A somatic hypermutation defective UNG mutant in antibody deficiency through destabilization of RPA interaction.

PROJECT SUMMARY: B cells require somatic hypermutation (SHM) and class switch recombination (CSR) to develop high affinity, isotype switched immunoglobulins (Igs). Human patients deficient in these processes suffer from hyper IgM syndrome, a primary immunodeficiency characterized by recurrent and severe infections. They have low IgG levels and are often unable to develop high-affinity antibodies. Both CSR and SHM are initiated by activation- induced cytidine deaminase (AID) which deaminates cytidines, creating uracils in Ig genes. Uracil is removed by Uracil DNA glycosylase (UNG) and repaired in an error-prone fashion to complete SHM and CSR. UNG is normally associated with high-fidelity base excision repair. The mechanism that directs uracil removal to error- prone repair during SHM is not understood. However, mis regulation could threaten genome integrity through inappropriate mutation or suppress SHM and the generation of high-affinity antibodies. To determine if UNG directly influences repair outcome, we analyzed various mutants of UNG in cell-line, AID induced mutation reporter assay. We found that UNG with point mutations in the Replication Protein (RPA) binding domain efficiently supported high-fidelity repair, while suppressing the frequency of mutation. Furthermore ,RPA mutants unable to interact with UNG also suppressed mutation frequency. Our hypothesis is that UNG-RPA interaction governs error-prone repair during SHM in B cells. The cell line models available do not completely recapitulate B cell SHM in terms of frequency and mutational spectrum. Therefore, our objective is to create a mouse model with a UNG mutant deficient in RPA binding to investigate error-prone repair. We will accomplish this in Aim 1 by using CRISPR gene editing technology to create a mouse with mutant UNG. In Aim2 this mouse will be used to define how UNG-RPA interaction in B cells results in SHM. We expect with the completion of these goals to have a mouse model deficient in normal SHM and a system to mechanistically investigate how UNG and RPA regulate error-free or mutation repair outcome during base excision repair of uracils.