Analysis of the mouse Msh2S637P mutation in DNA mismatch repair, apoptosis, recombination and cancer development
In yeast a mutation at position 656 of MSH2 that causes a serine to proline amino acid change has been shown to separate the repair and recombination functions of MSH2. My project involves analysis of the Msh2S637P equivalent mutation in the mouse system to determine whether it is possible to obtain a similar separation of function. More importantly, the separation of the mismatch repair and recombination function by this mutation would allow us to assess the importance of the suppression of homeologous recombination for tumorigenesis.;A mouse line designated Msh2S637P has been generated by gene targeting. RT-PCR and Western blot analysis demonstrate that the point mutation is expressed at the RNA level and that a stable protein is produced. Microsatellite instability (MSI) assays show an increase in genomic instability within repetitive sequences indicative of a significant mutator phenotype and pointing to a MMR defect in these mice. Interestingly, Msh2S673P mutant mice up to 18 months of age do not display increased cancer susceptibility, although the in vivo mutational frequency assessed at both the Hprt and cII locus was increased over wild type. Functional studies demonstrate that the Msh2S637P point mutant is still able to recognize and bind to a mismatched substrate. However, in the mutant protein extract, ATP binding/hydrolysis is affected, indicating an increase in resistance to the ATP-dependent mismatch release mechanism. Exposure of the Msh2S637P mutant mouse embryonic fibroblasts to various cytotoxic agents demonstrates that the Msh2S637P retains the ability to initiate apoptosis in response to DNA damage. Also, Msh2S637P mutant embryonic stem cells retain anti-recombination activity by suppressing recombination between homeologous sequences. Comparison between the MMR defects in Msh2S637P point mutant mice and mice with a similar mutation affecting the ATP binding/hydrolysis domain indicates that different missense mutations in this domain have different phenotypic presentations. Furthermore, our analysis shows that although there is MSI present in normal tissue, it causes only a weak cancer phenotype indicating that in mammals MSI by itself is not sufficient to drive tumorigenesis. (Abstract shortened by UMI.).
Source: Dissertation Abstracts International, Volume: 66-01, Section: B, page: 4700.;Advisors: Winfried Edelmann.