Ordered assembly of the V(D)J synaptic complex ensures accurate recombination
2002; Springer Nature; Volume: 21; Issue: 15 Linguagem: Inglês
10.1093/emboj/cdf394
ISSN1460-2075
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 August 2002free access Ordered assembly of the V(D)J synaptic complex ensures accurate recombination Jessica M. Jones Jessica M. Jones Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5/Room 241, Bethesda, MD, 20892 USA Search for more papers by this author Martin Gellert Corresponding Author Martin Gellert Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5/Room 241, Bethesda, MD, 20892 USA Search for more papers by this author Jessica M. Jones Jessica M. Jones Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5/Room 241, Bethesda, MD, 20892 USA Search for more papers by this author Martin Gellert Corresponding Author Martin Gellert Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5/Room 241, Bethesda, MD, 20892 USA Search for more papers by this author Author Information Jessica M. Jones1 and Martin Gellert 1 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5/Room 241, Bethesda, MD, 20892 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4162-4171https://doi.org/10.1093/emboj/cdf394 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recombination of gene segments at the immunoglobulin and T-cell receptor loci requires that the RAG1 and RAG2 proteins bring together DNA signal sequences (RSSs) with 12- and 23-bp spacers into a synaptic complex and cleave the DNA. A RAG1/2 multimer that can cleave both signals is shown to assemble on an isolated RSS, and the complementary RSS enters this complex as naked DNA. When RAG1/2 is allowed to bind 12 and 23 RSSs separately prior to their mixing, synaptic complex assembly and cleavage activity are greatly reduced, indicating that only a complex initially assembled on a single RSS leads to productive cleavage. RAG1/2 complexes assembled on 12 RSSs will only incorporate 23 partners, while complexes assembled on 23 RSSs show a 5- to 6-fold preference for 12 partners. Thus, initial assembly on a 12 RSS most accurately reflects the strict 12/23 coupled cleavage observed in the cell. Additional cellular factors such as chromatin may ensure that RAG1/2 first assembles on a 12 RSS, and then a free 23 RSS enters to activate cleavage. Introduction Unlike almost all known proteins, the variable regions of antigen receptor proteins are not encoded by intact germline genes (Tonegawa, 1983). Mature coding regions must be assembled from among multiple variable (V), joining (J) and sometimes diversity (D) gene segments. Rearrangement of these segments occurs through the site-specific V(D)J recombination reaction that is unique to jawed vertebrates. This process requires specific protein and DNA components as well as general DNA repair factors (reviewed in Gellert, 2002). In the germline, coding segments are flanked by recombination signal sequences (RSSs) comprising conserved heptamer and nonamer sequence motifs, which are separated by spacers of either 12 or 23 bp (called 12 and 23 RSSs). Segments to be recombined are flanked by RSSs with dissimilar spacer lengths (Tonegawa, 1983). For example, at the Igκ locus all V segments are flanked by 12 RSSs, while J segments are flanked by 23 RSSs. During recombination, specific DNA cleavage events introduce double-stranded breaks at one 12 and one 23 RSS (Roth et al., 1992a, 1992b, 1992c, 1993; Schlissel et al., 1993). The coding segments are fused to create coding joints, and the RSSs are then fused to one another to create signal joints. Depending on the germline arrangement, the DNA fragment containing the signal joints may be retained in the chromosome or may be permanently lost from the genome. Coding joints always remain in the genome, and can eventually become mature coding regions. Normally, the vast majority of coding joints in cells that undergo V(D)J recombination are the result of cleavage of a 12/23 RSS pair (Steen et al., 1997), a phenomenon referred to as the 12/23 rule (Tonegawa, 1983). Adherence to this rule is necessary to ensure production of functional joints. Cleavage is carried out by a