The alteration of somatic hypermutation and immunoglobulin class switching in mismatch repair deficient mice

Abstract

To gain insight into the molecular mechanism of somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR), we have examined the role of mismatch repair (MMR) proteins in mouse models. The MMR proteins have been shown to modulate the characteristics of V-region SHM and enable efficient CSR. Work from a number of labs has suggested that a two-phase process exists for both mutation and switching and that MMR proteins are required for the second phase. In this thesis, mice with defects in MMR proteins have been studied in order to gain a better understanding of the role of MMR in both of these processes.;The well-characterized SHM model system utilizing the anti-NP immune response has lent insight into the two-phase model. Using this assay, previous work in the lab has shown that Msh6-/- mice mutate V-regions at normal frequencies. However, in comparison to the normal process, G and C bases are mostly targeted for mutation in Msh6-/- mice. Other groups have shown a similar result in Msh2-/- mice. We will present data from a mouse line carrying a putative dominant negative mutant Msh2 protein as compared to Msh2-/- mice. It was also shown that Msh2-/- mice have a defect in CSR when ex vivo splenocytes are stimulated in culture. We will show that Msh6-/- mice also have isotype switching defects in vitro. In contrast, Msh3 -/- mice are normal for both SHM and CSR.;Nucleases involved in CSR or SHM have yet to be identified. Exo1 is an exonuclease that has been shown to interact with many of the protein factors involved in MMR. Mice deficient in Exo1 have been generated and are shown in this thesis to have perturbations in SHM and an impaired ability to undergo CSR. Further evidence that Exo1 is important in SHM was gained from the hypermutating cell line BL2. Chromatin immunoprecipitation experiments have localized Exo1 to a hypermutating Ig variable region. From these studies we hope to further clarify the role of MMR on these important immune processes. We have also developed a new method to screen V-region PCR products for single base changes.