Individual substitution mutations in the AID C terminus that ablate IgH class switch recombination

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Activation-induced cytidine deaminase (AID) is crucial for immunoglobulin gene diversification through class switch recombination (CSR) and somatic hypermutation (SHM).
• The C-terminal region of AID plays a known role in CSR, but its precise function and the mechanistic basis for its necessity in CSR, distinct from SHM, remain incompletely understood.

Purpose of the Study:

• To investigate the specific roles of individual amino acids within the C-terminus of AID in the process of CSR.
• To elucidate the molecular mechanisms by which C-terminal mutations affect DNA break formation, repair, and immunoglobulin gene diversification.

Main Methods:

• Utilizing retroviral transduction to introduce mutant AID proteins into AID-deficient mouse splenic B cells.
• Analyzing the impact of specific C-terminal AID mutations (R190K, A192K, L196S, F198S) on CSR efficiency, UNG and Msh2 binding to Ig Sμ regions, and DNA double-strand breaks (DSBs) within Sμ regions.
• Examining junctional sequences in CSR products to assess DNA repair pathways and analyzing mutations in unrearranged Sμ regions.

Main Results:

• Four specific C-terminal AID mutations significantly reduced CSR, comparable to or exceeding the effect of deleting the entire C-terminus.
• These mutants exhibited reduced binding of UNG and, in some cases, Msh2 to Ig Sμ regions, impacting S region DNA break introduction.
• Three substitution mutants showed decreased Sμ region DSBs, and reduced mutations in unrearranged Sμ regions, suggesting impaired repair or increased DNA degradation.
• Junctional analysis revealed increased microhomology in CSR products from mutant cells, indicating a shift towards alternative end-joining pathways.

Conclusions:

• Specific C-terminal amino acids of AID are critical for efficient CSR, likely by facilitating proper DNA break formation and/or repair.
• Impaired DNA break introduction and/or repair in Sμ regions, rather than altered recruitment of non-homologous end-joining (NHEJ) proteins, may explain the CSR defect.
• These findings provide mechanistic insights into the C-terminus's role in CSR and highlight the differential requirements of AID's C-terminus for CSR versus SHM.