recombinase composed of the RAG1 and RAG2 gene products (Oettinger et al., 1990; van Gent et al., 1995). RAG1 and RAG2 form a complex (RAG1/2) that can recognize both 12 and 23 RSSs (Hiom and Gellert, 1997). Binding to the 23 RSS is also facilitated by the high mobility group (HMG) 1 or 2 non-specific DNA binding proteins (van Gent et al., 1997), which are believed to bind within the spacer and bend the DNA (Mo et al., 2000). In the presence of Mn2+ or Mg2+, RAG1/2 introduces a single-stranded nick between the RSS and coding segment (McBlane et al., 1995; Hiom and Gellert, 1997), leaving a hydroxyl group on the 3′ end of the coding flank (Figure 1A). This hydroxyl can then attack the opposite DNA strand in a direct transesterification reaction (van Gent et al., 1996a) that leaves a hairpin on the end of the coding DNA and a blunt-cut RSS end (Figure 1A McBlane et al., 1995). RAG1 belongs to a family of tranposases and retroviral integrases characterized by similar biochemical mechanisms; in all cases examined cleavage or strand transfer is initiated by nicking events followed by transesterification reactions (reviewed in Rice and Baker, 2001). RAG1 contains the trio of conserved active site residues common to this family (Kim et al., 1999; Landree et al., 1999; Fugmann et al., 2000) that are involved in coordination of divalent metal ion co-factors. RAG1 and RAG2 form a complex in solution (Swanson and Desiderio, 1999). While the role of RAG2 in the complex is not clear, it is known to enhance RAG1 binding to the RSS and is absolutely required for all steps of cleavage (Li et al., 1997; Akamatsu and Oettinger, 1998; Swanson and Desiderio, 1999). Figure 1.Steps in RSS cleavage by RAG1/2. In Figures 1,2,3,4,5,6, 12 RSSs are depicted as open triangles and 23 RSSs are depicted as filled triangles, with both triangles pointing away from the coding flank. (A) RAG1/2 binds to the intact RSS and introduces a nick between the signal and coding DNA. The 3′ hydroxyl (OH) in the nicked species attacks the opposite strand in a direct transesterification reaction to cleave the DNA, leaving a 3′ OH on the blunt-ended RSS. RAG1/2 can also carry out transesterification on pre-nicked substrates. (B) RAG1/2, with the help of HMG1 or HMG2, assembles a synaptic complex including a pair of RSSs (canonically, one 12 RSS and one 23 RSS). The synaptic complex is competent to carry out coupled cleavage of both partners in Mg2+. This cartoon is not meant to reflect the stoichiometry of RAG1 and RAG2 protomers present in the synaptic complex. Download figure Download PowerPoint When cleavage is carried out in Mg2+, the most likely cellular co-factor, the transesterification step requires the presence of both RSSs (Eastman et al., 1996; van Gent et al., 1996b; West and Lieber, 1998). The core domains of RAG1/2, in recombinant, purified form, can recapitulate the 12/23 rule in that transesterification at a given RSS is strongly stimulated by the presence of the appropriate partner (van Gent et al., 1996b; Hiom and Gellert, 1998; West and Lieber, 1998). Transesterification takes place in a synaptic complex including the RSS pair (Figure 1B; Hiom and Gellert, 1998). RAG1/2, with the help of HMG proteins, has been shown to assemble such a complex in vitro from RSSs supplied on oligonucleotide substrates (Hiom and Gellert, 1998). Evidence suggests that the synaptic complex includes two RAG2 protomers (Mundy et al., 2002) and at least a dimer (Swanson and Desiderio, 1999), or possibly a trimer or tetramer (Landree et al., 2001; Mundy et al., 2002), of RAG1. There are many potential pathways leading to assembly of the synaptic complex. The observation that RAG1/2 binds to the individual 12 and 23 RSSs and nicks them in Mg2+ (Hiom and Gellert, 1997; van Gent et al., 1997; Kim and Oettinger, 1998; West and Lieber, 1998) implies that these may constitute 'half complexes' that come together to form the synaptic complex, activating coupled transesterification of the two partners. However, there is no formal evidence demonstrating that two single-RSS complexes are intermediates in assembly of the synaptic complex. It is possible that a complex capable of binding both partners