RAG1-DNA Binding in V(D)J Recombination

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 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 (1Tonegawa S. Nature. 1983; 302: 575-581Google Scholar). 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). 1The abbreviations used are: RSS, recombination signal sequence; aa, amino acid; EMSA, electrophoretic mobility shift assay; MBP, maltose-binding protein; SC, signal complex; BB, binding buffer; StrRAG1, Strep-RAG1; HMG, high mobility group; FD, frequency domain 1The abbreviations used are: RSS, recombination signal sequence; aa, amino acid; EMSA, electrophoretic mobility shift assay; MBP, maltose-binding protein; SC, signal complex; BB, binding buffer; StrRAG1, Strep-RAG1; HMG, high mobility group; FD, frequency domain 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 5′-CACAGTG-3′) and the nonamer (consensus 5′-ACAAAAACC-3′), 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. 2Lewis S.M. Adv. Immunol. 1994; 56: 27-150Google Scholar). Recognition of 12/23-RSSs and concerted cleavage at the RSS-coding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3Fugmann S.D. Lee A.I. Shockett P.E. Villey I.J. Schatz D.G. Annu. Rev. Immunol. 2000; 18: 495-527Google Scholar and 4Sadofsky M.J. Nucleic Acids Res. 2001; 29: 1399-1409Google Scholar), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5Oettinger M.A. Schatz D.G. Gorka C. Baltimore D. Science. 1990; 248: 1517-1523Google Scholar,6Schatz D.G. Oettinger M.A. Baltimore D. Cell. 1989; 59: 1035-1048Google Scholar). Binding and cleavage of DNA by the RAG1·RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins HMG1 and -2 (7Sawchuk D.J. Weis-Garcia F. Malik S. Besmer E. Bustin M. Nussenzweig M.C. Cortes P. J. Exp. Med. 1997; 185: 2025-2032Google Scholar, 8van Gent D.C. Hiom K. Paull T.T. Gellert M. EMBO J. 1997; 16: 2665-2670Google Scholar). Deletion mutagenesis has established the minimal “core” domains of murine RAG1 (residues 384–1008 of the 1040 aa RAG1 protein; Fig.1 A) and RAG2 (residues 1–383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9Cuomo C.A. Oettinger M.A. Nucleic Acids Res. 1994; 22: 1810-1814Google Scholar, 10Sadofsky M.J. Hesse J.E. Gellert M. Nucleic Acids Res. 1994; 22: 1805-1809Google Scholar, 11Sadofsky M.J. Hesse J.E. McBlane J.F. Gellert M. Nucleic Acids Res. 1993; 22: 5644-5650Google Scholar, 12Silver D.P. Spanopoulou E. Mulligan R.C. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6100-6104Google Scholar). Most biochemical studies of V(D)J recombination have been performed with the core RAG proteins because of their enhanced solubility compared with the full-length proteins. Surface plasmon resonance (13Spanopoulou E. Zaitseva F. Wang F.H. Santagata S. Baltimore D. Panayotou G. Cell. 1996; 87: 263-276Google Scholar) and in vivo one-hybrid (14Difilippantonio M.J. McMahan C.J. Eastman Q.M. Spanopoulou E. Schatz D.G. Cell. 1996; 87: 253-262Google Scholar) experiments demonstrated specific recognition of the nonamer by the RAG1 nonamer binding domain and a less significant contribution of the heptamer to binding. In the one-hybrid experiments, RAG2 only modestly enhanced RAG1 DNA binding (14Difilippantonio M.J. McMahan C.J. Eastman Q.M. Spanopoulou E. Schatz D.G. Cell. 1996; 87: 253-262Google Scholar), implying that RAG1 by itself was capable of significant, sequence-specific DNA binding. Subsequently, numerous studies making use of electrophoretic mobility shift assays (EMSAs) and, in most cases RAG proteins fused to large N-terminal tags (glutathione S-transferase (GST) or maltose-binding protein (MBP)), led to the identification of a complex the (for signal Oettinger M.A. Cell. 18: Scholar, K. Gellert M. Cell. 1997; Scholar, M.J. J. Scholar, S. Cell. Scholar). studies that RAG1 binds the with specificity and that RAG2 the affinity and specificity of the have led to the that the RAG1-RSS interaction is to significant in the absence of RAG2 Oettinger M.A. Cell. 18: Scholar). DNA binding by RAG2 has been M.J. J. Scholar), a interaction between RAG2 and DNA in the complex S.D. Schatz D.G. Cell. 2001; Scholar). experiments Oettinger M.A. Cell. 18: Scholar, F. K. A. A. Cell. 18: Scholar, S. Scholar) reveal that RAG1 the nonamer and the flanking of the with RAG the spacer and heptamer S. Scholar). experiments core proteins. The of the RAG proteins in the is experiments the of a dimer of RAG1 S. Cell. Scholar, Villey I.J. E. Schatz D.G. J.E. Nucleic