ABSTRACT
V(D)J recombination is targeted by short recombination signal (RS) sequences that are relatively conserved but exhibit natural sequence variations. To evaluate the potential of RS sequence variations to determine the primary and peripheral TCRbeta repertoire, we generated mice containing specific replacement of the endogenous Vbeta14 RS with the 3'Dbeta1 RS (Vbeta14/3'DbetaRS). These mice exhibited a dramatic increase in Vbeta14(+) thymocyte numbers at the expense of thymocytes expressing other Vbetas. In addition, the percentage of peripheral Vbeta14(+) alphabeta T lymphocytes was similarly increased. Strikingly, this altered Vbeta repertoire resulted predominantly from a higher relative level of primary Vbeta14/3'DbetaRS rearrangement to DbetaJbeta complexes, despite the ability of the 3'Dbeta1 RS to break B12/23 restriction and allow direct rearrangement of Vbeta14/3'DbetaRS to Jbeta segments.
Subject(s)
Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Recombination, Genetic , Animals , Base Sequence , DNA/genetics , Gene Targeting , Genes, T-Cell Receptor beta , Genetic Vectors , Hybridomas/immunology , Mice , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/immunologyABSTRACT
Radiosensitive severe combined immune deficiency in humans results from mutations in Artemis, a protein which, when coupled with DNA-dependent protein kinase catalytic subunit (DNA-PKcs), possesses DNA hairpin-opening activity in vitro. Here, we report that Artemis-deficient mice have an overall phenotype similar to that of DNA-PKcs-deficient mice-including severe combined immunodeficiency associated with defects in opening and joining V(D)J coding hairpin ends and increased cellular ionizing radiation sensitivity. While these findings strongly support the notion that Artemis functions with DNA-PKcs in a subset of NHEJ functions, differences between Artemis- and DNA-PKcs-deficient phenotypes, most notably decreased fidelity of V(D)J signal sequence joining in DNA-PKcs-deficient but not Artemis-deficient fibroblasts, suggest additional functions for DNA-PKcs. Finally, Artemis deficiency leads to chromosomal instability in fibroblasts, demonstrating that Artemis functions as a genomic caretaker.