normally assembles on a single RSS, with the partner RSS being incorporated free of additional protein. Recent work provided evidence that this is one possible route (Mundy et al., 2002), but did not examine whether pre-assembly on a single RSS is absolutely required for building an active complex or whether it is merely one of several possible pathways. Should assembly on a single RSS be the necessary route, it is unknown whether there would be any difference between initial assembly on the 12 or 23 RSS. We examined the steps leading to assembly of the synaptic complex in an in vitro system that accurately reflects the 12/23 rule. Pre-assembly of RAG1/2 complexes on both the 12 and 23 RSSs prior to their mixing was inhibitory to synaptic complex formation, suggesting that such half complexes are not direct precursors in assembly of the synaptic complex. Complexes pre-assembled on a 12 RSS could incorporate a 23 RSS provided that it was free of additional RAG1/2, and the reciprocal also appeared to be true. However, while complexes assembled first on a 12 RSS adhered strictly to the 12/23 rule, complexes assembled on a 23 RSS showed only a 5- to 6-fold preference for incorporation of a 12 versus a 23 RSS partner. Recent data indicate that on some chromatinized substrates, RAG1/2 is more active on 12 RSSs than on 23 RSSs (Kwon et al., 1998, 2000). In the cell, the presence of nucleosomes at recombining loci may reinforce adherence to the 12/23 rule by helping to ensure that RAG1/2 first assembles on a 12 RSS. Results Assembly of RAG1/2 on both RSSs prior to their mixing inhibits cleavage RAG1/2 can bind to individual 12 or 23 RSSs in the presence of Ca2+, Mg2+ or Mn2+ (Hiom and Gellert, 1997). We examined conditions that promoted assembly of synaptic complexes in which RAG1/2 bound to a pair of RSSs. For the experiments shown in Figures 2,3,4 cleavage (transesterification) in Mg2+ was used as a measure of synaptic complex assembly. Pre-nicked substrates were used so that transesterification could be specifically examined, as this is the only chemical step that strictly requires synaptic complex assembly (van Gent et al., 1996b; Hiom and Gellert, 1998; Yu and Lieber, 2000). Figure 2.Cleavage and RAG1/2 binding under two different assembly conditions. (A) Cleavage reactions (10 μl) were assembled in Ca2+ in one of two ways. (1) RAG1/2 at the concentration indicated in (B), HMG1 and pre-nicked 12 RSS substrate (the position of the label is indicated by an asterisk; the OH has been omitted) were first combined (5 min, 37°C), followed by the addition of pre-nicked 23 RSS partner (5 min, 37°C), and finally Mg2+ (15 min, 37°C). (2) Pre-nicked 12 RSS substrate and 23 RSS partner were each individually incubated with HMG1 and RAG1/2 at the concentration indicated in (B) (5 min, 37°C), these mixes were then combined (5 min, 37°C), and finally Mg2+ was added (15 min, 37°C). (B) Reaction products were separated on denaturing polyacrylamide gels as described in Materials and methods. Positions of pre-nicked substrate and hairpin (HP) cleavage products are shown to the right of the gel. Substrate (%) converted to HP product is indicated. (C) Binding reactions using intact RSS substrates were assembled as described in (A), omitting the addition of MgCl2 and the final 15 min incubation. Complexes assembled in these reactions were applied to polyacrylamide gels and separated as described in Materials and methods. The positions of the complex of RAG1/2 with a single RSS end and the synaptic complex are indicated to the left of the gel. Substrate (%) bound in the synaptic complex is indicated. Download figure Download PowerPoint Figure 3.Cleavage under various assembly conditions in the presence of specific competitor. (A) Assembly of cleavage reactions in Ca2+ was staged as indicated with reaction components being added in the order given; HMG1 and buffer components (see Materials and methods) were added prior to pre-nicked 12 RSS substrate (the position of the label is indicated by an asterisk). Competitor was intact 12 RSS, and intact 23 RSS acted as partner. Positions of pre-nicked substrate and hairpin (HP) cleavage products are shown to the right of the gel. Substrate (%) converted to product is indicated. (B) Reactions were staged as per (A), but pre-nicked 23 RSS was used as substrate, intact 23 RSS was used as competitor, and intact 12 RSS acted as partner. Download figure Download PowerPoint Figure 4.Cleavage of intact substrate in the presence of specific competitor without pre-incubation in Ca2+. Cleavage reactions were assembled as indicated with reaction components being added in the order given; HMG1 and buffer components (see Materials and methods) were added prior to intact 12 RSS (lanes 1–6) or 23 RSS (lanes 7–12) substrate (the position of the label is indicated by an asterisk). Reactions did not include Ca2+. Some reactions included specific competitor (intact 12 RSS, lanes 4–6; intact 23 RSS, lanes 10–12) and partner (intact 23 RSS, lanes 3–5; intact 12 RSS, lanes 9–11). Positions of substrates as well as nicked and hairpin (HP) products are shown on either side of the gel. Substrate (%) converted to HP product is indicated. Download figure Download PowerPoint We determined previously that brief (≤5 min) incubation at 37°C enhanced synaptic complex formation by ∼2-fold (Jones and Gellert, 2001; data not shown), and all incubations were carried out at this temperature; additives such as DMSO had no effect (Melek et al., 2002; data not shown). RAG1/2 was allowed to bind to a pre-nicked 12 RSS in Ca2+ followed by the addition of pre-nicked 23 RSS (Figure 2A, condition 1). RAG1/2–RSS complexes formed in this manner were competent for cleavage after the addition of Mg2+, indicating that synaptic complexes were assembled (Figure 2B, lanes 2–4). Under these assembly conditions, cleavage was enhanced at higher concentrations of RAG1/2 (Figure 2B, lanes 2–4). When RAG1/2 was allowed to bind to isolated 12 and 23 RSSs separately, and the two were then combined (Figure 2A, condition 2), cleavage was greatly reduced relative to pre-assembly on the 12 RSS alone (Figure 2B, lanes 5–7). This effect was most pronounced when the molar ratio of RAG1/2 to total RSS in the reaction was increased to ∼15:1 (Figure 2B, lane 7), conditions under which it is most likely that all the RSSs would be bound by a RAG1/2 complex during the initial incubation. The observation that relatively high concentrations of RAG1/2 did not inhibit cleavage when the proteins were pre-assembled on only one RSS (condition 1; Figure 2B, lanes 2–4) indicates that the inhibitory effect seen in assembly condition 2 could not be explained by factors such as non-specific protein aggregation, excess inactive protein or the presence of maltose-binding protein (MBP) tags fused to the RAG1 and RAG2 constructs. Similar results were obtained when intact (i.e. not pre-nicked) substrates were used (data not shown). These data indicated that at relatively high RAG1/2 concentrations, the complexes that assembled on individual 12 and 23 RSSs could not come together to form an active synaptic complex, and they suggested that the RAG1/2 complex first assembles on a single RSS followed by incorporation of naked partner DNA. These results were corroborated by band mobility-shift experiments. Conditions have been developed previously in which the synaptic complex can be distinguished from the RAG1/2 complex bound to a single RSS (Hiom and Gellert, 1998; Jones and Gellert, 2001; Mundy et al., 2002). The synaptic complex migrates more slowly than the complex of RAG1/2 with a single RSS (Hiom and Gellert, 1998; Jones and Gellert, 2001; Mundy et al., 2002; see also Figure 2C), and the presence of two RSSs in the synaptic complex has been confirmed using oligonucleotides of different lengths (Jones and Gellert, 2001). The synaptic complex was competent for coupled cleavage after incubation in Mg2+ (Jones and Gellert, 2001; Mundy et al., 2002), so we presume that it represents the active complex formed in solution in the experiments described above, and that it includes RAG1/2 components in the stoichiometry necessary to support coupled cleavage. Binding reactions were staged as described in Figure 2A, with the exception that the final step including Mg2+ was omitted and intact RSS substrates were used. When RAG1/2 