(A) Schematic of the Rag2 protein showing the core and non-core regions and enhancement of the C-terminal non-core region that includes the acidic hinge region, the PHD, and the noncanonical NLS overlapping the phosphorylation site at residue T490 (thin black vertical bar) and the cationic region between residues 499–508. Thick black vertical bars indicate residues mutated in OS/SCID patients; mutations analyzed in this study are in bold. Anchor residues forming the noncanonical PHD finger are indicated, and related interactions with Zinc ions (Zn1 and Zn2) are represented by gray bars; L1 and L2 indicate segments forming loops between pairs of zinc-coordinating residues, as previously described (18). C and H represent Zn⁺² coordinating cysteine and histidine residues present in the Rag2 PHD domain. (B) A mouse Rag2^–/– pro-B cell line was retrovirally transduced and selected to express constructs of interest: empty vector (Mock), FNT tag, FNT-tagged R2CR, C terminus of Rag2 (R2CT), R2FL, or full-length T-B-SCID/OS Rag2 mutants. Genomic DNA was harvested from the above cell lines, and Southern Blot analysis was performed on PCR-amplified products of IgH D-to-J, VH7183-to-DJ, VHQ52-to-DJ, and Vκ-to-Jκ rearrangements, as indicated to the left of each panel. Numbers of the left side of the figure (1 to 5) represent different recombination products. Triangles at the top of panels indicate PCR amplification of 400 ng, 200 ng, and 25 ng of DNA template. A fragment of Rag1 gene was amplified as a loading control. For each type of rearrangement, samples were loaded on the same gel, and detection intensity was identical. PCR from nontransduced cells was also used as control. The results shown are representative of 2 independent experiments. mRag2^–/–, mouse Rag2^–/–.