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The metallo-beta-lactamase/beta-CASP domain of Artemis constitutes the catalytic core for V(D)J recombination.

Poinsignon C, Moshous D, Callebaut I, de Chasseval R, Villey I, de Villartay JP - J. Exp. Med. (2004)

Bottom Line: Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates.Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity.The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

View Article: PubMed Central - PubMed

Affiliation: Développement Normal et Pathologique de Système Immunitaire, INSERM U429, Hôpital Necker Enfants Malades, 75015 Paris, France.

ABSTRACT
The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-beta-lactamase (beta-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the beta-Lact homology domain, to which is appended (b) the beta-CASP region, specific of members of the beta-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

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Complementation of the radiosensitivity of RS-SCID primary fibroblasts by Artemis. (A) Structure of the pMND-Arte-ires-GFP construct. (B) Index of GFP+ cells in transduced/untransduced mixed cell populations after irradiation. The index is calculated relative to the unirradiated cells. RS-SCID cells were transduced with empty virus (○), FL-Artemis–encoding virus (▪), or β-Lact/β-CASP–expressing virus (•). (C) WB analysis of Artemis expression in transduced mixed populations using ant-myc antibody.
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fig5: Complementation of the radiosensitivity of RS-SCID primary fibroblasts by Artemis. (A) Structure of the pMND-Arte-ires-GFP construct. (B) Index of GFP+ cells in transduced/untransduced mixed cell populations after irradiation. The index is calculated relative to the unirradiated cells. RS-SCID cells were transduced with empty virus (○), FL-Artemis–encoding virus (▪), or β-Lact/β-CASP–expressing virus (•). (C) WB analysis of Artemis expression in transduced mixed populations using ant-myc antibody.

Mentions: Artemis coding regions (FL or β-Lact/β-CASP domain) were subcloned into the pMND-MFG retroviral vector (provided by D. Kohn, Children's Hospital Los Angeles, Los Angeles, CA) upstream of an ires-GFP cassette under the regulation of the virus LTR (see Fig. 5 A). Retroviral supernatants were used to transduce a primary fibroblast cell line from an RS-SCID patient, resulting in a mixed population with a given percentage of transduced (GFP+) and untransduced (GFP−) cells. The mixed populations were subjected to increasing doses of γ ray irradiation (0–2 Gy) and the percentage of GFP+ cells was evaluated by FACS® analysis 15 d after irradiation to assess the selective advantage conferred by the introduction of the various forms of Artemis. The index of GFP+ cells after irradiation is calculated upon comparison with the percentage of GFP+ cells in the unirradiated samples.


The metallo-beta-lactamase/beta-CASP domain of Artemis constitutes the catalytic core for V(D)J recombination.

Poinsignon C, Moshous D, Callebaut I, de Chasseval R, Villey I, de Villartay JP - J. Exp. Med. (2004)

Complementation of the radiosensitivity of RS-SCID primary fibroblasts by Artemis. (A) Structure of the pMND-Arte-ires-GFP construct. (B) Index of GFP+ cells in transduced/untransduced mixed cell populations after irradiation. The index is calculated relative to the unirradiated cells. RS-SCID cells were transduced with empty virus (○), FL-Artemis–encoding virus (▪), or β-Lact/β-CASP–expressing virus (•). (C) WB analysis of Artemis expression in transduced mixed populations using ant-myc antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211804&req=5

fig5: Complementation of the radiosensitivity of RS-SCID primary fibroblasts by Artemis. (A) Structure of the pMND-Arte-ires-GFP construct. (B) Index of GFP+ cells in transduced/untransduced mixed cell populations after irradiation. The index is calculated relative to the unirradiated cells. RS-SCID cells were transduced with empty virus (○), FL-Artemis–encoding virus (▪), or β-Lact/β-CASP–expressing virus (•). (C) WB analysis of Artemis expression in transduced mixed populations using ant-myc antibody.
Mentions: Artemis coding regions (FL or β-Lact/β-CASP domain) were subcloned into the pMND-MFG retroviral vector (provided by D. Kohn, Children's Hospital Los Angeles, Los Angeles, CA) upstream of an ires-GFP cassette under the regulation of the virus LTR (see Fig. 5 A). Retroviral supernatants were used to transduce a primary fibroblast cell line from an RS-SCID patient, resulting in a mixed population with a given percentage of transduced (GFP+) and untransduced (GFP−) cells. The mixed populations were subjected to increasing doses of γ ray irradiation (0–2 Gy) and the percentage of GFP+ cells was evaluated by FACS® analysis 15 d after irradiation to assess the selective advantage conferred by the introduction of the various forms of Artemis. The index of GFP+ cells after irradiation is calculated upon comparison with the percentage of GFP+ cells in the unirradiated samples.

Bottom Line: Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates.Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity.The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

View Article: PubMed Central - PubMed

Affiliation: Développement Normal et Pathologique de Système Immunitaire, INSERM U429, Hôpital Necker Enfants Malades, 75015 Paris, France.

ABSTRACT
The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-beta-lactamase (beta-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the beta-Lact homology domain, to which is appended (b) the beta-CASP region, specific of members of the beta-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

Show MeSH
Related in: MedlinePlus