was allowed to bind the 12 RSS prior to the addition of 23 RSS partner, robust synaptic complex assembly was observed (Figure 2C, lanes 1–3). With these assembly conditions and this concentration of 23 RSS partner (12.5 nM), nearly all of the bound substrate was found in the synaptic complex (see below and data not shown). However, if RAG1/2 was incubated with both the 12 and 23 RSSs prior to their mixing, synaptic complex assembly was reduced as much as 15-fold (Figure 2C, lanes 4–6). The mobility of the complex of RAG1/2 with the single RSS did not change at relatively high RAG1/2 concentrations (Figure 2C, lanes 4–6), and in assays including a 12 or a 23 RSS alone we observed only one shifted complex, regardless of the concentration of RAG1/2 (see below and data not shown). This suggested that no potentially 'dead end' higher order complexes unable to assemble into the synaptic complex were formed at the RAG1/2 concentrations used in these experiments. Although in this assay the RSS substrate did not appear to be saturated even at 220 nM RAG1/2 (Figure 2C, lane 6), we have found that this level is competent to bind 100% of the substrate when binding is conducted at 25°C (see below). Unlike the synaptic complex, the complex of RAG1/2 bound to a single RSS appears to be less stable at 37°C, such that it cannot be captured with 100% efficiency in the gel mobility-shift assay. RAG1/2 bound to a 12 RSS can incorporate a 23 RSS in the absence of free RAG1/2 A competition experiment was designed to determine whether the RAG1/2 complex assembled on an isolated, pre-nicked 12 RSS included all of the protein components necessary for incorporation of an intact 23 RSS. Again, cleavage in Mg2+ was used as a measure of synaptic complex formation. Assembly in Ca2+ was performed in stages (Figure 3A), with RAG1/2, HMG1 and labeled 12 RSS always present during the first incubation. In the absence of specific competitor, robust cleavage was observed regardless of whether the 23 RSS partner was added during the first or second stage (Figure 3A, lanes 3 and 6), whereas very little cleavage was observed in the absence of partner (Figure 3A, lane 2). When competitor (100-fold molar excess of unlabeled 12 RSS) was present during the first stage, cleavage was nearly abolished (Figure 3A, lanes 4 and 7), indicating that this level of competitor was sufficient to bind all of the active RAG1/2 present in the reaction. When the addition of competitor was delayed until the second stage, cleavage was observed at 40–50% of the level seen in its absence (Figure 3A, lanes 5 and 8), indicating that this portion of the cleavage seen in the absence of competitor resulted from stable complexes formed during the first stage of incubation. Cleavage was observed at approximately the same level regardless of whether the 23 RSS partner was added during the first or second stage (Figure 3A, lanes 5 and 8). HMG1, which is vital for binding of RAG1/2 to the 23 RSS as well as for synaptic complex formation and cleavage, was present in these reactions at saturating concentrations (data not shown). If the competitor served to bind all of the HMG1 present, we would expect to see no cleavage when the 23 RSS and competitor were added in the second stage, which was not the case. These data indicated that the complex assembled on the 12 RSS during the first stage was sufficient to incorporate the 23 RSS partner even when no free RAG1/2 was present. While these experiments do not definitively demonstrate coupled cleavage, we and others have observed that neither partner in a synaptic complex can undergo transesterification before both are nicked, and that transesterification of both partners is usually highly coupled (Eastman et al., 1996; van Gent et al., 1996b; Eastman and Schatz, 1997; Kim and Oettinger, 1998). Therefore, it is very likely that the complex assembled on a single 12 RSS is capable of cleaving both partners once the 23 RSS has been bound. RAG1/2 complexes assembled on a 23 RSS were also able to incorporate a 12 RSS partner, but this binding was less resistant to a large excess of 23 RSS competitor (Figure 3B). As before, robust cleavage was observed in the