Abstract
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.