DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells

Abstract

Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V(D)J recombination and class switch recombination (CSR), two Ig gene–diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V(D)J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo–switched B cells from two DNA-PKcs–deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs–deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes.

High-throughput sequencing reveals similar molecular signatures for class switch recombination junctions for the γ and α isotypes

“…5 We also confirmed the previously reported slight increase in small insertions for S μ -S γ compared to S μ -S α . 8 Finally, we demonstrated that the slight increase in S μ -S α junctions with large micro-homology can be numerically reproduced using randomly generated synthetic junctions. As this simulation mimics a pure NHEJ process (linking two free DNA ends without any resection), it demonstrates that it is not necessary to invoke another molecular mechanism (such as A-EJ) to explain the observed structural alteration of S μ -S α junctions.…”

“…[3][4][5][6] Previous reports have suggested that IgG and IgA CSR might be differentially regulated, with preferential use of c-NHEJ for γ CSR and A-EJ for α CSR. 7,8 We recently reported a computational tool (CSReport) for automatic analysis of CSR junctions sequenced by high-throughput sequencing 9 and used it to analyze the rare S μ -σ δ junctions that formed during IgD CSR. 10,11 We thus used CSReport and high-throughput sequencing to analyze the molecular signature of S μ -S γ3 , S μ -S γ1 , and S μ -S α junctions in wt mice in greater detail.…”

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

“…DNA-PKcs deficiency has been linked to reduced CSR efficiency and a preference for MH in patients and mouse models(16, 19). The T2609 cluster phosphorylation is one of the most prominent post-translational modification of DNA-PKcs and has been associated with increased radiation sensitivity (22, 28–31), which its role in CSR remains elusive.…”

“…Nevertheless, in recent high-throughput sequence analyses, we found that CSR junctions recovered from DNA-PKcs -/- B cells contained increased chromosomal translocations and extensive end-resection, and preferentially used MHs (16), suggesting that the seemingly robust CSR achieved in DNA-PKcs -/- cells is primarily mediated by the Alt-EJ pathway like those in Lig4 -/- or Xrcc4 -/- B cells (17, 18). Accordingly, human patients with spontaneous mutations in DNA-PKcs show severe defects in both V(D)J recombination and CSR and increased MH in the residual CSR junctions(12, 19).…”

ZEB1 is Required for NHEJ-Mediated DSB Repair in Euchromatin

“…Extensive characterization of Class Switch (CSR) and Variable [Diversity] Joining (V[D]J) recombination have demonstrated an absolute requirement for 53BP1 in the former (53)(54)(55) and for a subset of recombination events in the latter (56). Both of these programmed, distal end-joining pathways also have strict requirement for DNA-PK activity for repair of their respective, enzymatically-induced DSBs (57,58). A role for ZEB1 in V[D]J recombination is further supported by a phenotypic outcome in a ZEB1 loss-of-function mutant mouse, which harbors a 99% reduction in the total thymic T cell population (59).…”