Acids Res. Scholar, M.J. Cell. Scholar, Cell. 22: Scholar), for the of or RAG1 M.A. Cell. 2001; Scholar, Oettinger M.A. Cell. 22: Scholar). RAG2 is in the either as a S. Cell. Scholar) or as a dimer M.J. Cell. Scholar, Oettinger M.A. Cell. 22: Scholar). The nonamer binding domain of RAG1 is to the a central domain of RAG1 has been to specificity for the heptamer J. 2001; Scholar). In a domain of RAG1 has been to to the coding M.J. Cell. 2001; Scholar). experiments indicate a of between RAG1 in the the the heptamer at the of cleavage M.A. Cell. 2001; Scholar, Cell. 2001; Scholar). the that the of RAG1 most DNA binding studies have been performed by EMSA, we the governing RAG1 or RAG1 in with RAG2 and proteins in with the in solution. The binding for the interaction of a core protein with or was by to to Villey I.J. E. Schatz D.G. J.E. Nucleic Acids Res. Scholar). In this the protein was capable of with protein and DNA binding and the the binding of the of have used fluorescence spectroscopy and a RAG1 protein a tag to the RAG1-RSS interaction in of the in the and by a large that the RAG1-RSS interaction is of affinity and specificity In we a conformational change in RAG1 by interaction with the RSS. we that the RAG1-RSS interaction may role in in of the that for a of the cell RAG1 in the absence of RAG2 protein S. Proc. Natl. Acad. Sci. U. S. A. 1994; Scholar). and and high by DNA The 12-RSS was by with The DNA was by with The was at the of the a that to the a of to DNA was performed in binding by the in for at followed by to core RAG1 was expressed as a in the of the The core of murine RAG1 from aa was that created at the of the and a at the products the in the Strep tag at the of the The protein was expressed in the by of and at with was was by the of to a of and to a of for at the was in and by and and by at for at in a was to in the the of the was with two of and RAG1 was with of to a in and with Strep-RAG1 was in The Strep-RAG1 protein was typically Strep-RAG1 was separated from and a the protein core and protein and performed as in Villey I.J. E. Schatz D.G. J.E. Nucleic Acids Res. S.D. Villey I.J. Schatz D.G. Cell. 2000; Scholar). cleavage of a 12-RSS and a 23-RSS, of RAG of and and for at A. Eastman Q.M. Schatz D.G. Nature. Scholar). products a To the 12-RSS DNA was and and purified a StrRAG1 or was with 12-RSS in the or absence of 12-RSS or DNA in with and at for and in at of the species by a was and a The bound at = is we expressed the bound as the between the bound at and the of the bound at The to a binding = the of protein expressed as The StrRAG1 intrinsic fluorescence a with a fluorescence measurements performed in at by a The StrRAG1 intrinsic fluorescence with of for the and the fluorescence between and and of StrRAG1 was in followed by a at experiments, of StrRAG1 with DNA or acrylamide was the followed by a at was M. J. Scholar) for and to a for the of protein was in with a of was that products are generated in the The fluorescence of expressed as a to a with the and was a at to of Scholar). the in DNA was with protein with a with the in the and for The at was at anisotropy measurements performed a with and a to the cell measurements performed the used for and at the at and was at for the was at and was at The for in and in anisotropy the in fluorescence at with a was used to To the 12-RSS either a StrRAG1 or with was in a less of the The together for and was initiated by either of DNA or DNA with StrRAG1 was a The used for was The at between and of the of DNA and StrRAG1 the DNA and the to upon the of StrRAG1 in fluorescence and anisotropy decay measured in the frequency domain at was used as the was in the to from the The fluorescence was in the a The cell was at a base for between and and at for a the decay we used a of in as the polarization used for decay measurements to from The fluorescence and from the a the in was used as the The for single double and decay with anisotropy to E. M. in Scholar), only the double with and between of anisotropy a of experiments was performed and the are was only the S.D. was less with to the The of was performed a binding and of and Steady-state Scholar), is the anisotropy of the is the anisotropy of the species in the absence of is the anisotropy at is the in the expressed as is the the of binding to the and is the interaction the at To was as a of of the and of the species in solution. The of the fluorescence anisotropy to The for StrRAG1 binding to 12-RSS DNA was with the = The and performed and experiments in anisotropy was because StrRAG1 changes with DNA binding we the the from the of fluorescence of StrRAG1, and is the of DNA bound StrRAG1 at Using the bound protein to anisotropy of Scholar, K. 2001; Scholar), with for DNA we the used for anisotropy in is the DNA = The of the of the performed the = the of the at is = the specific for the is the of = for = for the of most protein = and = the = the of the complex to between = and = quenching as the is a for fluorophores by and quenching The fluorescence acrylamide was to a C.A. Scholar, Scholar), K D and K are the and quenching that the of fluorophores and in In we used a expressed and purified murine RAG1 protein 377–1008) fused at to a short tag that affinity with in a StrRAG1 protein of The from the a at the for a StrRAG1 dimer a the of the is the purified protein was the only the dimer was that the protein associated in The intrinsic polarization anisotropy of the purified StrRAG1 at concentrations from to is with the of protein as a dimer at concentrations this range In a cleavage the StrRAG1 protein was as as RAG1 the DNA binding of StrRAG1 by of StrRAG1 for with a of DNA a 12-RSS The binding used this contains a and and was to (13Spanopoulou E. Zaitseva F. Wang F.H. Santagata S. Baltimore D. Panayotou G. Cell. 1996; 87: 263-276Google Scholar, Oettinger M.A. Cell. 18: Scholar). binding StrRAG1 to at of with a Villey I.J. E. Schatz D.G. J.E. Nucleic Acids Res. Scholar). Binding by StrRAG1 was and the to a single binding a of 12 To binding a was StrRAG1 with 12-RSS followed by the of either 12-RSS DNA or of the nonamer and heptamer sequences The 12-RSS is a of results in only in the complex may the of a of StrRAG1 that binds to DNA as as the of a high of by the of may the of the complex in the electrophoretic the with of to electrophoretic and electrophoretic may the results the M. M. Scholar, D. P. M. Rev. 56: Scholar). may the between two species is a major in the of the complex. have been in the RAG1-RSS interaction (13Spanopoulou E. Zaitseva F. Wang F.H. Santagata S. Baltimore D. Panayotou G. Cell. 1996; 87: 263-276Google Scholar, M.J. McMahan C.J. Eastman Q.M. Spanopoulou E. Schatz D.G. Cell. 1996; 87: 253-262Google Scholar). To the associated with we used fluorescence polarization to investigate between StrRAG1 and DNA in solution. anisotropy of the of in is in their and have and have anisotropy of Scholar). The experiments use of 12-RSS DNA or at the of strand with of the anisotropy of the DNA at or nm) was with of StrRAG1, and the polarization anisotropy was The for has a with anisotropy that StrRAG1 protein and of anisotropy of the in the absence of The of the = of = was a binding = and D = 41 protein for K to affinity of interaction that by The in 12-RSS DNA anisotropy in this is to the of the by the to the In the anisotropy are as a of of binding K. 2001; Scholar). The protein binding to 12-RSS In the of is a in to binding of StrRAG1 to The specificity of the StrRAG1-DNA interaction was by anisotropy was with StrRAG1, and either specific 12-RSS or DNA was in to the to the results of experiments, only the 12-RSS was capable of the complex and the anisotropy to of DNA a of DNA in only a in the anisotropy of the complex. In polarization anisotropy experiments 12-RSS that StrRAG1 binds D = 41 nm) and to DNA in solution. The in anisotropy a of protein to DNA of in the To investigate the of StrRAG1 in in we used frequency domain fluorescence spectroscopy domain fluorescence the changes in either or that in the a short the is with a of of Scholar). changes of the and the the fluorescence a is the of the of the of measured the of the to the 12-RSS or DNA and that was by the of StrRAG1 or DNA from the for in S. Scholar). measurements demonstrate that DNA protein amino with or change the proteins with the and measured by frequency domain measured by spectroscopy either for DNA or DNA in the of concentrations of StrRAG1 or The short for the of The anisotropy decay by to a double decay E. M. in Scholar) from the to the and to the or of in a measured by spectroscopy either for DNA or DNA in the of concentrations of StrRAG1 or The short for the of The anisotropy decay by to a double decay E. M. in Scholar) from the to the and to the or of used frequency domain and measurements to the anisotropy decay of the 12-RSS or DNA in the or absence of StrRAG1 or of the anisotropy decay was performed a double E. M. in Scholar, M.A. S. 2000; Scholar) In this the of the with to the and the the of the In is from the that the with the or the proteins during complex formation. change upon protein significant changes in the of DNA in with StrRAG1 of binding protein) or binding S. J. Sci. Scholar, S. M.A. 2001; 59: Scholar). are for DNA in the of StrRAG1, either at or protein the of species that in in the K D, that of the 12-RSS DNA is in in the with StrRAG1 the in this reaction that of the complex. Using the = binding and the see we the of the complex to between = and = the of J. J. Scholar). the of the protein and DNA we that StrRAG1 is most as a dimer in to by fluorescence anisotropy the of DNA to StrRAG1 as a the protein in solution. of polarization anisotropy experiments of the intrinsic fluorophores in the StrRAG1 protein to as the at experiments measured changes in the of the protein or bound to StrRAG1 nm) was with concentrations of and anisotropy was measured the anisotropy and a that of the protein The a with K D = K D the 12-RSS DNA in at in the protein binds as a D is to of the K D from the in the of StrRAG1 in the reaction The in anisotropy is to in the of a significant of StrRAG1 in the of 12-RSS either to of or to of associated with In the of is in anisotropy to the for the and the is and to DNA the of the StrRAG1 we the intrinsic fluorescence at at nm) of StrRAG1 protein is by of StrRAG1 with of 12-RSS a of fluorescence at of the fluorescence in the absence of DNA with the in polarization anisotropy this in fluorescence by 12-RSS DNA to a the the DNA binding is to that the quenching occurs at concentrations of DNA to a significant of is that interaction with the 12-RSS a change in protein The in fluorescence by 12-RSS is associated with a shift of the the of the from to in fluorescence was in the of DNA and the quenching was and are changes of in this a for the quenching of the of DNA we used of that a fluorescence to that of the of of 12-RSS DNA in minimal quenching we performed experiments to quenching and binding 12-RSS with was with StrRAG1, and the change in DNA anisotropy was measured with anisotropy from the and for that are In the StrRAG1 was with 12-RSS or with and intrinsic StrRAG1 fluorescence was measured with The 12-RSS a quenching of StrRAG1 from that by The was by a in In the between and fluorescence because of changes in by protein conformational StrRAG1 intrinsic fluorescence quenching at a the of StrRAG1 intrinsic fluorescence is that DNA binding occurs fluorescence quenching that fluorescence quenching conformational changes by the 12-RSS To investigate the by 12-RSS DNA as a of StrRAG1 intrinsic we the of acrylamide the StrRAG1 fluorescence in the or absence of is a that and fluorescence and has to of a protein a DNA acrylamide complex the intrinsic protein fluorophores to the the of a conformational or less to are in either with DNA or with amino to the protein fluorophores in the absence of concentrations of acrylamide to StrRAG1 in the absence or of specific 12-RSS or and protein intrinsic fluorescence was In the the between the fluorescence in the absence of acrylamide and the fluorescence at the of acrylamide the C.A. Scholar). In we the of the quenching from to the and The of the the of and quenching The of the is by the of DNA by 12-RSS that DNA of the amino fluorophores to to the as in the of their quenching is with the shift of the that occurs upon the of StrRAG1 has a of fluorophores and and to in StrRAG1 quenching In experiments indicate role for quenching of intrinsic fluorophores of StrRAG1 upon interaction with and that this is with specific and quenching expressed as from of the acrylamide quenching to StrRAG1 StrRAG1 in a the conformational change in StrRAG1 by fluorescence by circular the and of StrRAG1 protein or in the of of 12-RSS The at and are of significant in StrRAG1 S. 1994; Scholar). 