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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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V(D)J recombination on chromosomal substrates. (A) structure of the MX-RSS-EGFP/ires-HuCD4 construct before and after inversional V(D)J recombination. In the germline configuration, an inverted EGFP cassette is flanked by two RSS. Upon V(D)J recombination, the EGFP gene is reoriented and productively transcribed from the LTR promoter. (B) Wild-type OTEL- and Artemis-deficient GUETEL cells were transduced with MX-RSS-EGFP/ires-HuCD4 resulting in 43 and 80% CD4+ cells, respectively. Recombination of the substrates is initiated by transient transfection of RAG1/RAG2 expression constructs with or without Artemis. (C) Mean results of 6 V(D)J recombination experiments in GUETEL/RSS cells. The V(D)J recombination activity is calculated relative to the recombination frequency obtained with RAG1 (R1), RAG2 (R2), and wild-type Artemis (Arte wt).
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fig2: V(D)J recombination on chromosomal substrates. (A) structure of the MX-RSS-EGFP/ires-HuCD4 construct before and after inversional V(D)J recombination. In the germline configuration, an inverted EGFP cassette is flanked by two RSS. Upon V(D)J recombination, the EGFP gene is reoriented and productively transcribed from the LTR promoter. (B) Wild-type OTEL- and Artemis-deficient GUETEL cells were transduced with MX-RSS-EGFP/ires-HuCD4 resulting in 43 and 80% CD4+ cells, respectively. Recombination of the substrates is initiated by transient transfection of RAG1/RAG2 expression constructs with or without Artemis. (C) Mean results of 6 V(D)J recombination experiments in GUETEL/RSS cells. The V(D)J recombination activity is calculated relative to the recombination frequency obtained with RAG1 (R1), RAG2 (R2), and wild-type Artemis (Arte wt).

Mentions: We undertook deletional and single amino acid mutagenesis analyses to define the core catalytic region of Artemis in vivo. Given recent studies demonstrating a different RAG1/RAG2 requirement for V(D)J recombination on extrachromosomal versus chromosomal substrates (12, 15–17), we analyzed the function of Artemis in the context of in-chromosome V(D)J recombination by using the experimental strategy developed by Liang et al. (12). A chromosomal V(D)J recombination substrate was stably integrated in the Artemis-deficient GUETEL (GUETEL/RSS cells) and control OTEL (OTEL/RSS) cell lines by means of retroviral infection (Fig. 2). pMX-RSS-GFP/ires-huCD4 (Fig. 2 A) is a retroviral construct in which an RSS-flanked GFP cassette is inserted in reverse transcriptional orientation relative to the 5′ LTR promoter. Cells carrying the V(D)J reporter cassette are detected through the cell surface expression of huCD4 (Fig. 2 B). V(D)J recombination of the construct is induced through the transient transfection of RAG1/RAG2 expression plasmids, which results in the inversion of the GFP cassette leading to green fluorescence expression. The control OTEL/RSS cells recombine the substrate (1.44% GFP+/CD4+) in the presence of both RAG1 and RAG2 but not in the sole presence of RAG1 (0.07%) as expected, and the addition of exogenous Artemis does not increase the recombination frequency (1.79%). In contrast, the Artemis-deficient GUETEL/RSS cells poorly rearrange the substrate in the presence of RAG1 and RAG2 (0.22%), a defect that is fully complemented by the addition of exogenous Artemis (1.36%). Interestingly, the recombination frequency of GUETEL/RSS cells in the absence of Artemis is significantly above background level (0.22 vs. 0.03%) in accord with the previously reported leakiness of Artemis-deficient cells in mice (4, 18). Sequencing the V(D)J coding joints from GUETEL/RSS cells transfected in the absence of Artemis revealed a high frequency of 1–8-bp-long P nucleotide addition (Fig. 3 B), a situation previously associated with Artemis deficiency in murine ES cells but not found in control, V(D)J-proficient, OTEL/RSS cells (Fig. 3 A). The addition of Artemis completely restores the quality of the V(D)J junctions in GUETEL/RSS cells (Fig. 3 C). The integrated results of six experiments show that the relative level of V(D)J recombination (Fig. 2 C) and the quality of the resulting CJ (Fig. 3 D) is identical whether FL or β-Lact/β-CASP domain-only forms of Artemis are used to complement the GUETEL/RSS cells. Therefore, we conclude that the β-Lact/β-CASP region of Artemis carries the catalytic activity required for V(D)J recombination.


