Rag1_R142* is a null mutant and Rag1_V779M is a hypomorphic mutant.

<p>A. Western analysis of Flag-tagged full-length Rag1 proteins expressed in Br3neo human fibroblast cells confirms that the wild-type (Rag1) and mutant (Rag1_V779M) proteins are expressed at comparable levels <i>in vivo</i>. B. Representative recombination data from using the indicated constructs for transient V(D)J recombination assays in Br3neo cells. C. Absolute recombination activity using wild-type Rag1 (hatched) or the p.V779M mutant (shaded) with signal-joint substrates (left) or coding-joint substrates (right). Results represent the mean ±s.d. of six independent experiments. D. Normalized recombination activity of the p.V779M mutant. Recombination activity of the p.V779M mutant on each substrate was normalized to the activity of wild-type Rag1. Results represent the mean ± s.d. of six independent experiments.</p

p.R142* maternal and p.V779M paternal mutations.

<p>P1 harbors a maternally inherited c.424C>T mutation, resulting in a premature stop codon. <b>A.</b> Sequencing chromatogram demonstrating the presence of a heterozygous c.424C>T mutation. <b>B.</b> Alignment of the wildtype and mutant Rag1 cDNA and protein sequences. c.424C>T creates a premature stop codon at position 142 of the protein. <b>C.</b> P1 harbors a paternally inherited c.2335G>A mutation, resulting in the non-synonymous coding mutation p.V779M. Sequencing chromatogram demonstrating the presence of a heterozygous c.2335G>A mutation. <b>D.</b> Alignment of the wildtype and mutant Rag1 cDNA and protein sequences. c.2335G>A creates a missense p.V779M mutation in the Rag1 protein.</p

Peripheral blood analysis of P1 at time of initial presentation (age 4½ months) is consistent with Omenn’s Syndrome.

<p>Normal values from the Children’s Hospital Laboratory, the Cincinnati Children’s Hospital Laboratory, or from reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121489#pone.0121489.ref030" target="_blank">30</a>].</p><p>Peripheral blood analysis of P1 at time of initial presentation (age 4½ months) is consistent with Omenn’s Syndrome.</p

Collapsed T cell repertoire in Omenn Syndrome Patient.

<p>TCR Vβ spectratype analysis of CDR3 reveals profound oligoclonality and monoclonality, consistent with Omenn Syndrome.</p

Overall domain structure of the human RAG1 protein.

<p>The human RAG1 protein is 1043 amino acid long and consists of a core region (aa 387–1011; yellow) and non-core regions (aa 1–386 and 1012–1043; white). There are two potential domains within the core region of Rag1: the central domain (aa 531–763; purple bar); and the C-terminal domain (aa 764–983; orange bar). Rag1 contains four basic regions (BI: aa 142–147; BII: aa 219–237; BIII: aa 244–252; BIV: aa 829–843; gray), a RING finger (aa 293–331; red), two zinc fingers (ZFA: aa 356–379; ZFB: aa 728–753; blue), a nonamer DNA-binding region (NBR: 387–457; dark yellow), a nuclear localization signal (NLS: aa 972–976; green), an Asp-Asp-Glu active site motif (D603, D711, E965), and two C-terminal zinc-binding sites (C905/C907 and H940/H945). The four basic regions serve as binding sites for the nuclear transport proteins Srp1 and Rch1. The RING finger functions as an E3 ubiquitin ligase and, together with zinc finger A, mediates Rag1 multimerization. Zinc finger B is thought to function as a Rag2 binding site. The positions of R142 and V779 are indicated.</p

Rag1_V779M has wild-type V(D)J cleavage activity.

<p><b>A</b>, Wild-type and mutant Rag1 proteins express and purify equally well. Coomassie stained gel of wild-type (Rag1) and mutant (Rag1_V779M) recombinant core Rag1 proteins purified from E. coli. A serial dilution series (5-fold dilutions between lanes) is shown for each protein. <b>B</b>, The p.V779M mutant protein catalyzes wild-type V(D)J cleavage <i>in vitro</i>. Cleavage reactions were performed with recombinant wild-type core Rag1 and core Rag1_V779M in the presence of recombinant full-length Rag2 and resolved by denaturing polyacrylamide gel electrophoresis. The positions of the substrate (S) and cleavage products (hairpin (H) and nick (N)) are indicated. <b>C</b>, Absolute V(D)J cleavage activity of wild-type Rag1 (left) and the p.V779M mutant (right). Results represent the mean ± s.d. of four independent experiments.</p

Overexpression of <i>SOD3</i> from a heterologous promoter significantly rescues ROS hypersensitivity of cells lacking Pho84.

<p>Cells were grown on YPD agar medium without or with 50 ng/ml doxycycline for 39 hrs, and were maintained in these doxycycline concentrations throughout the course of the experiment. Cells were inoculated at OD₆₀₀ 0.1 in YPD (glucose-containing medium that represses transcription from <i>pMAL2</i>), with vehicle DMSO (V) or 21 μM Plumbagin (P). OD₆₀₀ was monitored every 15 minutes for strains (A) wild type background: +/+, JKC915; +/+ <i>tetO-SOD3/SOD3</i>, JKC1738; +/+ <i>pMAL2-SOD3/SOD3</i>, JKC1776; (B) <i>pho84</i> null mutant background: -/-, JKC1450; -/- <i>tetO-SOD3/SOD3</i>, JKC1745; -/- <i>pMAL2-SOD3/SOD3</i>, JKC1780. A, B are representative of 3 biological replicates, error bars SD of 3 technical replicates.</p

Pho84 is required for resistance to killing by whole blood or neutrophils, in dependence on neutrophil ROS.

