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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 and in vitro nuclease 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.

SUBMITTER: Niewolik D

PROVIDER: S-EPMC5336168 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.

Niewolik Doris D Peter Ingrid I Butscher Carmen C Schwarz Klaus K

The Journal of biological chemistry 20170112 8

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). ...[more]

PMID: 28082683

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Project description:BackgroundThe Gag capsid (CA) is one of the most conserved proteins in highly-diversified human and simian immunodeficiency viruses (HIV and SIV). Understanding the limitations imposed on amino acid sequences in CA could provide valuable information for vaccine immunogen design or anti-HIV drug development. Here, by comparing two pathogenic SIV strains, SIVmac239 and SIVsmE543-3, we found critical amino acid residues for functional interaction between the N-terminal and the C-terminal domains in CA.ResultsWe first examined the impact of Gag residue 205, aspartate (Gag205D) in SIVmac239 and glutamate (Gag205E) in SIVsmE543-3, on viral replication; due to this difference, Gag206-216 (IINEEAADWDL) epitope-specific cytotoxic T lymphocytes (CTLs) were previously shown to respond to SIVmac239 but not SIVsmE543-3 infection. A mutant SIVmac239, SIVmac239Gag205E, whose Gag205D is replaced with Gag205E showed lower replicative ability. Interestingly, however, SIVmac239Gag205E passaged in macaque T cell culture often resulted in selection of an additional mutation at Gag residue 340, a change from SIVmac239 valine (Gag340V) to SIVsmE543-3 methionine (Gag340M), with recovery of viral fitness. Structural modeling analysis suggested possible intermolecular interaction between the Gag205 residue in the N-terminal domain and Gag340 in the C-terminal in CA hexamers. The Gag205D-to-Gag205E substitution in SIVmac239 resulted in loss of in vitro core stability, which was recovered by additional Gag340V-to-Gag340M substitution. Finally, selection of Gag205E plus Gag340M mutations, but not Gag205E alone was observed in a chronically SIVmac239-infected rhesus macaque eliciting Gag206-216-specific CTL responses.ConclusionsThese results present in vitro and in vivo evidence implicating the interaction between Gag residues 205 in CA NTD and 340 in CA CTD in SIV replication. Thus, this study indicates a structural constraint for functional interaction between SIV CA NTD and CTD, providing insight into immunogen design to limit viral escape options.

Dataset Information

Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.

Publications

Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.

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