rag-1 and rag-2 Are Components of a High-Molecular-Weight Complex, and Association of rag-2 with This Complex Is rag-1 Dependent

Leu, Thomas; Schatz, David G.

doi:10.1128/mcb.15.10.5657

Cited by 72 publications

(57 citation statements)

References 55 publications

(69 reference statements)

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“…3B, lanes 4 and 8), in agreement with previously published results (37). Similar levels of mRAG1 wild-type and C325Y proteins could be found in the soluble nuclear fraction (Fig.…”

Section: Normal Mrag1 C325y Expression and Cellular Distributionsupporting

confidence: 92%

“…FACS analysis of transiently transfected pro-B cells indicated that the levels of mRAG1-EGFP wild-type and C325Y expression were indistinguishable from one another but that both were expressed at a much lower level than EGFP alone (data not shown), again suggesting that the wild-type and mutant mRAG1 proteins are regulated in a similar manner. We cannot explain why mRAG1 was present in the cytoplasmic extract although the microscopy clearly indicated its nuclear localization, but this discordant pattern has been noted previously (37). Taken together, these data indicate that inappropriate compartmentalization could not account for the mRAG1 C325Y recombination defect.…”

Section: Normal Mrag1 C325y Expression and Cellular Distributionsupporting

confidence: 42%

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

Simkus

Anand

Bhattacharyya

et al. 2007

The Journal of Immunology

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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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“…3B, lanes 4 and 8), in agreement with previously published results (37). Similar levels of mRAG1 wild-type and C325Y proteins could be found in the soluble nuclear fraction (Fig.…”

Section: Normal Mrag1 C325y Expression and Cellular Distributionsupporting

confidence: 92%

Section: Normal Mrag1 C325y Expression and Cellular Distributionsupporting

confidence: 42%

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

Simkus

Anand

Bhattacharyya

et al. 2007

The Journal of Immunology

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“…In developing lymphocytes in the G 1 phase of the cell cycle, RAG1 and RAG2 are co-expressed, with RAG2 apparently in molar excess (50). In the S, G 2 , and M phases of the cell cycle, however, RAG2 is rendered unstable by phosphorylation by a cyclin-dependent kinase (51,52), and RAG2 levels drop dramatically, whereas RAG1 levels are almost unchanged (30).…”

Section: Discussionmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K D ‫؍‬ 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.The genes encoding the variable domains of immunoglobulins or T cell receptors are generated during lymphocyte differentiation by a somatic recombination reaction known as V(D)J recombination (1). The reaction is initiated by DNA double strand breaks created at the junction between two coding segments (termed V, D, or J) and their flanking recombination signal sequences (RSSs). 1 Cleavage is followed by a complex repair process that results in imprecise joining of the two coding segments and typically precise joining of the two RSSs. The RSSs consist of two conserved sequence elements, the heptamer (consensus 5Ј-CACAGTG-3Ј) and the nonamer (consensus 5Ј-ACAAAAACC-3Ј), separated by a poorly conserved spacer sequence of either 12 or 23 base pairs. Efficient recombination occurs only between a 12-RSS and a 23-RSS, a phenomenon known as the 12/23 rule (for review, see Ref.2). Recognition of 12/23-RSSs and concerted cleavage at the RSScoding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3 and 4), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5, 6). Binding and cleavage of DNA by the RAG1⅐RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins 8).Deletion mutagenesis has established the minimal "core" domains of murine RAG1 (residues 384 -1008 of the 1040 aa RAG1 protein; Fig. 1A) and RAG2 (residues 1-383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9 -12). Most bi...

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“…The function of many RING-containing proteins can be mediated through DNA binding or chromatin association. RAG1 is involved in V(D)J recombination complex, and RAD-16 participates in DNA repair (6). RING finger-containing polycomb group proteins Psc, Su(z)2, Bmi-1, and RING1 are involved in the maintenance of the transcriptionally repressed state of genes by regulating chromatin structure (7)(8)(9), and Mel-18 is shown to act as a transcriptional repressor via binding to specific DNA sequence (10).…”

Section: Ringmentioning

confidence: 99%

The RING Finger Protein SNURF Is a Bifunctional Protein Possessing DNA Binding Activity

Häkli

Karvonen

Jänne

et al. 2001

Journal of Biological Chemistry

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The small nuclear C 3 HC 4 finger protein (SNURF), RNF4, acts as transcriptional coactivator for both steroiddependent and -independent promoters such as those driven by androgen response elements and GC boxes, respectively. However, SNURF does not possess intrinsic transcription activation function, and the precise molecular mechanism of its action is unknown. We have studied herein the interaction of SNURF with DNA in vitro. SNURF binds to linear double-stranded DNA with no apparent sequence specificity in a cooperative fashion that is highly dependent on the length of the DNA fragment used. SNURF interacts efficiently with both supercoiled circular and four-way junction DNA, and importantly, it also recognizes nucleosomes. An intact RING structure of SNURF is not mandatory for DNA binding, whereas mutations of specific positively charged residues in the N terminus (amino acids 8 -11) abolish DNA binding. Interestingly, the ability of SNURF to interact with DNA is associated with its capability to enhance transcription from promoters containing GC box elements. Because SNURF can interact with both DNA and protein (transcription) factors, it may promote assembly of nucleoprotein structures. RING1 (really interesting new gene) finger is a motif of conserved cysteines and histidines that coordinate two zinc atoms in a "cross-brace" system, a ligation scheme distinct from those of the classical zinc fingers (1, 2). The RING motifs can be classified into two subgroups according to the presence of a cysteine or histidine in the fifth position: C 3 HC 4 (RING-HC) and C 3 H 2 C 3 (RING-H2) fingers. Otherwise their composition and length can vary substantially. The RING finger has been found in a variety of eukaryotic proteins of diverse evolutionary origin that are involved in various cellular processes such as oncogenesis, development, signal transduction, and apoptosis (1-3). RING fingers have been shown to mediate protein-protein interactions and formation of multi-protein complexes. The RING motif of promyelocytic leukemia gene product is important in the assembly of protein complexes linked to SUMO-1 (a small ubiquitin-like modifier protein) modifications (4). RING finger has also been suggested to act as a DNAbinding motif (5). The function of many RING-containing proteins can be mediated through DNA binding or chromatin association. RAG1 is involved in V(D)J recombination complex, and RAD-16 participates in DNA repair (6). RING finger-containing polycomb group proteins Psc, Su(z)2, Bmi-1, and RING1 are involved in the maintenance of the transcriptionally repressed state of genes by regulating chromatin structure (7-9), and Mel-18 is shown to act as a transcriptional repressor via binding to specific DNA sequence (10). Nuclear receptor mediator TIF␣ is tightly associated with euchromatin (11), whereas BRCA1 appears to be associated with the RNA polymerase II holoenzyme (12). However, the RING structures of these latter proteins have not been implicated in mediating their binding to chromatin or DNA.Recent intrig...

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rag-1 and rag-2 Are Components of a High-Molecular-Weight Complex, and Association of rag-2 with This Complex Is rag-1 Dependent

Cited by 72 publications

References 55 publications

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

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

RAG1-DNA Binding in V(D)J Recombination

The RING Finger Protein SNURF Is a Bifunctional Protein Possessing DNA Binding Activity

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