<p>(A) Percent survival of <i>C</i>. <i>albicans</i> cells after incubation with whole blood from healthy human volunteers; cells of genotypes <i>+/+</i>, JKC915; -/-, JKC1450 and -/-/+, JKC1588 were inoculated into blood and plated onto agar medium at the indicated time points for calculation of CFU/ml. <i>-/-</i> versus <i>+/+</i> at 5 h <i>p</i> = 0.008. (B) Percent survival of <i>C</i>. <i>albicans</i> cells after incubation with neutrophils, strains as in A, at M.O.I. 2 for 2 hrs and 5 hrs. <i>-/-</i> versus <i>+/+</i> at 5 h <i>p</i> = 0.04. (C) Human peripheral blood-derived neutrophils pretreated with different concentrations of N-acetyl-l-cysteine (NAC) were incubated for 90 min with strains as in A at M.O.I. 2. <i>-/-</i> versus <i>+/+</i> with vehicle <i>p</i><0.0001. (D) Human peripheral blood-derived neutrophils pretreated with 10 μM Diphenyleneiodonium (DPI) were incubated for 90 min with strains as in A at M.O.I. 2. Vehicle alone, DPI alone and neutrophil alone groups are controls. (E) Chronic Granulomatous Disease patient-derived neutrophils (CGD) were incubated for 2 hours with strains as in A at M.O.I. 2. <i>p</i><0.0001 for <i>-/-</i> in healthy control neutrophils (control) versus <i>+/+</i> in control, all others non-significant. <i>p</i> values per Student’s t-test. *<i>p</i><0.01; +<i>p</i><0.05; ns is non-significant. A-D representative of at least 3 biological replicates.</p

ROS management and Sod3 expression are defective in cells lacking <i>PHO84</i>.

<p>(A) DCFDA-detectable ROS of cells unexposed to extrinsic oxidative stress, of strains <i>+/+</i>, JKC915; -/-, JKC1450 and -/-/+, JKC1588 diluted into SC medium with 0.22 mM, 1 mM and 11 mM Pi. Fluorescence intensity was measured after staining cells with 50 μM DCFH-DA. <i>-/-</i> versus +/+ at 0.22 mM Pi <i>p</i> = 0.0149. <i>-/-</i> versus +/+ at 1 mM Pi <i>p</i><0.0001. <i>-/-</i> versus <i>+/+</i> at 11 mM Pi <i>p</i><0.0001. <i>p</i> values per Student’s t-test. (B) DCFDA-detectable ROS production during exposure to 100 μM menadione (Mena). Strains as in A cultured overnight were diluted into SC medium (Loflo) and fluorescence intensity was measured as in A. <i>p</i> = 0.0002 for <i>-/-</i> versus <i>+/+</i>. (C) Superoxide dismutase (SOD) activity of strains as in A, grown in YPD medium with vehicle, 50 μM Menadione (Mena) and 0.8 mM bathocuproine disulfonic acid (BCS) for 8 hours; cell lysate in non-denaturing gel stained with nitroblue tetrazolium to detect SOD activity and with Coomassie blue to assess loading. (D) Western blot of strains as in A, grown in normal SC medium with vehicle, 3 mM MnCl₂ and 3 mM CuSO₄ for 13 hours, probed for Sod3 and loading control tubulin. <i>p</i> values were calculated using Student’s t-test. *<i>p</i><0.01; +<i>p</i><0.05; ns non-significant. A-D show representatives of at least 3 biological replicates; error bars SD of 3 technical replicates.</p

TORC1 activity contributes to ROS management and Sod3 expression.

<p>(A) DCFDA-detectable ROS measurement of strains, (1) <i>+/+</i> containing vector (JKC1594), (2) <i>-/-</i> containing vector (JKC1598), (3) <i>+/+</i> overexpressing <i>GTR1</i> (JKC1596), (4) <i>-/-</i> overexpressing <i>GTR1</i> (JKC1600). Cells cultured overnight in YPD were diluted into SC medium (Loflo) with vehicle or 8 ng/ml rapamycin for 1 hour, and fluorescence intensity was determined after staining cells with 50 μM DCFH-DA. <i>p</i> = 0.0252 for ratio of <i>-/-</i> with vector and <i>-/-</i> overexpressing <i>GTR1</i> versus ratio of wild type with vector and wild type overexpressing <i>GTR1</i>; all cells exposed to vehicle. <i>p</i> = 0.0014 for ratio of <i>-/-</i> with vector and <i>-/-</i> overexpressing <i>GTR1</i> versus ratio of wild type with vector and wild type overexpressing <i>GTR1</i>; all cells exposed to rapamycin. <i>p</i> values per Student’s t-test. (B) Western blot of strains as in A, grown in SC medium (Loflo) for 1 hour, probed for Sod3 and loading control tubulin. (C) Wild type (SC5314) cells were exposed to vehicle (v), 0.5 mM foscarnet (F) or 1 mM PAA (P) for 1 hour, and fluorescence intensity was determined as in A. p<0.001 for both 0.5 mM foscarnet versus vehicle and 1 mM PAA versus vehicle. A-C show representatives of at least 3 biological replicates; error bars SD of 3 technical replicates.</p