absence of competitor regardless of the order of addition (Figure 3B, lanes 3 and 6), and competitor added in the first stage abolished this activity (Figure 3B, lanes 4 and 7). Unlike the results with labeled 12 RSS, low but detectable levels of cleavage occurred in the absence of partner (Figure 3B, lane 2). When 12 RSS partner was present during the first stage and competitor was added in the second, cleavage was restored (Figure 3B, lane 5). However, when both 12 RSS partner and competitor were added in the second stage, no partner-dependent cleavage was observed. This indicates that when a large excess of free 23 RSS was present, the 12 RSS was not able to efficiently enter the complex to activate robust cleavage. We confirmed that these observations were not an artifact of assembly in Ca2+ or the use of pre-nicked labeled substrates (Figure 4). In an alternative protocol, RAG1/2 was incubated in Mg2+ with labeled, intact 12 or 23 RSS substrate in the absence of partner for 10 min, at which point the nicking reaction is nearly complete (data not shown). Competitor and/or partner was then added, and incubation was continued for an additional 30 min. As before, the addition of complementary partner during the second stage in the absence of competitor strongly stimulated cleavage on both the 12 and 23 RSS substrates (Figure 4, lanes 3 and 9). When competitor was added during the first stage, both nicking and cleavage were abolished (Figure 4, lanes 4 and 10). On the 12 RSS substrate, when competitor was added in the second stage, the addition of 23 RSS partner restored moderate levels of cleavage (Figure 4, lane 5). This indicated that the complex assembled on the 12 RSS could incorporate a partner and carry out both steps of cleavage. Competitor added to the 12 RSS in the absence of partner had no stimulatory effect (Figure 4, lane 6). In contrast, on the 23 RSS substrate, competitor added during the second stage had a small stimulatory effect on cleavage. This was true regardless of whether 12 RSS partner was present (Figure 4, compare lanes 11 and 12 with lane 8). This suggested that under these conditions, excess unlabeled 23 RSS could stimulate cleavage of the labeled 23 RSS to which RAG1/2 was already bound, and that the RAG1/2 complex bound to the 23 RSS did not have as strong a preference for the canonical partner as RAG1/2 bound to a 12 RSS. RAG1/2 binding to a 12 RSS leads to high affinity binding of a 23 RSS A RAG1/2 complex assembled on a single RSS always showed some preference for the canonical partner. However, whereas a complex assembled on a 12 RSS appeared to be locked in a conformation that guaranteed incorporation of a 23 RSS, a complex assembled on a 23 RSS showed only a 5- to 6-fold preference for incorporation of a 12 RSS partner over a 23 RSS (see below). Assembly of synaptic complexes with RAG1/2 bound to a pair of RSSs was directly observed by gel mobility-shift experiments. When RAG1/2 and HMG1 were incubated with either a 12 or 23 RSS, one major shifted complex including a single DNA molecule was evident across a range of RAG1/2 concentrations (Figure 5A; data not shown). With HMG1 present, RAG1/2 bound to the individual RSSs with roughly the same affinity to form this complex (Figure 5A). To examine synaptic complex assembly, binding in Ca2+ was staged in a manner similar to cleavage (Figure 5B). RAG1/2 was allowed to bind to labeled 12 or 23 RSSs for 5 min, followed by 5 min incubation in the presence of partner. In the absence of partner, a very small amount of synaptic complex was observed after pre-incubation on the 23 RSS, but not on the 12 RSS (Figure 5B, compare lanes 2 and 8), consistent with the cleavage results obtained in solution. When partner was added, robust synaptic complex assembly was observed across an 8-fold partner concentration range regardless of whether RAG1/2 was pre-assembled on the 12 or 23 RSS (Figure 5B, lanes 3–6 and 9–12). This suggested that complexes assembled on either a 12 or 23 RSS could readily incorporate the appropriate partner, and that there was not a large difference in canonical partner binding affinity between 23 and 12 RSS partners in the concentration