Ataxia Telangiectasia Mutated and MSH2 Control Blunt DNA End Joining in Ig Class Switch Recombination.

Class-switch recombination (CSR) produces secondary Ig isotypes and requires activation-induced cytidine deaminase (AID)-dependent DNA deamination of intronic switch regions within the IgH (Igh) gene locus. Noncanonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal switch regions. Ataxia telangiectasia mutated (ATM)-dependent phosphorylation of AID at serine 38 (pS38-AID) promotes its interaction with apurinic/apyrimidinic endonuclease 1 (APE1), a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also function in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, Atm-/- mice were bred to mice deficient for the MMR gene mutS homolog 2 (Msh2). Surprisingly, the predicted Mendelian frequencies of Atm-/-Msh2-/- adult mice were not obtained. To generate ATM and MSH2-deficient B cells, Atm was conditionally deleted on an Msh2-/- background using a floxed ATM allele (Atmf) and B cell-specific Cre recombinase expression (CD23-cre) to produce a deleted ATM allele (AtmD). As compared with AtmD/D and Msh2-/- mice and B cells, AtmD/DMsh2-/- mice and B cells display a reduced CSR phenotype. Interestingly, Sµ-Sγ1 junctions from AtmD/DMsh2-/- B cells that were induced to switch to IgG1 in vitro showed a significant loss of blunt end joins and an increase in insertions as compared with wild-type, AtmD/D, or Msh2-/- B cells. These data indicate that the absence of both ATM and MSH2 blocks nonhomologous end joining, leading to inefficient CSR. We propose a model whereby ATM and MSH2 function cooperatively to regulate end joining during CSR through pS38-AID.

AID Phosphorylation Regulates Mismatch Repair-Dependent Class Switch Recombination and Affinity Maturation.

Activation-induced cytidine deaminase (AID) generates U:G mismatches in Ig genes that can be converted into untemplated mutations during somatic hypermutation or DNA double-strand breaks during class switch recombination (CSR). Null mutations in UNG and MSH2 demonstrate the complementary roles of the base excision repair (BER) and mismatch repair pathways, respectively, in CSR. Phosphorylation of AID at serine 38 was previously hypothesized to regulate BER during CSR, as the AID phosphorylation mutant, AID(S38A), cannot interact with APE1, a BER protein. Consistent with these findings, we observe a complete block in CSR in AIDS38A/S38AMSH2-/- mouse B cells that correlates with an impaired mutation frequency at 5'Sµ. Similarly, somatic hypermutation is almost negligible at the JH4 intron in AIDS38A/S38AMSH2-/- mouse B cells, and, consistent with this, NP-specific affinity maturation in AIDS38A/S38AMSH2-/- mice is not significantly elevated in response to NP-CGG immunization. Surprisingly, AIDS38A/S38AUNG-/- mouse B cells also cannot complete CSR or affinity maturation despite accumulating significant mutations in 5'Sµ as well as the JH4 intron. These data identify a novel role for phosphorylation of AID at serine 38 in mismatch repair-dependent CSR and affinity maturation.