Autoinhibition of the Nuclease ARTEMIS is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains [Immunology]

January 12th, 2017 by Doris Niewolik, Ingrid Peter, Carmen Butscher, Klaus Schwarz

The nuclease ARTEMIS is essential for the development of B and T lymphocytes. It is required for opening DNA hairpins generated during antigen receptor gene assembly from variable (V), diversity (D) and joining (J) subgenic elements (V(D)J recombination). As a member of the non-homologous end joining pathway it is also involved in repairing a subset of pathological DNA double-strand breaks. Loss of ARTEMIS function therefore results in radiosensitive severe combined immunodeficiency (RS-SCID). The hairpin-opening activity is dependent on the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which can bind to and phosphorylate ARTEMIS. The ARTEMIS C-terminus is dispensable for cellular V(D)J recombination and in vitro nuclease assays with C-terminally truncated ARTEMIS show DNA PKcs-independent hairpin-opening activity. Therefore it has been postulated that ARTEMIS is regulated via autoinhibition by its C-terminus. To obtain evidence for the autoinhibition model, we performed co-immunoprecipitation experiments with combinations of ARTEMIS mutants. We show that an N-terminal fragment comprising the catalytic domain can interact both with itself and with a C-terminal fragment. Amino acid exchanges N456A+S457A+E458Q in the C-terminus of full-length ARTEMIS resulted in unmasking of the N-terminus and in increased ARTEMIS activity in cellular V(D)J recombination assays. Mutations in ARTEMIS-deficient patients impaired the interaction with the C-terminus and also affected protein stability. The interaction between the N- and C-terminal domains was not DNA-PKcs dependent and phosphomimetic mutations in the C-terminal domain did not result in unmasking of the catalytic domain. Our experiments provide strong evidence that a physical interaction between the C-terminal and catalytic domains mediates ARTEMIS autoinhibition.