Alternative end-joining in BCR gene rearrangements and translocations

Abstract

Programmed DNA double-strand breaks (DSBs) occur during antigen receptor gene recombination, namely V(D)J recombination in developing B lymphocytes and class switch recombination (CSR) in mature B cells. Repair of these DSBs by classical end-joining (c-NHEJ) enables the generation of diverse BCR repertoires for efficient humoral immunity. Deletion of or mutation in c-NHEJ genes in mice and humans confer various degrees of primary immune deficiency and predisposition to lymphoid malignancies that often harbor oncogenic chromosomal translocations. In the absence of c-NHEJ, alternative end-joining (A-EJ) catalyzes robust CSR and to a much lesser extent, V(D)J recombination, but the mechanisms of A-EJ are only poorly defined. In this review, we introduce recent advances in the understanding of A-EJ in the context of V(D)J recombination and CSR with emphases on DSB end processing, DNA polymerases and ligases, and discuss the implications of A-EJ to lymphoid development and chromosomal translocations.

Keywords: DSB end resection; V(D)J recombination; alternative end-joining; chromosomal translocation; classical nonhomologous end-joining; endonuclease.

Figure 1

The DSB repair pathways in eukaryotic cells NHEJ re-ligates broken DNA ends with the help of DDR factors binding to and protecting ends from nucleolytic degradation. NHEJ operates in all phases of the cell cycle. On the contrary, HR is only active in S/G2 phase of the cell cycle due to requirement for DSB end resection-generated ssDNA for homology searching and invasion. SSA and A-EJ also require DSB end resection and homology sequence annealing. SSA and A-EJ mainly differ by homology length requirement and likely in essential components after annealing.

Figure 2

BCR gene rearrangements by V(D)J recombination and class switch recombination (A) Overview of V(D)J recombination. The mouse IgH locus contains multiple V, D, and J exons, and RSS is located adjacent to each coding segment. RAG1/2 initiates cleavage right at the RSS that is converted into DSBs on the coding end, and joining D to J followed by V- to DJ assembles a full exon for the variable region of an antibody. (B) Overview of mouse IgH class switch recombination. Stimulation of mature B cells by cytokines and ligands turns on AID expression and germline transcription of specific S regions, which facilitates targeting of AID to initiate S region DSBs. End-joining of the donor Sμ DSB and downstream S region DSB juxtaposes downstream constant exons to the assembled VDJ exon to express a different isotype of antibody without altering its specificity.

Figure 3

Summary of c-NHEJ and A-EJ during CSR and other DSB repairs In wild-type cells, c-NHEJ dominates DSB repair. DSB response kinase ATM and downstream factors such as 53BP1 enforce repair by c-NHEJ largely by end-tethering and preventing DSB end resection. Ku70/80 complex initially binds to DSBs to recruit DNA-PKcs/Artemis (in case complex end processing is needed) and XRCC4/Lig4 for ligation. XRCC4 paralogs (XLF, PAXX, etc.) play redundant roles possibly in end-tethering for efficient c-NHEJ. In cells deficient in c-NHEJ and thus with deprotected ends, PARP1 functions to hold DSBs in proximity and recruits other downstream processing and ligation proteins like Mre11 and XRCC1/Lig3. End resection by both the short-range and long-range resection machineries generates 3′ and potentially 5′ ssDNA (in case of AID-induced breaks) that anneal with each other through imbedded MH under the help of Rad52 and HMCES proteins. Polθ also participates in MH annealing and ensuing repair synthesis by endonucleolytic trimming-off of unmatched 3′-termini. The removal of nonhomologous flaps is mainly carried out by the structure-specific endonuclease XPF/ERCC1, likely redundantly by the Mus81-EME1/2 complex or FEN1. DNA polymerase Polδ, Polλ and Polθ function in gap fill-in synthesis, while both Lig1 and Lig3 are functional in the last ligation step. As a result, A-EJ in mouse B cells and other cells promotes the deletion of sequences, utilization of MH and chromosomal translocations.