A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2

Rodgers, Karla K.; Villey, Isabelle; Ptaszek, Leon M.; Corbett, Elizabeth L.; Schatz, David G.; Coleman, Joseph E.

doi:10.1093/nar/27.14.2938

Cited by 63 publications

(116 citation statements)

References 25 publications

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“…3a) gives rise to one predominant shifted species, whereas MBP-RAG1 (Fig. 3b) yields at least three complexes of distinct mobility, consistent with a previous study (22). Binding by StrRAG1 was quantified, and the data points were fit to a single Hill binding isotherm equation (Equation 1; "Experimental Procedures"), giving a dissociation constant of 92 Ϯ 12 nM.…”

Section: Resultssupporting

confidence: 83%

“…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%

“…Some experiments indicated the presence of a dimer of RAG1 (18,22,23,24), whereas others argue for the presence of three or four RAG1 subunits (25,26). RAG2 is present in the SC either as a monomer (18) or as a dimer (23,26).…”

mentioning

confidence: 99%

“…Furthermore, since most DNA binding studies have been performed by EMSA, we do not know the equilibrium constants governing reactions involving RAG1 alone or RAG1 in conjunction with RAG2 and HMG proteins in forming complexes with the RSS in solution. The equilibrium dissociation binding constant for the interaction of a MBP-RAG1 core (aa 384 -1008) fusion protein with 12-or 23-RSS oligonucleotides was determined by EMSA to be close to 100 nM (22). In this study, however, the MBP-RAG1 fusion protein was capable of forming several protein-DNA complexes with various protein stoichiometries and various DNA binding affinities, and the determined dissociation constant reflected the overall binding without specification of the individual components of reaction.…”

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confidence: 99%

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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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Section: Resultssupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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“…PCR products were digested with BamHI and XhoI and ligated into the pMH6 expression vector [ 30 ] linearized with BamHI and XhoI to yield the expression vectors in pMH6-SpPHD1, pMH6-SpPHD2, pMH6-MR2-PHD and pMH6-ING2-PHD, respectively.…”

Section: Plasmidsmentioning

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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An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex

et al. 2015

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V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex. Here, we combined limited proteolysis and mass spectrometry methods to identify regions of RAG1 that are sequestered upon association with RAG2. These results show that RAG2 bridges an interdomain boundary in the catalytic region of RAG1. In a second approach, mutation of RAG1 residues within the interdomain boundary were tested for disruption of RAG1:RAG2 complex formation using fluorescence-based pull down assays. The core RAG1 mutants demonstrated varying effects on complex formation with RAG2. Interestingly, two mutants showed opposing results for the ability to interact with core versus full length RAG2, indicating that the non-core region of RAG2 participates in binding to core RAG1. Significantly, all of the RAG1 interdomain mutants demonstrated altered stoichiometries of the RAG complexes, with an increased number of RAG2 per RAG1 subunit compared to the wild type complex. Based on our results, we propose that interaction of RAG2 with RAG1 induces cooperative interactions of multiple binding sites, induced through conformational changes at the RAG1 interdomain boundary, and resulting in formation of the DNA cleavage active site.

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A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2

Cited by 63 publications

References 25 publications

RAG1-DNA Binding in V(D)J Recombination

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

An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex

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