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Abstract Mismatch repair (MMR) is a highly conserved DNA repair pathway that promotes genome stability by directing the repair of errors in DNA replication. InSaccharomyces cerevisiae, MMR is initiated by either Msh2-Msh3 or Msh2-Msh6, via recognition of insertion deletion loops (IDLs; up to ∼ 17 nucleotides) and misincorporation events, respectively. Both complexes recognize and bind small (1-2 nucleotide) IDLs. Once bound, MSH complexes recruit one or more downstream MLH complexes to continue repair: Mlh1-Pms1, Mlh1-Mlh2 and/or Mlh1-Mlh3. Msh2-Msh3 also promotesCAGtrinucleotide repeat (TNR) expansions through specific DNA-binding to TNR DNA structures, followed by recruitment of MLH complexes. These expansions lead to genome instability that causes neurodegenerative diseases such as Huntington’s Disease in humans. Here, we defined a hierarchy of MLH function in these Msh2-Msh3-mediated pathwaysin vivoinS. cerevisiae. We determined that Mlh1-Pms1 is the primary MLH complex required in Msh2-Msh3-mediated MMR. In contrast, all three MLH complexes were required to promoteCAGexpansions, with loss of Mlh1-Pms1 or Mlh1-Mlh2 exhibiting the strongest effects. Mutations inPMS1andMLH3were synergistic. We propose a model in which Mlh1-Pms1 is primarily responsible for “appropriate” Msh2-Msh3-mediated MMR, while all three MLH complexes collaborate specifically in the presence ofCAGstructure, to promote a “pathogenic” Msh2-Msh3-mediated pathway that leads to expansions. Our model highlights the importance of DNA structure-dependent conformations in modulating MLH function.
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This content will become publicly available on January 1, 2027
Novel African American Colorectal Cancer MSH3 Variants Associate With Major Genomic Instability
Pathogenic variants ofMSH3can increase mutational load within colorectal cells, which may drive initiation and progression of colorectal cancer (CRC). We identified several variants withinMSH3among CRCs from African Americans (AA). To predict and assess the functional significance of theseMSH3variants, we employed a combination of in silico analyses and in vitro functionality assays. Our objective was to elucidate the correlation between computational predictions and functional outcomes. MethodsA CRISPR‐Cas9 knock‐in approach was used to introduce and generate specific point mutations in Exons 21, 22, and 23 ofMSH3in the wild‐type (WT)MSH3CRC cell line SW620, which was confirmed with Sanger sequencing. We employed cell proliferation, microsatellite instability assays, and whole genome sequencing to assess biological and genetic consequences. We utilized immunofluorescence, Western blot, and coimmunoprecipitation methods to assess subcellular localization and differences in heterodimer MSH2 binding between WT and variant MSH3 proteins. ResultsWe previously identified six novel, potentially pathogenic nonsynonymous variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, and c.G3241A) within six exons (Exons 8 (E413K), 9 (S466N), 20 (S920F), 21 (E976K), 22 (H1010Y), and 23 (E1081K), respectively) ofMSH3among AA CRCs as assessed by computational bioinformatic and molecular dynamic simulation analysis. We successfully knocked in three of the MSH3 variants (Exons 21, 22, and 23). Biological phenotypic assays revealed no observable changes in cell morphology or proliferation between WT andMSH3‐variant knocked‐in cells, and no differences were observed in microsatellite assays. Subcellular localization of variant MSH3 protein was unaffected compared with WT, whereas the interaction between MSH3 and MSH2 was not impacted. Short tandem repeats (STRs), or microsatellites, are short DNA motifs of two to six base pairs repeated consecutively, and they serve as powerful genetic markers for studying inheritance patterns and disease‐associated repeat instability. In our analysis, STR profiling revealed both repeat expansions and contractions across multiple motifs, with tetranucleotide repeats showing the most pronounced alterations. Notably, loci flankingLINC00550(ATTT),FBXL7(AGAT), andDUSP28(CTTT/GTTT) displayed consistent repeat expansions (+1.5 to +2), whereas intronic regions withinGPC5,TMEM232, andABCA13exhibited contractions. Trinucleotide STRs revealed a mix of instability, with repeat gains atSLC25A12and losses nearGTF3C3andAKAP12. Pentanucleotide and hexanucleotide motifs were more stable overall, but expansions were still noted atCNTN5,CPEB1, andCBSL, and contractions atSGO1andFER. These findings highlight the bidirectional and motif‐specific nature of STR instability driven byMSH3deficiency. Our results support the utility of STR‐based assays as sensitive tools to detect nonclassical MSI events and deepen our understanding ofMSH3′s role in preserving microsatellite integrity across the genome. ConclusionThis study investigated the functional consequences of MSH3 variants identified among AA CRCs. While CRISPR‐Cas9 knock‐in of MSH3 variants (Exons 21, 22, and 23) in SW620 cells did not alter cell morphology, proliferation, protein localization, or MSH2 binding, we observed genetic changes that collectively underscore the bidirectional nature of STR instability inMSH3‐deficient cells and reinforce this protein′s pivotal role in suppressing slippage at longer repeat motifs. This study advances our understanding of howMSH3deficiency contributes to genomic instability beyond canonically defined MSI loci, offering novel insights into the mutational landscapes of MMR‐deficient tumors and how theseMSH3mutations can potentially contribute to the outcome of AA CRC patients.
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- Award ID(s):
- 2325415
- PAR ID:
- 10710940
- Editor(s):
- Macrae, Finlay
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Human Mutation
- Volume:
- 2026
- Issue:
- 1
- ISSN:
- 1059-7794
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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