A functional analysis of the spacer of V(D)J recombination signal sequences

During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems.     

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.     

V(D)J recombination frequencies can be profoundly affected by changes in the spacer sequence

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two V(H)S107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is approximately 9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.     

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.     

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.     

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.     

The central domain of core RAG1 preferentially recognizes single-stranded recombination signal sequence heptamer

RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double strand breaks between each selected gene segment and its bordering recombination signal sequence (RSS) in a two-step mechanism in which the DNA is first nicked, followed by hairpin formation. The RSS consists of a conserved nonamer and heptamer sequence, in which the latter borders the site of DNA cleavage. A region within RAG1, referred to as the central domain (residues 528-760 of 1040 in the full-length protein), has been shown previously to bind specifically to the double-stranded (ds) RSS heptamer, but with both weak specificity and affinity. However, additional investigations into the RAG1-RSS heptamer interaction are required because the DNA substrate forms intermediate conformations during the V(D)J recombination reaction. These include the nicked and hairpin products, as well as likely base unpairing to produce single-stranded (ss) DNA near the cleavage site. Here, it was determined that although the central domain showed substantially higher binding affinity for ss and nicked versus ds substrate, the interaction with ss RSS was particularly robust. In addition, the central domain bound with greater sequence specificity to the ss RSS heptamer than to the ds form. This study provides important insight into the V(D)J recombination reaction, specifically that significant interaction of the RSS heptamer with RAG1 occurs only after the induction of conformational changes at the RSS heptamer.     

Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks.

In V(D)J recombination, double-strand breaks (DSBs) are introduced at recombination signal sequences (RSSs) which consist of three distinct elements: a heptamer, a 12 or 23 nucleotide spacer and a nonamer. Efficient DSB formation requires a 12/23 RSS pair and occurs at both RSS in a temporally coupled fashion (coupled cleavage). It remains unknown which RSS elements are important for coupled cleavage. Furthermore, it has not been established whether some RSS components are critical only for cleavage in cis, with others mainly promoting cleavage in trans at the partner RSS. We investigated these questions by analyzing the effects of RSS mutations on the formation of DSBs in vivo. The abundance of DSBs in cis (at the mutant RSS) and in trans (at the consensus RSS) was determined using an established ligation-mediated PCR assay. We also developed a Southern blotting approach that allows the first direct measurement of dual and single RSS cleavage in vivo. Our results demonstrate that the heptamer, spacer and nonamer elements are all required for coupled cleavage in vivo. These studies also provide evidence for cleavage events involving a single RSS both in mutant substrates and in substrates containing a consensus 12/23 RSS pair.     

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.     

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

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.     

RAG-2 promotes heptamer occupancy by RAG-1 in the assembly of a V(D)J initiation complex

V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.     

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.     

Detection of RAG protein-V(D)J recombination signal interactions near the site of DNA cleavage by UV cross-linking

V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.     

Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination

V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of the recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residues 384-1008 of 1040) alone displays binding specificity for the conserved heptamer and nonamer sequences of the RSS. The nonamer-binding region lies near the N terminus of core RAG1, whereas the heptamer-binding region has not been identified. Here, potential domains within core RAG1 were identified using limited proteolysis studies. An iterative procedure of DNA cloning, protein expression, and characterization revealed the presence of two topologically independent domains within core RAG1, referred to as the central domain (residues 528-760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span the remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant binding site for RAG2. The C-terminal domain bound DNA cooperatively but did not show specificity for either conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RAG1.     

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.     

The role of components of recombination signal sequences in immunoglobulin gene segment usage: a V81x model.

It has long been appreciated that some immunoglobulin (and T-cell receptor) gene segments are used much more frequently than others. The VHsegment V81x is a particularly striking case of overusage. Its usage varies with the stage of B-cell development and with the strain of mice, but it is always high in B cell progenitors. We have found that the coding sequence and the recombination signal sequences (RSS) are identical in five mouse strains, including CAST/Ei, a strain derived from the species Mus castaneus. Thus, the strain differences cannot be attributed to sequences within V81x itself. V81x RSS mediated recombination at rates significantly higher than another VHRSS. Although the V81x nonamer differs at one base pair from the consensus sequence, an RSS with this nonamer and a consensus heptamer recombines as well as the consensus RSS. When the V81x spacer is replaced by that of VA1, the frequency of recombination decreases by approximately 5-fold; thus, the contribution of variation in natural spacers to variability in VHusage in vivo is likely to be more than has been previously appreciated. Furthermore, the contribution of the heptamer and nonamer to differential VHusage in our assay is correlated inversely with their conservation throughout the VHlocus.     

Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences

The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.     

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.     

DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences.

Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination.     

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex,making ends available to alternative DNA repair pathways

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC’s ability to restrict repair pathway choice.