RAG1-DNA Binding in V(D)J Recombination

Abstract

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...

Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsis

“…Both gel filtration and multi-angle laser light scattering methods confirmed that the NBD exists as a dimer in solution in the absence of DNA (data not shown). Consistent with the structure, RAG1 core also forms a stable dimer that is resistant to dissociation20,23. Perturbations in the NBD dimer interface, such as alanine substitutions of residues 423–433, substantially reduced RAG1 core protein expression in vivo 30, suggesting the importance of NBD dimerization in RAG1 protein folding and dimerization.…”

“…The Hin/RAG1 hybrid mutant is similar to the previously published mutant in which RAG1 residues 389–444 have been replaced with Hin DBD residues 138–19018, except the substitution was done in the context of the above mentioned MBP-RAG1 core construct. Wild-type and mutant MBP-RAG1 core proteins were purified from bacteria using nickel-chelating resin, amylose beads, and gel filtration as described previously23, with the dimer peak fractions isolated and used in all experiments. GST-tagged murine RAG2 core (residues 1–383) expressed in 293T cells and His-tagged full-length human HMGB1 expressed in bacteria were purified as described previously18,53.…”

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

“…However, because AFM measures the volume (shape) of the proteins rather than their molecular masses, and the molecular mass estimates were obtained based on volume measurements in which a protein is modeled as a hemisphere, it is possible that differences between the anticipated and observed masses may reflect deviations from a hemispheric model that are intrinsic to the proteins themselves. Biochemical evidence in support of the latter scenario has been reported (34), and it is therefore reasonable to speculate that geometric characteristics of free and bound RAG proteins may differ.…”

Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex

“…The first, FRET, relies on the distance dependence of the excited-state interactions between two different fluorophores, and is widely used as a ‘spectroscopic ruler’ to investigate the structure and dynamics of nucleic acids and proteins (44,45). The second, steady-state fluorescence anisotropy, relies on the measurement of the polarization state of the fluorescence emission and has been previously employed to characterize the interaction between cRAG1 and oligonucleotide RSS substrates (46,47). …”