Synapsis of recombination signal sequences located in cis and DNA underwinding in V(D)J recombination

V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.     

RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160 Å in the linear substrate to <80 Å in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule.     

Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis

The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein–DNA complexes that support this process have mainly been studied using RAG–RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's ‘12/23 rule’ and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the postcleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.     

The architecture of the 12RSS in V(D)J recombination signal and synaptic complexes

V(D)J recombination is initiated by RAG1 and RAG2, which together with HMGB1 bind to a recombination signal sequence (12RSS or 23RSS) to form the signal complex (SC) and then capture a complementary partner RSS, yielding the paired complex (PC). Little is known regarding the structural changes that accompany the SC to PC transition or the structural features that allow RAG to distinguish its two asymmetric substrates. To address these issues, we analyzed the structure of the 12RSS in the SC and PC using fluorescence resonance energy transfer (FRET) and molecular dynamics modeling. The resulting models indicate that the 12RSS adopts a strongly bent V-shaped structure upon RAG/HMGB1 binding and reveal structural differences, particularly near the heptamer, between the 12RSS in the SC and PC. Comparison of models of the 12RSS and 23RSS in the PC reveals broadly similar shapes but a distinct number and location of DNA bends as well as a smaller central cavity for the 12RSS. These findings provide the most detailed view yet of the 12RSS in RAG–DNA complexes and highlight structural features of the RSS that might underlie activation of RAG-mediated cleavage and substrate asymmetry important for the 12/23 rule of V(D)J recombination.     

HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

Background  

V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element.     

The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination

The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell receptor (TCR) beta variable region exon and is manifest by a failure of direct Vbeta-to-Jbeta gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRbeta gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRbeta locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRbeta locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jbeta substrates and to some extent through poor synapsis of Vbeta and Jbeta substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jbeta substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo.     

A RAG-1/RAG-2 tetramer supports 12/23-regulated synapsis,cleavage, and transposition of V(D)J recombination signals

Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.     

An updated definition of V(D)J recombination signal sequences revealed by high-throughput recombination assays

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.     

Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.     

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg²⁺ cations with Ca²⁺ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments (1). This DNA rearrangement process, known as V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called RAG1 and RAG2, assemble a multiprotein synaptic complex with a pair of antigen receptor gene segments and subsequently introduce a DNA double strand break at the end of each gene segment (2). A recombination signal sequence (RSS)³ that abuts each participating gene segment serves as the binding site of the RAG proteins and directs the location of DNA cleavage. Each RSS contains conserved heptamer and nonamer sequences that are separated by either 12 or 23 bp of DNA of more varied sequence (12RSS and 23RSS, respectively); efficient V(D)J recombination generally only occurs between two RSSs in which the lengths of DNA separating the heptamer and nonamer differ (the 12/23 rule). The RAG proteins mediate DNA cleavage via a nick-hairpin mechanism, breaking the DNA between the RSS heptamer and the coding segment; these reaction products are subsequently processed and repaired by the non-homologous end-joining pathway (1, 3).Previous studies suggest that RAG synaptic complexes are assembled through the stepwise binding of a 12RSS followed by the capture of a 23RSS (4–6). In vitro biochemical studies suggest synapsis is mediated by a RAG1/2 heterotetramer, but there remains disagreement over the stoichiometry of RAG1 in these complexes (7). In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientation of DNA strands within the synaptic complex. The data obtained from these experiments led the authors to favor a model in which the RSSs adopt a bent and crossed configuration in the synaptic complex, although an alternative model in which synaptic complexes containing RSSs in parallel and antiparallel configurations assemble with similar frequency could not be formally excluded (8). For most in vitro biochemical studies, the synaptic complex has been assembled by incubating the RAG proteins with a pair of oligonucleotide substrates, one containing a 12RSS, and one containing a 23RSS. Whether the RAG proteins and the RSSs adopt the same configuration in synaptic complexes assembled with oligonucleotide substrates as those assembled with longer, more physiological substrates remains to be verified, but some studies suggest there are DNA length-dependent differences in RAG-mediated RSS binding and cleavage activity (9, 35).To directly observe and analyze RAG-RSS synaptic complexes assembled on long DNA substrates that more closely model an RSS embedded in chromosomal DNA, we used atomic force microscopy (AFM), given its previously demonstrated success for visualizing synaptic complexes in other systems (10–14), and its ability to reveal structural details for synaptic complexes that correlate well with independently obtained crystallographic data (15). AFM has also been recently applied to study the bending of 12RSS substrates by RAG1 and RAG2 (16). We report here the first successful visualization of RAG-RSS synaptic complexes by AFM, and describe their characterization with respect to DNA arrangement and the protein stoichiometry within the complexes. These data provide new and important insight into how RAG-RSS synaptic complexes are assembled and organized.     

