Orientation-Specific Joining of AID-initiated DNA Breaks Promotes Antibody Class Switching

During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons(1,2). In mice, six additional sets of constant region exons (C(H)s) lie 100-200 kb downstream in the same transcriptional orientation as V(D)J and Cμ exons(2). Long repetitive switch (S) regions precede Cμ and downstream C(H)s. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream C(H)(2). Activation-Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region(2,3); these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors(3). Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB (Fig. 1a). However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high-throughput genome-wide translocation sequencing (HTGTS)(4) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor.