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

Simkus, Carrie; Anand, Priyanka; Bhattacharyya, Anamika; Jones, Jessica M.

doi:10.4049/jimmunol.179.12.8332

Cited by 28 publications

(76 citation statements)

References 53 publications

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“…The ring-finger domain's E3 activity prepares full-length RAG1 for the initiation of cleavage via the ubiquitylation of histone H3. Our results suggest that ubiquitylation of histone H3 spatially releases RAG1, however, we could not rule out the possibility that this transition into cleavage may also involve a conformational change in RAG1 [16]. The spatial release and an active conformation could be triggered simultaneously, and they are not necessarily mutually exclusive.…”

Section: Discussionmentioning

confidence: 90%

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RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

Deng

Liu

2015

Cell Res

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The adaptive immune system in vertebrates relies on an enormous repertoire of antigen receptors. The variable domains of antigen receptors are encoded by extensive arrays of variety, diversity and joining (V, D, and J) gene segments flanked by conserved recombination signal sequences (RSSs) [1]. During the early lymphoid-specific process known as V(D)J recombination, RAG1 and RAG2 recognize and pair two RSSs based on the 12/23 rule and introduce a pair of double-strand breaks (DSB) between each RSS and its adjoining coding segment [2]. The non-homologous DNA end-joining pathway then rejoins the coding segments together and generates functional receptors [3].Murine RAG1 contains 1 040 amino acids, and RAG2 contains 527 amino acids. Biochemical studies have confirmed that the core regions of RAG1 (aa 384-1 008) and RAG2 (aa 1-387) are fundamental for the cleavage reaction in vitro [4]. Core RAG1 is capable of recognizing RSSs and can form tetramers with RAG2 in which the DDE triad is responsible for the DNA cleavage activity [5]. Core RAG2 contains Kelch motifs that assist RAG1 binding and cutting of the RSSs [6]. The plant homology domain within the RAG2 non-core region has recently been shown to bind histone H3K4Me3 and activate the recombination reaction [7,8]. Regarding the non-core region, especially the N-terminal region of RAG1 (aa 1-383), several conserved clusters of basic residues have been identified within residues 1-264 [9]. This region is thought to bind to a zinc ion and associate with KPNA1 and SRP1 [10][11][12]. A recently defined WW-like domain (aa 179-205) might mediate protein-protein interactions [13]. Residues 265-383 of RAG1 contain a C3HC4 ring-finger domain harboring E3 ubiquitin ligase activity [14]. However, the roles of these domains or motifs in V(D)J recombination have not been fully elucidated.The impaired V(D)J recombination in core RAG1 knock-in mice suggests that non-core RAG1 increases the efficiency and accuracy of recombination, yet quite a number of lymphocytes can mature, indicating that non-core RAG1 is dispensable for basic recombination activity [15]. Nevertheless, in some Omenn syndrome Cell Research (2015) 25:181-192.

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Section: Discussionmentioning

confidence: 90%

“…Previous studies of the RAG1 ring-finger domain have revealed that the loss of E3 ubiquitin ligase activity correlates with decreased V(D)J recombination of episomal substrates [16,17]. Histone H3 was recently defined as a ubiquitylation substrate of the RAG1 ring-finger domain, suggesting a chromatin-mediated regulation of V(D)J recombination [18,19].…”

mentioning

confidence: 99%

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

Deng

Liu

2015

Cell Res

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“…A naturally occurring human mutation in this RING finger motif (C328Y) was found to cause the primary immunodeficiency disease Omenn's syndrome (20). A study of the equivalent mutation in mouse RAG1 (C325Y) showed that it greatly reduced recombination of an extrachromosomal plasmid, as did mutation of the neighboring residue (P326G) (21). Other RING finger residues critical for ubiquitin ligase activity appeared to contribute to robust recombination of extrachromosomal substrates (22).…”

Section: Significancementioning

confidence: 99%

Role of RAG1 autoubiquitination in V(D)J recombination

Singh

Gellert

2015

Proc. Natl. Acad. Sci. U.S.A.

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The variable domains of Ig and T-cell receptor genes in vertebrates are assembled from gene fragments by the V(D)J recombination process. The RAG1-RAG2 recombinase (RAG1/2) initiates this recombination by cutting DNA at the borders of recombination signal sequences (RSS) and their neighboring gene segments. The RAG1 protein is also known to contain a ubiquitin E3 ligase activity, located in an N-terminal region that is not strictly required for the basic recombination reaction but helps to regulate recombination. The isolated E3 ligase domain was earlier shown to ubiquitinate one site in a neighboring RAG1 sequence. Here we show that autoubiquitination of full-length RAG1 at this specific residue (K233) results in a large increase of DNA cleavage by RAG1/2. A mutational block of the ubiquitination site abolishes this effect and inhibits recombination of a test substrate in mouse cells. Thus, ubiquitination of RAG1, which can be promoted by RAG1's own ubiquitin ligase activity, plays a significant role in governing the level of V(D)J recombination activity.J recombination plays a central role in the production of antigen receptors by recombining V, D, and J gene segments from their genomic clusters to give rise to the highly varied populations of immunoglobulins and T-cell receptors (1). Recombination starts with the introduction of double-strand breaks by the RAG1/RAG2 protein complex at a pair of recombination signal sequences (RSS) (2, 3), distinguished by the length of the spacer DNA separating their conserved heptamer and nonamer elements. Recombination requires one RSS with a 12-base pair spacer and another with a 23-base pair spacer. Each pair of breaks is then processed by the nonhomologous DNA end-joining group of proteins to produce a junction of two segments of coding sequence (a coding joint) and a junction of the two RSSs (a signal joint) (4). The purified RAG1/2 protein complex displays the correct specificity for pairs of RSSs (5, 6), and has thus been used as a model for the initiation of V(D)J recombination. Until recently, the RAG proteins used for these studies have generally been minimal "core" regions of RAG1 and RAG2 (amino acids 384-1,008 of 1,040 in mouse RAG1 and 1-387 of 527 in RAG2), which are sufficient for specific binding and cleavage activity in a purified cell-free system. Ectopic expression of these truncated proteins supports V(D)J recombination in suitable cell lines, although with differences from the full-length proteins that will be discussed here.A complex composed of core RAG1 and RAG2 is more active than its full-length counterpart in cleavage of extrachromosomal substrates in a hamster cell line, but overall recombination is reported to be lower (7), indicating a defect in the stages of recombination subsequent to DNA cleavage. Similarly, mice or pre-B cells missing the RAG2 C-terminal noncore region are defective in the V to DJ recombination step of Ig heavy chain joining, although the earlier D to J joining step is normal (8). The mice also display an increased prevalen...

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“…cRAG1 is also partially defective in chromosomal recombination (Roman et al 1997), and the knock-in mouse displays a phenotype consistent with an overall reduced level of rearrangement and an increase in aberrant joints Talukder et al 2004). In an otherwise intact protein, mutations in the nc regions can lead to reductions in recombination far more severe than that seen with deletion of the entire nc regions (Roman et al 1997;Sadofsky et al 1994;Sadofsky et al 1993;Silver et al 1993;Simkus et al 2007). Furthermore, defects associated with mutations in ncRAG1 become more pronounced when the protein is present at limiting levels (Roman et al 1997;Simkus et al 2007), a situation likely to mimic normal recombination conditions (Leu and Schatz 1995).…”

Section: Rag1mentioning

confidence: 99%

“…<1% (Simkus et al 2007) P326G~W.T.~10%, <1% under limiting conditions (Silver et al 1993;Simkus et al 2007) C328S~W.T.~6% (Silver et al 1993) hC328Y OS (Villa et al 2001) hK136Q OS (Sobacchi et al 2006) hC192Y OS (Sobacchi et al 2006) hR314W B/T reduced immunodeficiency with granulomas (Schuetz et al 2008) All substitutions refer to the murine protein unless designated human (h). up~40-100% on extra-chromosomal substrates (Silver et al 1993;Sadofsky et al 1994) block in IgH V to DJ rearrangement in mouse (Liang et al 2002;Akamatsu et al 2003) RAG2∆388-413~100% (Silver et al 1993 T490A up supports recombination no G1/S-specific destruction of RAG2 accumulation of aberrant products in mouse thymocytes (Lee and Desiderio1999) persistence of SEC throughout cell cycle (Jiang et al 2004) hW416L OS (Sobacchi et al 2006) hK440L OS (Sobacchi et al 2006) hW453R OS (Noordzij et al 2002) hN474S SCID with maternal fetal engraftment (Villa et al 2001) hC478Y SCID (Villa et al 2001) hH481P SCID (Noordzij et al 2002) M502V OS (Sobacchi et al 2006) All substitutions refer to the murine protein unless designated human (h).…”

Section: Rag1mentioning

confidence: 99%

The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

Jones

Simkus

2009

Arch. Immunol. Ther. Exp.

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The enormous repertoire of the vertebrate specific immune system relies on the rearrangement of discrete gene segments into intact antigen receptor genes during the early stages of B-and T-cell development. This V(D)J recombination is initiated by a lymphoid-specific recombinase comprising the RAG1 and RAG2 proteins, which introduces double-strand breaks in the DNA adjacent to the coding segments. Much of the biochemical research into V(D)J recombination has focused on truncated or "core" fragments of RAG1 and RAG2, which lack approximately one third of the amino acids from each. However, genetic analyses of SCID and Omenn syndrome patients indicate that residues outside the cores are essential to normal immune development. This is in agreement with the striking degree of conservation across all vertebrate classes in certain non-core domains. Work from multiple laboratories has shed light on activities resident within these domains, including ubiquitin ligase activity and KPNA1 binding by the RING finger domain of RAG1 and the recognition of specific chromatin modifications as well as phosphoinositide binding by the PHD module of RAG2. In addition, elements outside of the cores are necessary for regulated protein expression and turnover. Here the current state of knowledge is reviewed regarding the non-core regions of RAG1 and RAG2 and how these findings contribute to our broader understanding of recombination.

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

Cited by 28 publications

References 53 publications

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

Role of RAG1 autoubiquitination in V(D)J recombination

The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

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