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 C mu constant region exons(1,2). In mice, six additional sets of constant region exons (C(H)s) lie 100-200 kilobases downstream in the same transcriptional orientation as V(D) J and C mu exons(2). Long repetitive switch (S) regions precede C mu and downstream C(H)s. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing C mu with a downstream C-H (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within S mu 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 S mu DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has 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 is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing(4) into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR 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 factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.