range used in our experiments. Figure 5.Gel mobility-shift assay for detection of synaptic complex assembly. (A) Binding reactions were assembled in Ca2+ and analyzed as described in Materials and methods. Intact 12 RSS (squares) or 23 RSS (circles) substrate (2 nM) was combined with RAG1/2 at the concentrations indicated and HMG1 (5 min, 25°C). Complexes assembled in these reactions were applied to polyacrylamide gels and separated as described. Substrate (%) bound in the single end complex was determined. (B) Intact 12 RSS (lanes 1–6) or 23 RSS (lanes 7–12) substrate (the position of the label is indicated by an asterisk) was incubated with RAG1/2 (110 nM) and HMG1 (5 min, 37°C), followed by the addition of 23 or 12 RSS partner, respectively, at the concentrations indicated (5 min, 37°C). Complexes assembled in these reactions were applied to polyacrylamide gels and separated as described. The positions of the complex of RAG1/2 with a single RSS end and the synaptic complex are indicated to the left of the gel. Substrate (%) bound in the synaptic complex is indicated. Download figure Download PowerPoint Competition experiments confirmed that RAG1/2 complexes assembled on a 12 RSS bound to the 23 RSS partner with high affinity. Those assembled on a 23 RSS showed a preference for a 12 RSS partner, but could bind a second 23 RSS under certain conditions. Binding was again staged, with partner being added in the second stage, and competitor being added in either the first or second. When RAG1/2 was assembled on a 12 RSS, a synaptic complex could be formed when 23 RSS partner was present (Figure 6A, lane 3), even when excess 12 RSS competitor was also added (Figure 6A, lane 5). No synaptic complex was assembled when the 23 RSS partner was omitted (Figure 6A, lanes 2 and 6). The complex of RAG1/2 bound to a 23 RSS could incorporate a 12 RSS to form a synaptic complex (Figure 6A, lane 9). However, when a large excess of 23 RSS competitor was present, the 12 RSS partner did not stimulate assembly of the synaptic complex beyond what was seen with competitor alone (Figure 6A, lanes 11 and 12). We consistently observed some competitor-dependent reduction in total substrate binding (Figure 6) and cleavage (Figures 3 and 4), even when competitor was added in the second stage, implying that complexes formed in the first stage are not completely resistant to a large excess of competitor. Figure 6.Gel mobility-shift assay for detection of synaptic complex assembly in the presence of specific competitor. (A) Binding reactions were assembled as indicated with reaction components being added in the order given; HMG1 and buffer components (see Materials and methods) were added prior to intact 12 RSS (lanes 1–6) or 23 RSS (lanes 7–12) substrate (the position of the label is indicated by an asterisk). Some reactions included specific competitor (intact 12 RSS, lanes 4–6; intact 23 RSS, lanes 10–12) and partner (intact 23 RSS, lanes 3–5; intact 12 RSS, lanes 9–11). The positions of the complex of RAG1/2 with a single RSS end and the synaptic complex are indicated to the left of the gel. Substrate (%) bound in the synaptic complex is indicated. (B) Binding reactions were assembled as in (A) with labeled 23 RSS substrate present in the first stage (5 min, 37°C) and 12 RSS (squares) or 23 RSS (circles) partner, at the concentrations indicated added in the second (5 min, 37°C). Complexes were separated and quantified as described. Substrate (%) bound in the synaptic complex is indicated. Download figure Download PowerPoint The amounts of synaptic complex formed in the presence of 12 RSS partner versus excess 23 RSS competitor suggested that incorporation of a second 23 RSS was less efficient than incorporation of the 12 RSS partner (Figure 6A, compare lanes 9 and 12). The relative affinity of a complex assembled on a 23 RSS for a 12 or 23 RSS partner was examined using the gel mobility-shift assay. In the first stage, RAG1/2 was allowed to assemble on the labeled 23 RSS substrate in the absence of partner; various concentrations of unlabeled 12 or 23 RSS partner were then added in the second stage (
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