MutSß abundance and Msh3 ATP hydrolysis activity are important drivers of CTG•CAG repeat expansions.

CTG•CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutSß (Msh2-Msh3 heterodimer). To evaluate properties of MutSß that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutSß, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutSß. Msh3-/- cells are severely defective for CTG•CAG repeat expansions but show full activity on contractions. Msh3-/- cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutSß on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutSß complex, indicating that MutSß abundance drives expansions. An ATPase-defective Msh3 expressed at normal levels was as defective in expansions as Msh3-/- cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG•CAG expansions are limited by the abundance of MutSß and rely heavily on Msh3 ATPase function.

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents.

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

Inflammation-Associated Microsatellite Alterations Caused by MSH3 Dysfunction Are Prevalent in Ulcerative Colitis and Increase With Neoplastic Advancement.

OBJECTIVES: Inflammation-associated microsatellite alterations (also known as elevated microsatellite alterations at selected tetranucleotide repeats [EMAST]) result from IL-6-induced nuclear-to-cytosolic displacement of the DNA mismatch repair (MMR) protein MSH3, allowing frameshifts of dinucleotide or longer microsatellites within DNA. MSH3 also engages homologous recombination to repair double-strand breaks (DSBs), making MSH3 deficiency contributory to both EMAST and DSBs. EMAST is observed in cancers, but given its genesis by cytokines, it may be present in non-neoplastic inflammatory conditions. We examined ulcerative colitis (UC), a preneoplastic condition from prolonged inflammatory duration. METHODS: We assessed 70 UC colons without neoplasia, 5 UC specimens with dysplasia, 14 UC-derived colorectal cancers (CRCs), and 19 early-stage sporadic CRCs for microsatellite instability (MSI) via multiplexed polymerase chain reaction capable of simultaneous detection of MSI-H, MSI-L, and EMAST. We evaluated UC specimens for MSH3 expression via immunohistochemistry. RESULTS: UC, UC with dysplasia, and UC-derived CRCs demonstrated dinucleotide or longer microsatellite frameshifts, with UC showing coincident reduction of nuclear MSH3 expression. No UC specimen, with or without neoplasia, demonstrated mononucleotide frameshifts. EMAST frequency was higher in UC-derived CRCs than UC (71.4% vs 31.4%, P = 0.0045) and higher than early-stage sporadic CRCs (66.7% vs 26.3%, P = 0.0426). EMAST frequency was higher with UC duration >8 years compared with ≤8 years (40% vs 16%, P = 0.0459). DISCUSSION: Inflammation-associated microsatellite alterations/EMAST are prevalent in UC and signify genomic mutations in the absence of neoplasia. Duration of disease and advancement to neoplasia increases frequency of EMAST. MSH3 dysfunction is a potential contributory pathway toward neoplasia in UC that could be targeted by therapeutic intervention.