Alternative End-Joining and Classical Nonhomologous End-Joining Pathways Repair Different Types of Double-Strand Breaks during Class-Switch Recombination

Abstract

Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ–defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

Cyclin-Dependent Kinases Regulate Ig Class Switching by Controlling Access of AID to the Switch Region

“…In the absence of some c-NHEJ factor, CSR is still able to occur, although with reduced efficiency and with increased microhomology in the S-S junctions, a process called alternative end joining. Furthermore, Cortizas et al (21) recently showed that factors of the C-NHEJ and A-EJ have the same kinetics of recruitment to the S regions. Therefore, CSR may occur at the G 1 /S border possibly because this is the only time that factors involved in NHEJ, A-EJ, and HR pathways can simultaneously participate.…”

“…3D and Ref. 21). LMB treatment alone caused a strong increase of nuclear AID accumulation, in agreement with a previous report (24) (Fig.…”

“…Chromatin immunoprecipitation (ChIP) was performed as previously described (21 inhibitors) and sonicated on a Bioruptor (Diagenode). For immunoprecipitation, 0.5 mg (2 mg/ml) of protein extract was precleared for 2 h with 30 ml 50% protein G-Sepharose slurry before addition of Ab.…”

A Novel Mouse Model for the Hyper-IgM Syndrome: A Spontaneous Activation-Induced Cytidine Deaminase Mutation Leading to Complete Loss of Ig Class Switching and Reduced Somatic Hypermutation

“…Chromatin immunoprecipitation was performed as previously described (11). In short, activated or nonactivated B cells were cross-linked with 1% formaldehyde for 20 min at room temperature, and the reaction was stopped by addition of glycine to 125 mM final concentration.…”

“…2D), we were interested in how the mutation influenced nuclear translocation and chromatin association. To address this, we performed chromatin immunoprecipitation in combination with real-time PCR using primers specific for the AID binding site in the Ig Sm locus (11). DNA was isolated from wild-type and AID R112H B cells at 0 or 48 h When assessing peripheral B cell development and function, before and after immunization with TNP-SRBCs, AID R112H mice had an increased number of total B cells in the spleen compared with wild-type littermate mice (Fig.…”