NHEJ and Other Repair Factors in V(D)J Recombination

V(D)J recombination requires the formation and resolution of programmed DNA double-strand breaks (DSBs) to effect the gene rearrangements necessary for immunoglobulin and T cell receptor formation. Improper repair of DNA DSBs can lead to deleterious consequences for the cell, including loss of genetic information, cell death, and formation of chromosomal translocations. The classical nonhomologous end-joining (C-NHEJ) pathway is a major DNA DSB repair pathway that maintains cellular genomic integrity in response to DNA DSBs. The V(D)J recombination reaction absolutely requires C-NHEJ for DSB repair. In addition to this requirement, C-NHEJ also contributes to the diversity of antigen receptor molecules through the processing of DNA DSBs prior to their repair. In this article, we discuss the C-NHEJ pathway in the context of the V(D)J recombination reaction. We outline the major phases of C-NHEJ, how C-NHEJ handles the different species of DNA DSBs generated during V(D)J recombination, and the protein players known to be involved in C-NHEJ. The joining phase of V(D)J recombination has provided a rich context for understanding not only how antigen receptors form but also how DNA DSB repair occurs more generally in cells. Classical Nonhomologous End-Joining and DNA Double-Strand Break Repair Our cells are subject to a variety of stresses that can damage the integrity of our genome, the most lethal of which is the DNA double-strand break (DSB). Effective repair of a DSB involves two broadly defined pathways: homologous recombination, which requires a template for repair and is most active during the S/G2 phases of the cell cycle, and classical nonhomologous end-joining (C-NHEJ), which operates throughout interphase and involves the ligation of DSBs without the need for homologous template (Lieber, 2010; Moynahan and Jasin, 2010). B lymphocytes and T lymphocytes exclusively utilize the ubiquitously expressed C-NHEJ pathway to resolve the requisite DSB intermediates generated during V(D)J recombination and, in mature B cells, the class switch recombination (CSR) reaction largely uses this pathway. Another end-joining pathway (or pathways) has been discovered by looking in cells that lack C-NHEJ and is broadly referred to as alternative end-joining (A-EJ) (Lieber, 2010; Boboila et al., 2012). While V(D)J recombination absolutely requires C-NHEJ for completion, CSR, as well as joining of more general DSBs (Guirouilh-Barbat et al., 2004), can utilize A-EJ to complete a subset of joining events (we refer to other references (Boboila et al., 2012; Deriano and Roth, 2013) for extensive discussions of A-EJ and end joining during CSR). We focus our subsequent discussion on the specific DNA repair aspects of the V(D)J recombination reaction, studies of which have led to the discovery of C-NHEJ pathway components (Taccioli et al., 1993) and the greater role of C-NHEJ in the maintenance of genomic stability in mammalian cells. C-NHEJ of V(D)J Recombination DNA DSBs By virtue of the biochemical mechanism, recombinationactivating gene (RAG) 1/2 employs to generate DNA DSBs between a recombination signal sequence (RSS) and a coding gene segment (reviewed extensively by Schatz and Swanson, 2011); two distinct types of DNA DSB ends are generated between coding gene segments and their corresponding RSSs (Figure 1): a pair of hairpin-sealed coding ends (CEs), and two blunt, 50 phosphorylated signal ends (SEs) (Roth et al., 1993; McBlane et al., 1995). These DSBs are initially held by RAG in a postcleavage synaptic complex (PSC) in which RAG, rather than allowing simple rejoining of the DSBs to generate ‘open-and-shut’ joins (Lewis and Hesse, 1991; Lewis et al., 1988), directs the physiologic joining of CE to CE to generate coding joins (CJs) and SE to SE to generate signal joins (SJs) (Agrawal and Schatz, 1997; Alt et al., 2013). After RAG cleavage, the SE and CE DSBs are exclusively repaired by the C-NHEJ machinery (Taccioli et al., 1993). Why only C-NHEJ is involved in V(D)J recombination is not fully known, but may be mediated by RAG itself (Gigi et al., 2014; Corneo et al., 2007). The blunt 50 phosphorylated SEs can simply be religated, while the hairpinned CEs must be processed prior to ligation. In addition, the dependence on C-NHEJ is further cemented by the cell-cycle timing of V(D)J recombination, which occurs entirely in G1-phase lymphocytes where homologous recombination is unavailable (Lin and Desiderio, 1994). As the V(D)J recombination reaction is wholly dependent on the presence of both RAG1 and RAG2, this G1 restriction is enforced by the ubiquitin-mediated degradation of RAG2 at the G1/S transition (Desiderio, 2010). Accordingly, V(D)J recombination is absent in mice or cells deficient for C-NHEJ, due to complete inability to repair RAG-generated CE DSBs (Gao et al., 1998a,b; Zhu et al., 1996; Gu et al., 1997; Rooney et al., 2002; Frank et al., 1998). Seven major proteins are currently known to function in the C-NHEJ pathway, including the Ku70/Ku80 heterodimer, Artemis nuclease, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray repair cross-complementing protein 4 (XRCC4), ligase IV, and XRCC4-like factor (XLF) Encyclopedia of Immunobiology, Volume 2 http://dx.doi.org/10.1016/B978-0-12-374279-7.05013-X 107 Author's personal copy