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)

V(D)J recombination on chromosomal substrates. (A) structure of the MX-RSS-EGFP/ires-HuCD4 construct before and after inversional V(D)J recombination. In the germline configuration, an inverted EGFP cassette is flanked by two RSS. Upon V(D)J recombination, the EGFP gene is reoriented and productively transcribed from the LTR promoter. (B) Wild-type OTEL- and Artemis-deficient GUETEL cells were transduced with MX-RSS-EGFP/ires-HuCD4 resulting in 43 and 80% CD4+ cells, respectively. Recombination of the substrates is initiated by transient transfection of RAG1/RAG2 expression constructs with or without Artemis. (C) Mean results of 6 V(D)J recombination experiments in GUETEL/RSS cells. The V(D)J recombination activity is calculated relative to the recombination frequency obtained with RAG1 (R1), RAG2 (R2), and wild-type Artemis (Arte wt).
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Related In: Results  -  Collection

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fig2: V(D)J recombination on chromosomal substrates. (A) structure of the MX-RSS-EGFP/ires-HuCD4 construct before and after inversional V(D)J recombination. In the germline configuration, an inverted EGFP cassette is flanked by two RSS. Upon V(D)J recombination, the EGFP gene is reoriented and productively transcribed from the LTR promoter. (B) Wild-type OTEL- and Artemis-deficient GUETEL cells were transduced with MX-RSS-EGFP/ires-HuCD4 resulting in 43 and 80% CD4+ cells, respectively. Recombination of the substrates is initiated by transient transfection of RAG1/RAG2 expression constructs with or without Artemis. (C) Mean results of 6 V(D)J recombination experiments in GUETEL/RSS cells. The V(D)J recombination activity is calculated relative to the recombination frequency obtained with RAG1 (R1), RAG2 (R2), and wild-type Artemis (Arte wt).
Mentions: We undertook deletional and single amino acid mutagenesis analyses to define the core catalytic region of Artemis in vivo. Given recent studies demonstrating a different RAG1/RAG2 requirement for V(D)J recombination on extrachromosomal versus chromosomal substrates (12, 15–17), we analyzed the function of Artemis in the context of in-chromosome V(D)J recombination by using the experimental strategy developed by Liang et al. (12). A chromosomal V(D)J recombination substrate was stably integrated in the Artemis-deficient GUETEL (GUETEL/RSS cells) and control OTEL (OTEL/RSS) cell lines by means of retroviral infection (Fig. 2). pMX-RSS-GFP/ires-huCD4 (Fig. 2 A) is a retroviral construct in which an RSS-flanked GFP cassette is inserted in reverse transcriptional orientation relative to the 5′ LTR promoter. Cells carrying the V(D)J reporter cassette are detected through the cell surface expression of huCD4 (Fig. 2 B). V(D)J recombination of the construct is induced through the transient transfection of RAG1/RAG2 expression plasmids, which results in the inversion of the GFP cassette leading to green fluorescence expression. The control OTEL/RSS cells recombine the substrate (1.44% GFP+/CD4+) in the presence of both RAG1 and RAG2 but not in the sole presence of RAG1 (0.07%) as expected, and the addition of exogenous Artemis does not increase the recombination frequency (1.79%). In contrast, the Artemis-deficient GUETEL/RSS cells poorly rearrange the substrate in the presence of RAG1 and RAG2 (0.22%), a defect that is fully complemented by the addition of exogenous Artemis (1.36%). Interestingly, the recombination frequency of GUETEL/RSS cells in the absence of Artemis is significantly above background level (0.22 vs. 0.03%) in accord with the previously reported leakiness of Artemis-deficient cells in mice (4, 18). Sequencing the V(D)J coding joints from GUETEL/RSS cells transfected in the absence of Artemis revealed a high frequency of 1–8-bp-long P nucleotide addition (Fig. 3 B), a situation previously associated with Artemis deficiency in murine ES cells but not found in control, V(D)J-proficient, OTEL/RSS cells (Fig. 3 A). The addition of Artemis completely restores the quality of the V(D)J junctions in GUETEL/RSS cells (Fig. 3 C). The integrated results of six experiments show that the relative level of V(D)J recombination (Fig. 2 C) and the quality of the resulting CJ (Fig. 3 D) is identical whether FL or β-Lact/β-CASP domain-only forms of Artemis are used to complement the GUETEL/RSS cells. Therefore, we conclude that the β-Lact/β-CASP region of Artemis carries the catalytic activity required for V(D)J recombination.

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