title: Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.
abstract: 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 andin vitronuclease assays with C-terminally truncated ARTEMIS showing 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.
authors.affiliation: Institute for Transfusion Medicine, University of Ulm. Electronic address: doris.niewolik@uni-ulm.de.
authors.forename: Doris
authors.initials: D
authors.lastname: Niewolik
authors.affiliation: Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Service Baden-Wuerttemberg-Hessen, Ulm, Germany 89081.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Peter
authors.affiliation: Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Service Baden-Wuerttemberg-Hessen, Ulm, Germany 89081.
authors.forename: Carmen
authors.initials: C
authors.lastname: Butscher
authors.affiliation: Institute for Transfusion Medicine, University of Ulm; Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Service Baden-Wuerttemberg-Hessen, Ulm, Germany 89081.
authors.forename: Klaus
authors.initials: K
authors.lastname: Schwarz
mesh_terms: D020134:Catalytic Domain; D002478:Cells, Cultured; D000072481:DNA Ligase ATP / Q000378:metabolism; D004268:DNA-Binding Proteins; D004720:Endonucleases; D005347:Fibroblasts / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D060152:V(D)J Recombination
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C426344:DCLRE1C protein, human; D004720:Endonucleases; D000072481:DNA Ligase ATP
keywords: ARTEMIS; DNA repair; DNA-dependent serine/threonine protein kinase (DNA-PK); NHEJ; V(D)J recombination; autoinhibition; immunodeficiency; protein phosphorylation; protein-protein interaction