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

Abstract

Double‐strand DNA breaks (DSBs) are critical for Ig class switch (S) DNA recombination (CSR). They would be generated through deamination of dC by AID, subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and nicking by apurinic/apyrimidic endonuclease of nearby abasic sites on opposite DNA strands. We found that in human and mouse B cells, S region DSBs can be generated in an AID‐ and Ung‐independent fashion. These DSBs were blunt and 5′‐phosphorylated, and could be readily detected in human and mouse AID‐ or Ung‐deficient B cells. In B cells undergoing CSR, blunt DSBs were processed in an AID‐ and Ung‐dependent fashion to yield staggered DNA ends. Accordingly, we readily detected blunt‐DSB in B cells from AID‐ or Ung‐deficient patients and AID‐ or Ung‐deficient mice. These B cells were CSR defective, but showed evidence of intra‐S region recombination. Enforced expression of AID in AID‐negative B cells converted S region blunt DSBs to staggered DSBs. Conversely, enforced expression of dominant negative AID or inhibition of Ung by Ung inhibitor (Ugi) in switching B cells abrogated 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.Supported by NIH grants AI 45011, AI 60573 and AR 40908.