The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

Abstract

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

Keywords: DNA double-strand break; DNA double-strand break repair pathway choice; DNA end resection; V(D)J recombination; homology-directed repair; non-homologous end-joining.

FIGURE 1

V(D)J recombination and NHEJ Basics: Generating antigen receptor diversity. (A) V(D)J recombination at the immunoglobulin heavy chain locus (depicted as an example) consists in a sequential 2-step rearrangement of V, D and J segments. This combinatorial process generates the diversity of antigen receptors. (B) After RAG cleavage, the NHEJ repair pathway is initiated by binding of the Ku70/80 heterodimer (Ku) to DNA ends. Ku together with DNA-PKcs form the DNA-PK holoenzyme. RAG DNA ends are then processed by the endonuclease Artemis and polymerases (e.g., Pol µ), specifically the terminal deoxynucleotidyl transferase (TdT), resulting in increased junctional diversity (in gray). This additional diversity is generated, prior to joining, in two forms: 1) P- palindromic sequences, produced through the endonuclease action of Artemis at RAG-induced hairpin-sealed ends and 2) N-nucleotide sequences, the addition of non-templated nucleotides by TdT. Finally, the ligation complex composed of Ligase IV, XRCC4 and XLF joins the processed ends. Joining of DNA ends via NHEJ further participates to generating indels, moreover favoring junctional diversity. NHEJ: non-homologous end-joining, indels: insertions or deletions.

FIGURE 2

Major parameters restricting DNA repair pathway choice to NHEJ during V(D)J recombination. (A) RAG-induced breakage generates a covalently sealed hairpin end (coding end) and a blunt end (signal end). This facilitates the loading of Ku, which acts as a scaffold for other NHEJ factors, as it has a high affinity for blunt or hairpin sealed ends. In addition, hairpin sealed ends require to be opened by another NHEJ factor Artemis, which renders ends compatible for ligation. Thus, this DNA end topology contributes to the establishment of a NHEJ-prone environment. (B) (i) Upon binding an RSS, RAG scans the adjacent chromatin by a loop extrusion mechanism. Breakage is induced only upon reaching a compatible RSS, ensuring the induction of DSBs in close proximity despite the large size of the immunoglobulin locus. (ii) Following DSB induction, RAG remains bound to DNA ends in a post-cleavage complex (PCC). The PCC together with NHEJ and ATM-dependent chromatin-bound DNA factors (e.g., phosphorylated H2AX and 53BP1) favor DNA ends tethering and stabilization. This likely prevents the search for distant partner DNA ends and channels broken DSB ends to NHEJ for safe repair. (C) (i) V(D)J recombination is a G1-restricted process, as RAG is degraded upon entry in the S phase. In G1, HR cannot operate as pre-replicative cells do not harbor a sister chromatid, used as a template for repair. In addition, several factors required for HR are transcriptionally repressed in G0/G1. Similarly, Pol θ, an important factor for alt-EJ, is poorly expressed in G1 consequently limiting the use of this repair pathway. Furthermore, alt-EJ is blocked by Ku upon binding DNA ends, yet again promoting processing and repair by NHEJ. (ii) Chromatin DSB-response factors γH2AX, 53BP1 and possibly additional downstream effectors contribute to the protection of RAG-DSB ends by blocking the activity of nucleases such as CtIP or acting via transcriptional repressors such as KAP-1. This protection prevents DNA end resection, an essential intermediate step for (micro)homology-directed repair (e.g., alt-EJ, HR, etc.), hence promoting NHEJ. NHEJ: non-homologous end-joining, RSS: Recombination Signal Sequence, Alt-EJ: alternative end-joining, HR: homologous recombination.