Identification of Two Catalytic Residues in RAG1 that Define a Single Active Site within the RAG1/RAG2 Protein Complex

Fugmann, Sebastian D.; Villey, Isabelle; Ptaszek, Leon M.; Schatz, David G.

doi:10.1016/s1097-2765(00)80406-2

Cited by 155 publications

(135 citation statements)

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“…In support of this possibility, we report here that mutation of a single residue in RAG-1, E649A, specifically enhances the ability of the RAG complex to catalyze hairpin formation in vitro and facilitate greater cleavage of plasmid V(D)J recombination substrates containing an unpaired RSS or a mispaired (12/12 or 23/23) RSS in vivo, in violation of the 12/23 rule. The E649A RAG-1 mutation is located within the central domain of RAG-1 (8), which encompasses two residues critical for the catalytic activity of the recombinase, D600 and D708 (12,21,24). This colocalization suggests that the central domain plays an active role not only in recognition and catalysis at the site of DNA cleavage but also in perceiving 12/23-regulated synapsis.…”

Section: Discussionmentioning

confidence: 99%

“…These studies have defined regions of RAG-1 and RAG-2 dispensable for supporting the rearrangement of plasmid V(D)J recombination substrates in cell culture (7,22,43,44); identified various mutations in the RAG proteins that impair RSS binding (9,11,49,52), one or both cleavage steps (12,20,24,28,39), or the joining phase of V(D)J recombination (21,39,47,54); and refined structural elements required for RAG-1/RAG-2 association or interactions with other proteins (2,33,60). RAG mutants that enhance the cleavage or joining phases of V(D)J recombination have not yet been reported, although mutations in RAG-2 that impair its degradation at the G 1 /S transition of the cell cycle enable the RAG complex to cleave DNA beyond the G 0 to G 1 phases, where it is normally restricted (25).…”

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confidence: 99%

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Identification and Characterization of a Gain-of-Function RAG-1 Mutant

Kriatchko

Anderson

Swanson

2006

Molecular and Cellular Biology

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RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis. V(D)J recombination is the process by which noncontiguous antigen receptor gene coding segments, called variable (V), diversity (D), and joining (J), are assembled during lymphocyte development to produce the variable region exon of a mature antigen receptor gene (3). V(D)J recombination occurs in two distinct phases. In the first phase, two lymphoid cell-specific proteins called RAG-1 and RAG-2 assemble a multiprotein synaptic complex with two different gene segments through interactions with a conserved recombination signal sequence (RSS) that adjoins each gene segment. Each RSS contains a conserved heptamer and nonamer sequence, separated by either 12 or 23 base pairs of intervening DNA of more varied composition (12-RSS and 23-RSS, respectively). Generally, synaptic complexes are assembled with two RSSs whose spacer lengths are different (the 12/23 rule). Subsequently, the RAG proteins catalyze a DNA double-strand break at each RSS (for reviews, see references 10 and 13), yielding a postcleavage complex containing four DNA ends: two blunt, 5Ј phosphorylated recombination signal ends and two coding ends terminating in DNA hairpin structures (41,42,46). The RAG proteins generate these recombination intermediates by nicking the DNA at the junction between the RSS and the coding sequence and then transferring the resulting 3Ј-OH to the opposing DNA strand by direct transesterification (32,...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

Kriatchko

Anderson

Swanson

2006

Molecular and Cellular Biology

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“…Fractions containing the dimeric protein were dialyzed against BB. MBP-RAG1 core (aa 384 -1008) and glutathione S-transferase-RAG2 (aa 1-383) protein expression and purification were performed as described in (22,31).…”

Section: Methodsmentioning

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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“…While the active site of the V(D)J recombinase resides entirely in RAG1, [7][8][9], RAG2 acts as an essential accessory factor enhancing the recognition of the RSS and the stability of the RAG1/2-RSS complex [10][11][12][13]. RAG2 can conceptually be divided into two regions, the N-terminal core region (murine amino acids 1-383) essential for all catalytic activities, and the C-terminal non-core region (murine amino acids 384-527) dispensable for recombination on artificial DNA substrates [ 14,15 ].…”

Section: Introductionmentioning

confidence: 99%

The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

Wilson

Norton

Fugmann

2008

Developmental & Comparative Immunology

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V(D)J recombination is a somatic gene rearrangement process that assembles antigen receptor genes from individual segments during lymphocyte development. The access of the RAG1/RAG2 recombinase to these gene segments is regulated at the level of chromatin modifications, in particular histone tail modifications. Trimethylation of lysine 4 in histone H3 (H3K4me3) correlates with actively recombining gene elements, and this mark is recognized and interpreted by the plant homeodomain (PHD) of RAG2. Here we report that the PHD domain of the only known invertebrate homolog of RAG2, the SpRAG2L protein of the purple sea urchin (Strongylocentrotus purpuratus) also binds to methylated histones, but with a unique preference for H3K4me2. While the cognate substrate for the sea urchin RAG1L/RAG2L complex remains elusive, the affinity for histone tails and the nuclear localization of ectopically expressed SpRAG2L strongly support the model that this enzyme complex exerts its activity on DNA in the context of chromatin.

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Identification of Two Catalytic Residues in RAG1 that Define a Single Active Site within the RAG1/RAG2 Protein Complex

Cited by 155 publications

References 51 publications

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

Identification and Characterization of a Gain-of-Function RAG-1 Mutant

RAG1-DNA Binding in V(D)J Recombination

The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

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