AID- and Ung-dependent generation of staggered double-strand DNA breaks in immunoglobulin class switch DNA recombination: A post-cleavage role for AID

Class switch DNA recombination (CSR) substitutes an immunoglobulin (Ig) constant heavy chain (C_H) region with a different C_H region, thereby endowing an antibody with different biological effector functions. CSR requires activation-induced cytidine deaminase (AID) and occurrence of double-strand DNA breaks (DSBs) in S regions of upstream and downstream C_H region genes. DSBs are critical for CSR and would be generated through deamination of dC by AID, subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and nicking by apurinic/apyrimidic endonuclease (APE) of nearby abasic sites on opposite DNA strands. We show here that in human and mouse B cells, S region DSBs can be generated in an AID- and Ung-independent fashion. These DSBs are blunt and 5′-phosphorylated. In B cells undergoing CSR, blunt and 5′-phosphorylated DSBs are processed in an AID- and Ung-dependent fashion to yield staggered DNA ends. Blunt and 5′-phosphorylated DSBs can be readily detected in human and mouse AID- or Ung-deficient B cells. These B cells are CSR defective, but show evidence of intra-S region recombination. Forced expression of AID in AID-negative B cells converts blunt S region DSBs to staggered DSBs. Conversely, forced expression of dominant negative AID or inhibition of Ung by Ung inhibitor (Ugi) in switching B cells abrogates the emergence of staggered DSBs and concomitant CSR. Thus, AID and Ung generate staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends, whose generation is AID- and Ung-independent, thereby outlining a post-cleavage role for AID in CSR.