12-RSS the at of at a DNA of DNA was the changes and the StrRAG1 fluorescence and changes by 12-RSS DNA to the that sequences major conformational changes in In because changes are with 12-RSS sequence-specific of the In in vivo one-hybrid core RAG1 was to with a of 12-RSS elements, and RAG2 to binding only modestly (14Difilippantonio M.J. McMahan C.J. Eastman Q.M. Spanopoulou E. Schatz D.G. Cell. 1996; 87: 253-262Google Scholar). this and was that recognition a process of RAG1 binding followed by of RAG2 (13Spanopoulou E. Zaitseva F. Wang F.H. Santagata S. Baltimore D. Panayotou G. Cell. 1996; 87: 263-276Google Scholar, M.J. McMahan C.J. Eastman Q.M. Spanopoulou E. Schatz D.G. Cell. 1996; 87: 253-262Google Scholar). The results of in at two of the of this was that the of DNA cleavage enhanced by with RAG2 by with the DNA P. S. Cell. 1997; Scholar). studies and RAG1 proteins with a tag that the RAG1-RSS interaction was specific to the The only to investigate the binding of RAG1 core protein used and specificity for the and a reasonably high affinity D = nm) Villey I.J. E. Schatz D.G. J.E. Nucleic Acids Res. Scholar). as the core protein used in experiments species with the in the protein is in that a RAG1 core protein with only a short amino tag is and in in the absence of Both and DNA binding assays demonstrate that this protein binds the as a dimer and that and with high affinity D = 41 the complex that with the 12-RSS is to with DNA and by a of is from that upon interaction with to the of the for recognition In in the of the cell RAG1 and RAG2 are with RAG2 in Schatz D.G. Cell. Scholar). In the and of the cell RAG2 is by by a J. S. Scholar, S. Science. 1993; Scholar), and RAG2 RAG1 are S. Proc. Natl. Acad. Sci. U. S. A. 1994; Scholar). to that in and RAG1 to RSSs in the absence of we that as and and RAG2 to RAG2 to RSSs by a complex. the that and the of recognition is performed by RAG1·RAG2 results to the that a of RAG1-RSS in may to the of the recombination has been that RAG2 a conformational change in RAG1 that binding of RAG1 to the and the of the RAG1 S.D. Lee A.I. Shockett P.E. Villey I.J. Schatz D.G. Annu. Rev. Immunol. 2000; 18: 495-527Google Scholar, S. Cell. Scholar, J. 2001; Scholar). the experiments have been studies of conformational changes in the RAG proteins either by or by have been to the of StrRAG1 with DNA in by protein intrinsic circular and fluorescence studies have been less RAG1 been to or tags to the of the of the that DNA a conformational change in The of DNA to StrRAG1 results in quenching of intrinsic StrRAG1 protein fluorescence and a in the of the protein of in The in fluorescence and shift of StrRAG1 with 12-RSS DNA have quenching of the fluorophores and of the protein a change in from the hydrophobic core to the of the are to quenching by The shift of StrRAG1 was with 12-RSS DNA is in itself that the of the of of the protein fluorophores with the shift for the in M. Scholar). only by a conformational change in the protein changes of the protein fluorophores to the and acrylamide quenching for DNA for is is in quenching of the fluorophores of StrRAG1 in with of DNA the DNA upon binding to StrRAG1, a protein conformational change that amino to with or fluorophores that occurs in the decay of the and quenching by DNA a protein conformational In acrylamide quenching for StrRAG1 for the complex fluorophores in quenching by is in with the that we in was in the and acrylamide quenching K in as of the The of quenching of StrRAG1 fluorescence a complex and occurs complex formation. the 12-RSS and StrRAG1 intrinsic with the at DNA concentrations only a of the protein is in are by a in that from the DNA either the or their the in fluorescence in and, are as or to quenching bound to is that the 12-RSS as a to major conformational changes in StrRAG1 bound to the DNA or that the of DNA-binding proteins as S. C. S. 1996; Scholar) and E. P. J.F. C. 2000; Scholar) has been to by the of In the of anisotropy measurements of that exists in in and and DNA of the to the that is in complex with DNA E. P. J.F. C. 2000; Scholar, E. P. J.F. C. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). The of 12-RSS DNA and to a DNA results in a change in the of StrRAG1 for the in and of J. is the of the from to the a with to a change in the protein a of the has been to the of of proteins with M.C. Scholar). The of is with two that two of RAG1 M.A. Cell. 2001; Scholar, Oettinger M.A. Cell. 22: Scholar), and that only a single dimer of RAG1 Cell. 22: Scholar). the is StrRAG1 to with StrRAG1 is that of two of the RAG1 core with The for the full-length RAG1 contains domain Schatz D.G. J.E. 1997; Scholar). In we demonstrate that the core RAG1 protein a tag binds the as a dimer and undergoes a conformational change upon results a for studies of conformational changes in and for of the for and to and for use of a with and during the of this performed by the M. at