Ordered assembly of the V(D)J synaptic complex ensures accurate recombination

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.     

Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA

During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG–RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1–RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1–DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1–DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG–DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1–DNA interactions.     

Joining mutants of RAG1 and RAG2 that demonstrate impaired interactions with the coding-end DNA

In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12- and 23-RSSs, form a complex with the protein products of recombination activating genes, RAG1 and RAG2. DNaseI footprinting demonstrates that the interaction of RAG proteins with substrate RSS DNA is not just limited to the signal region but involves the coding sequence as well. Joining mutants of RAG1 and RAG2 demonstrate impaired interactions with the coding region in both pre- and postcleavage type complexes. A possible role of this RAG coding region interaction is discussed in the context of V(D)J recombination.     

A highly ordered structure in V(D)J recombination cleavage complexes is facilitated by HMG1

Central to understanding the process of V(D)J recombination is appreciation of the protein–DNA complex which assembles on the recombination signal sequences (RSS). In addition to RAG1 and RAG2, the protein HMG1 is known to stimulate the efficiency of the cleavage reaction. Using electrophoretic mobility shift analysis we show that HMG1 stimulates the in vitro assembly of a stable complex with the RAG proteins on each RSS. We use UV crosslinking studies of this complex with azido-phenacyl derivatized probes to map the contact sites between the RAG proteins, HMG1 derivatives and the RSS. We find that the RAG proteins make contacts at the nonamer, heptamer and adjacent coding region. The HMG1 protein by itself appears to localize at the 3′ side of the nonamer, but a cooperative complex with the RAG proteins is positioned at the 3′ side of the heptamer and adjacent spacer in the 12RSS. In the complex with RAG proteins, HMG1 is positioned primarily in the spacer of the 23RSS. We suggest that bends introduced into these DNA substrates at specific locations by the RAG proteins and HMG1 may help distinguish the 12RSS from the 23RSS and may therefore play an important role in the coordinated reaction.     

Identification and characterization of a gain-of-function RAG-1 mutant

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.     

Stimulation of V(D)J recombination by histone acetylation

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.     

Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.     

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction.     

Fine structure and activity of discrete RAG-HMG complexes on V(D)J recombination signals

Two lymphoid cell-specific proteins, RAG-1 and RAG-2, initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences (RSSs). Although the RAG proteins themselves bind and cleave DNA substrates containing either a 12-RSS or a 23-RSS, DNA-bending proteins HMG-1 and HMG-2 are known to promote these processes, particularly with 23-RSS substrates. Using in-gel cleavage assays and DNA footprinting techniques, I analyzed the catalytic activity and protein-DNA contacts in discrete 12-RSS and 23-RSS complexes containing the RAG proteins and either HMG-1 or HMG-2. I found that both the cleavage activity and the pattern of protein-DNA contacts in RAG-HMG complexes assembled on 12-RSS substrates closely resembled those obtained from analogous 12-RSS complexes lacking HMG protein. In contrast, 23-RSS complexes containing both RAG proteins and either HMG-1 or HMG-2 exhibited enhanced cleavage activity and displayed an altered distribution of cleavage products compared to 23-RSS complexes containing only RAG-1 and RAG-2. Moreover, HMG-dependent heptamer contacts in 23-RSS complexes were observed. The protein-DNA contacts in RAG-RSS-HMG complexes assembled on 12-RSS or 23-RSS substrates were strikingly similar at comparable positions, suggesting that the RAG proteins mediate HMG-dependent heptamer contacts in 23-RSS complexes. Results of ethylation interference experiments suggest that the HMG protein is positioned 5' of the nonamer in 23-RSS complexes, interacting largely with the side of the duplex opposite the one contacting the RAG proteins. Thus, HMG protein plays the dual role of bringing critical elements of the 23-RSS heptamer into the same phase as the 12-RSS to promote RAG binding and assisting in the catalysis of 23-RSS cleavage.     

Nucleosome structure completely inhibits in vitro cleavage by the V(D)J recombinase.

Lineage specificity and temporal ordering of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In this report, we investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. We found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. We suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure.