Individual Substitution Mutations In The Aid C Terminus That Ablate Igh Class Switch Recombination

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The C terminus of AID is required for CSR but not for SHM, but the reason for this is not entirely clear. By retroviral transduction of mutant AID proteins into aid(-/-) mouse splenic B cells, we show that 4 amino acids within the C terminus of mouse AID, when individually mutated to specific amino acids (R190K, A192K, L196S, F198S), reduce CSR about as much or more than deletion of the entire C terminal 10 amino acids. Similar to Delta AID, the substitutions reduce binding of UNG to Ig S mu regions and some reduce binding of Msh2, both of which are important for introducing S region DNA breaks. Junctions between the IgH donor switch (S)mu and acceptor S alpha regions from cells expressing Delta AID or the L196S mutant show increased microhomology compared to junctions in cells expressing wild-type AID, consistent with problems during CSR and the use of alternative end-joining, rather than non-homologous end-joining (NHEJ). Unlike deletion of the AID C terminus, 3 of the substitution mutants reduce DNA double-strand breaks (DSBs) detected within the S mu region in splenic B cells undergoing CSR. Cells expressing these 3 substitution mutants also have greatly reduced mutations within unrearranged S mu regions, and they decrease with time after activation. These results might be explained by increased error-free repair, but as the C terminus has been shown to be important for recruitment of NHEJ proteins, this appears unlikely. We hypothesize that S mu DNA breaks in cells expressing these C terminus substitution mutants are poorly repaired, resulting in destruction of S mu segments that are deaminated by these mutants. This could explain why these mutants cannot undergo CSR.