During lymphocyte development, variable (V), diversity (D) and joining (J) gene segments recombine to form the variable regions of antigen receptors. Recently, Artemis — a member of the metallo-β-lactamase superfamily — was implicated in this process when it was found to be mutated in a subset of patients with severe combined immunodeficiency who have a complete block in V(D)J recombination (patients with RS-SCID). Now, a new study by Rooney and colleagues has clarified the in vivo functions of Artemis.
An early step in V(D)J recombination is the generation of double-strand breaks between the coding gene segments and the conserved recombination signal sequences that flank them, leading to the generation of two intermediates — a blunt recombination signal (RS) end and a hairpin coding end. Two coding ends can then be ligated through non-homologous end-joining (NHEJ) — a generic mechanism of double-strand break repair for which DNA-dependent protein kinase (DNA-PK) is an essential component.
Opening of the coding-end hairpins is a prerequisite for joining, but the identity of the molecule(s) that mediate this crucial step has been an important unresolved issue. In vitro studies have shown that Artemis can associate with the catalytic subunit of DNA-PK (DNA-PKcs) to form a complex that has hairpin-opening activity. But, whether this occurs in vivo has been a subject of debate.
To determine the physiological role of Artemis in V(D)J recombination and DNA repair, Artemis-deficient mice were generated by introducing mutations that mimic those in humans with RS-SCID (referred to as ArtN/N mice). Overall, the phenotype of these mice was similar to that of patients with RS-SCID and of DNA-PKcs-deficient mice — B-cell and T-cell development were blocked and sensitivity to ionising radiation was increased.
Similar to DNA-PKcs-deficient mice, ArtN/N mice had normal RS end joining but no V(D)J recombination, and unresolved hairpin intermediates accumulated in ArtN/N thymocytes. Together, these data support previous proposals that Artemis co-operates with DNA-PKcs in the processing of hairpin coding ends.
Surprisingly, however, the fidelity of RS joining was reduced in DNA-PKcs-deficient, but not ArtN/N, mice, which indicates that DNA-PKcs has a broader role in V(D)J recombination. Moreover, unlike DNA-PKcs-deficient mice, a proportion of ArtN/N mice do develop some T cells, which shows that a low level of V(D)J recombination can occur independently of Artemis. These are the first functional differences to be uncovered between Artemis and DNA-PKcs.
ORIGINAL RESEARCH PAPER
Rooney, S. et al. Leaky Scid phenotype associated with defective V(D)J coding end processing in Artemis-deficient mice. Mol. Cell 10, 1379–1390 (2002)
Ma, Y. et al. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in non-homologous end joining and V(D)J recombination. Cell 108, 781–794 (2002)