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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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Analysis of artemis protein sequence. (A) Artemis is composed of three identifiable regions, the β-Lact homologous region (amino acids 1–155), the associated β-CASP domain (amino acids 156–385), and the COOH-terminal region (amino acids 386–692). Regions of the Artemis sequence used for functional experiments are indicated. (B) Critical residues (in bold) that compose the catalytic site of bacterial β-Lacts are conserved in Artemis. The numbering is according to the Artemis protein sequence. Three residues (D165, H319, and V341) represent the signature of the β-CASP domain.
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fig1: Analysis of artemis protein sequence. (A) Artemis is composed of three identifiable regions, the β-Lact homologous region (amino acids 1–155), the associated β-CASP domain (amino acids 156–385), and the COOH-terminal region (amino acids 386–692). Regions of the Artemis sequence used for functional experiments are indicated. (B) Critical residues (in bold) that compose the catalytic site of bacterial β-Lacts are conserved in Artemis. The numbering is according to the Artemis protein sequence. Three residues (D165, H319, and V341) represent the signature of the β-CASP domain.

Mentions: We defined three separate regions in the Artemis protein (Fig. 1 A) based on analysis of its sequence (3, 11). The most NH2-terminal region, from the initiation methionine up to R155, encoded by exons 1–6, corresponds to the β-Lact homology domain. This region presents four out of the five conserved motives (Fig. 1 B, I to IV), constituting the β-Lact–active center and mostly consisting of histidine and aspartic acid residues that participate in zinc coordination and hydrolysis reaction characteristic of enzymes of this superfamily (14). This region is followed by the β-CASP region (11), which is encoded by exons 7–13, up to S385. This region is always associated with β-Lact motives I to IV within nucleic acid–processing enzymes of the β-CASP family such as murine SNM1, yeast PSO2, or the cleavage- and polyadenylation-specific factor. Two highly conserved residues (D165 and H319 in Artemis) constitute an outstanding signature of the β-CASP domain, whereas a third one (V341 in Artemis) appears to be specific of the kind of substrate that is highly conserved as a valine or a histidine in proteins acting on DNA or RNA, respectively (Fig. 1 B). Importantly, D165 or H319 in the β-CASP–specific region could represent the fifth motif of the β-Lact signature, which is not present in the β-Lact homology region per se. We propose that the β-CASP region participates with the β-Lact region in forming the catalytic site of Artemis. Lastly, the exon 14–encoded carboxy terminal half of the protein, from E386 to T692, designated hereafter as “C-Ter,” appears as a separate domain.


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)

Analysis of artemis protein sequence. (A) Artemis is composed of three identifiable regions, the β-Lact homologous region (amino acids 1–155), the associated β-CASP domain (amino acids 156–385), and the COOH-terminal region (amino acids 386–692). Regions of the Artemis sequence used for functional experiments are indicated. (B) Critical residues (in bold) that compose the catalytic site of bacterial β-Lacts are conserved in Artemis. The numbering is according to the Artemis protein sequence. Three residues (D165, H319, and V341) represent the signature of the β-CASP domain.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211804&req=5

fig1: Analysis of artemis protein sequence. (A) Artemis is composed of three identifiable regions, the β-Lact homologous region (amino acids 1–155), the associated β-CASP domain (amino acids 156–385), and the COOH-terminal region (amino acids 386–692). Regions of the Artemis sequence used for functional experiments are indicated. (B) Critical residues (in bold) that compose the catalytic site of bacterial β-Lacts are conserved in Artemis. The numbering is according to the Artemis protein sequence. Three residues (D165, H319, and V341) represent the signature of the β-CASP domain.
Mentions: We defined three separate regions in the Artemis protein (Fig. 1 A) based on analysis of its sequence (3, 11). The most NH2-terminal region, from the initiation methionine up to R155, encoded by exons 1–6, corresponds to the β-Lact homology domain. This region presents four out of the five conserved motives (Fig. 1 B, I to IV), constituting the β-Lact–active center and mostly consisting of histidine and aspartic acid residues that participate in zinc coordination and hydrolysis reaction characteristic of enzymes of this superfamily (14). This region is followed by the β-CASP region (11), which is encoded by exons 7–13, up to S385. This region is always associated with β-Lact motives I to IV within nucleic acid–processing enzymes of the β-CASP family such as murine SNM1, yeast PSO2, or the cleavage- and polyadenylation-specific factor. Two highly conserved residues (D165 and H319 in Artemis) constitute an outstanding signature of the β-CASP domain, whereas a third one (V341 in Artemis) appears to be specific of the kind of substrate that is highly conserved as a valine or a histidine in proteins acting on DNA or RNA, respectively (Fig. 1 B). Importantly, D165 or H319 in the β-CASP–specific region could represent the fifth motif of the β-Lact signature, which is not present in the β-Lact homology region per se. We propose that the β-CASP region participates with the β-Lact region in forming the catalytic site of Artemis. Lastly, the exon 14–encoded carboxy terminal half of the protein, from E386 to T692, designated hereafter as “C-Ter,” appears as a separate domain.

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