The recent advances in non-homologous end-joining through the lens of lymphocyte development

Abstract

Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocyte-specific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.

Keywords: ATM; Class switch recombination; DNA-PK; Non-homologous end-joining; V(D)J recombination.

Figure 1. The overview of V(D)J recombination and class switch recombination

The Immunoglobulin (Ig) gene product, an antibody, is shown at the top. An antibody is formed by a pair of Ig Heavy chain gene products (IgH, in orange and green) and a pair of Ig light chain (IgL, in white and blue). The V(D)J recombination (on the lower left) assembles the variable gene exon that encodes the antigen-specific portion of the antibody (orange) from individual V, D or J gene segments in a two-step process, D to J first, then V to DJ. The reaction is initiated by RAG endonucleases, which introduces DNA double-strand breaks. An insert above shows the pair of RAG cleavage products at Dh and Jh with the hairpin coding ends and blunt signal ends adjacent to RSSs (triangles). Upon ligation by the cNHEJ pathway, the intermediate sequence is removed and the participating V, D, and J segments are fused to form the variable region exon that is spliced with downstream constant region exons (on the right) to form the IgH. While we only depict the IgH here, V(D)J recombination occurs in all 3 Ig (IgH, Igλ, and Igκ) and 4 T cell receptor (TCRσ/δ, TCRγ, TCRβ) gene loci. In naïve B cells, the IgH undergoes two additional modifications initiated by activating induced deaminase (AID). Somatic hypermutation (SHM) introduces point mutations in the variable region exon without creating DNA double-strand breaks. Class switch recombination (CSR) occurs in the constant region (on the right) and joins DNA double-strand breaks from two different switch (S) regions and effectively replaces the initially expressed IgM constant region (Cμ) with a downstream constant region, such as the Cγ1 for IgG1 depicted in the illustration. The constant region exons dictate the effector function of the antibody. CSR only occurs in the IgH locus of B cells, not in IgL or TCR loci. In contrast to RAG, which directly introduces DNA breaks, AID introduces U:G mismatches, which are processed by the base excision repair and mismatch repair pathways to generate mutations for somatic hypermutation (SHM) and DNA double-strand breaks for CSR.

Figure 2. The features of V(D)J recombination and the cNHEJ pathway

(A) RAG1/2 endonuclease cleavage occurs after synapsing two compatible RSSs (with different spacer length, 12 and 23bp, illustrated as white or black triangles) together and generates a pair of 5’ phosphorylated blunt signal ends (SE) and a pair of covalently sealed hairpinned coding ends (CE) (red and blue hairpins). The two SEs can be directly joined by KU together with the LIG4-XRCC4-XLF complex to form a signal join (SJ). The two CEs have to be opened by KU-DNA-PKcs and Artemis endonuclease before they can be ligated to form a coding join (CJ). (B) A cartoon of the RAG post-cleavage complex shows two RAG1 and two RAG2 proteins synapsing two compatible RSSs (with 12 bp, 12RSS or 23 bp, 23RSS spacers) together and also holding the resulting SEs (solid lines, open) and CEs (dash lines, hairpinned) in close proximity to facilitate ligation^,,. The precise position of the CEs is yet to be firmly determined. (C) V(D)J recombination can occur in a deletional (upper) or inversional (middle) configuration depending on the relative orientation of the participating RSSs (open or filled triangles). The vast majority of V(D)J recombination occurs in the deletional configuration (e.g., IgH in Figure 1) with the two RSSs facing each other, and the recombination removing the SJ-containing internal sequence as an excised circular DNA (top). In rare cases, such as the TCRβ locus diagrammed at the bottom, the V(D)J recombination occurs between a pair of RSSs in the same orientation, and, as such, the recombination leads to the inversion of the internal sequence (grey box) to form an SJ on the chromosome in addition to the CJ (bottom left of the middlebox). In the case of ATM deficiency, the internal sequence is lost in ~50% of the cases, and, as such, the CE and SE on the genome join together to form a hybrid join (HJ) accompanied by the deletion of the internal sequence, indicated on the bottom right of the middlebox. A diagram of the murine TCRβ locus, including the Vβs with deletional rearrangements and the Vβ14 that undergoes inversional V(D)J recombination, is shown at the bottom. (D) A cartoon illustration of non-homologous end-joining at a CE. The KU heterodimer binds to the hairpin sealed CEs, and then recruits DNA-PKcs, which, upon phosphorylation by itself or by ATM kinase, serves as a platform to recruit and activate Artemis endonuclease. In the absence of kinase activation, DNA-PKcs at the ends precludes the DNA ligation complex from accessing the ends as well. The successful activation of DNA-PKcs triggers a conformational change that then allows end ligation by the LIG4-XRCC4-XLF complex.

Figure 3. The features of class switch recombination and the DNA damage response

(A) The switch region sequence is highly repetitive and GC rich. A collection of representative murine Sμ regions, (chr12 113424221–113424368, mm10) is shown. The genomic coordinates of the first bp of each sequence segment are listed in the first column and the GC% within the segment is noted in the last column. The overall GC% is 61%. (B) A cartoon of cNHEJ vs Alt-EJ during CSR. The cNHEJ mediated by KU heterodimers and the LIG4-XRCC4-XLF ligation complex directly ligate both strands of a double-strand break. Alternatively, annealing between complementary single-strand overhangs converts one double-strand break into two single-strand nicks, which can then be ligated by LIG3 or LIG1 through Alt-EJ. (C) A simplified cartoon illustrating the main DDR players that have been implicated in CSR. Upon DNA damage, ATM kinase phosphorylates histone H2AX to form γH2AX, which recruits MDC1. MDC1 subsequently recruits E3 ubiquitin ligases RNF8 and RNF168 to modify adjacent H2A. Histone Ub modifications, together with H4K20me2, recruit 53BP1 and leads to hyperphosphorylation of 53BP1. This phosphorylated 53BP1 recruits RIF1 and the SHIELDIN (SHLD1–3 and REV7) complex. Among the SHIELDIN complex, SHLD2 serves as a scaffold. The N-terminus of SHLD2 binds to SHLD3 and REV7, while the C-terminus of SHLD2 interacts with SHLD1 and single-strand DNA. These factors work together to prevent excessive end-resection and promote productive CSR.