH2 revealed that the transcriptional induction in response to MMS is required to maintain induced levels of Msh2. Turnover experiments confirmed an elevated rate of degradation in the presence of MMS. Taken together, the data show that the DNA damage regulation of Msh2 occurs at the transcriptional and post-transcriptional levels. The transcriptional and translational control elements identified are conserved in mammalian cells, underscoring the use of yeast as a model system to examine the regulation of MSH2.

Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2.

MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies.