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Biochemical and Folding Defects in a RAG1 Variant Associated with Omenn Syndrome¹

Department of Biochemistry and Molecular and Cellular Sciences, Georgetown University, Washington, DC 20057

The RAG1 and RAG2 proteins are required to assemble mature Ag receptor genes in developing lymphocytes. Hypomorphic mutations in the gene encoding RAG1 are associated with Omenn syndrome, a primary immunodeficiency. We explored the biochemical defects resulting from a mutation identified in an Omenn syndrome patient which generates an amino acid substitution in the RAG1 RING finger/ubiquitin ligase domain (C325Y in murine RAG1) as well as an adjacent substitution (P326G). RAG1 C325Y demonstrated a 50-fold reduction in recombination activity in cultured pro-B cells despite the fact that its expression and localization to the nucleus were similar to the wild-type protein. The C325Y substitution severely abrogated ubiquitin ligase activity of the purified RAG1 RING finger domain, and the tertiary structure of the domain was altered. The P326G substitution also abrogated ubiquitin ligase activity but had a less severe effect on protein folding. RAG1 P326G also demonstrated a recombination impairment that was most pronounced when RAG1 levels were limiting. Thus, a correctly folded RAG1 RING finger domain is required for normal V(D)J recombination, and RAG1 ubiquitin ligase activity can contribute when the protein is present at relatively low levels.

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¹ This work was supported by grants to J.M.J. from the National Institutes of Health (AI062854-01) and American Cancer Society (IRG-97-152-11). P.A. and A.B. are Ph.D. candidates at Georgetown University, and this work is submitted in partial fulfillment of the requirements for the Ph.D.

² C.S. and P.A. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Jessica M. Jones, Department of Biochemistry and Molecular and Cellular Sciences, Georgetown University, Basic Science Building Room 323, 3900 Reservoir Road Northwest, Washington, DC 20057. E-mail address: jonesj5{at}georgetown.edu

⁴ Abbreviations used in this paper: OS, Omenn syndrome; SJ, signal joint; CJ, coding joint; N-TR, N-terminal region; mRAG1, murine RAG1; hRAG1, human RAG1; EGFP, enhanced GFP; H6, histidine 6.