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Eastman, Q. M. & Schatz, D. G. Nicking is asynchronous and stimulated by synapsis in 12/23 rule-regulated V(D)J cleavage. Nucleic Acids Res. 25, 4370-4378
Abstract
The first step in DNA cleavage at V(D)J recombination signals by RAG1 and RAG2 is creation of a nick at the heptamer/coding
flank border. Under proper conditions in vitro the second step, hairpin formation, requires two signals with spacers of 12 and 23 bp, a restriction referred to as the 12/23
rule. Under these conditions hairpin formation occurs at the two signals at or near the same time. In contrast, we find that
under the same conditions nicking occurs at isolated signals and hence is not subject to the 12/23 rule. With two signals
the nicking events are not concerted and the signal with a 12 bp spacer is usually nicked first. However, the extent and rate
of nicking at a given signal are diminished by mutations of the other signal. The appearance of DNA nicked at both signals
is stimulated by more than an order of magnitude by the ability of the signals to synapse, indicating that synapsis accelerates
nicking and often precedes it. These observations allow formulation of a more complete model of catalysis of DNA cleavage
and how the 12/23 rule is enforced.
... In principle, such deletions produce episomal hybrid joints that contain a single RSS or cRSS. Although in vitro assays have shown that RAG1/2 can induce breaks at single RSSs, the extent to which this occurs in vivo is unclear (McBlane et al., 1995;Eastman and Schatz, 1997;Yu and Lieber, 2000;Rahman et al., 2006). Hence, similar to coding-end insertions, those with hybrid ends likely derive from linear deletion products. ...
The RAG recombinase (RAG1/2) plays an essential role in adaptive immunity by mediating V(D)J recombination in developing lymphocytes. In contrast, aberrant RAG1/2 activity promotes lymphocyte malignancies by causing chromosomal translocations and DNA deletions at cancer genes. RAG1/2 can also induce genomic DNA insertions by transposition and trans-V(D)J recombination, but only few such putative events have been documented in vivo. We used next-generation sequencing techniques to examine chromosomal rearrangements in primary murine B cells and discovered that RAG1/2 causes aberrant insertions by releasing cleaved antibody gene fragments that subsequently reintegrate into DNA breaks induced on a heterologous chromosome. We confirmed that RAG1/2 also mobilizes genomic DNA into independent physiological breaks by identifying similar insertions in human lymphoma and leukemia. Our findings reveal a novel RAG1/2-mediated insertion pathway distinct from DNA transposition and trans-V(D)J recombination that destabilizes the genome and shares features with reported oncogenic DNA insertions.
... RSSs consist of highly conserved heptamer (canonical sequence: 5' CACAGTG 3') and nonamer (canonical sequence: 5' ACAAAAACC 3') elements separated by a lesser conserved spacer sequence of 12 or 23 bp. The different spacer lengths ensure fidelity in joining and prevent variable segments from joining to each other (Eastman et al., 1996;Eastman and Schatz, 1997;Gellert, 2002;Goldsby, 2003;Jones and Gellert, 2002). ...
... Importantly, these PCs were all purified well above the 3% threshold. We conclude that RSS nicking can indeed be stimulated by synapsis, as previously proposed (40), and that the magnitude of this effect is highly dependent on the sequence of the RSS, its coding flank, and the partner RSS involved. ...
At the Tcrb locus, Vβ-to-Jβ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the “beyond 12/23” rule (B12/23 rule). Previous work showed that Vβ recombination signal sequences
(RSSs) do not recombine with Jβ RSSs because Jβ RSSs are crippled for either nicking or synapsis. This result raised the following
question: how can crippled Jβ RSSs recombine with Dβ RSSs? We report here that the nicking of some Jβ RSSs can be substantially
stimulated by synapsis with a 3′Dβ1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact
of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking
RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly
suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations
indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial
RAG binding can occur on RSSs with either 12- or 23-bp spacers (12- or 23-RSSs, respectively).
... This hydroxyl group subsequently attacks the opposing strand in a direct transesterification reaction, generating a double-strand break at the coding gene:RSS border [2]. Under physiological conditions, hairpin formation requires that the RAG proteins bind to both a 12-RSS and 23-RSS in a paired complex345678. The generation of double-strand breaks is therefore coordinated at the two RSSs undergoing recombination, assuring that doublestrand breaks are not made at isolated RSSs. ...
The repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity. Though not absolutely required, non-core regions of RAG1 and RAG2 have been shown to influence the efficiency and fidelity of the recombination reaction.
Using a partial proteolysis approach in combination with bioinformatics analyses, we identified the domain boundaries of a structural domain that is present in the 380-residue N-terminal non-core region of RAG1. We term this domain the Central Non-core Domain (CND; residues 87-217).
We show how the CND alone, and in combination with other regions of non-core RAG1, functions in nuclear localization, zinc coordination, and interactions with nucleic acid. Together, these results demonstrate the multiple roles that the non-core region can play in the function of the full length protein.
... However, nicking at the 23-RSS, along with hairpin formation at both RSSs, occurs in the context of the paired complex. 9,10 Processing and joining of the appropriate coding and signal ends is mediated by components of the nonhomologous end-joining machinery. 11 The core regions of the murine RAG proteins include residues 384-1008 of 1040 in RAG1 and residues 1-387 of 527 in RAG2. 2 The majority of biochemical studies with RAG1 and RAG2 are accomplished with these truncated proteins, which are the minimal catalytically active regions required for double-stranded DNA cleavage activity and also are more soluble than their full-length counterparts. ...
The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn(2+) ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn(2+) ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn(2+) ions. Stripping the full complement of bound Zn(2+) ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn(2+) core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn(2+) ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn(2+) ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn(2+) coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn(2+) serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.
... This outcome cannot be attributed to differential activity of the complexes, because under conditions used for complex assembly, WT cMR1/cMR2 and FLMR1/cMR2 nick the 12/23 substrate at very low, but comparable levels ( Figure 2A; 12/23 TOP, compare lanes 5 and 6). It is worth pointing out that nicking activity under these conditions shows clear evidence of regulation by the 12/23 rule, as RAG complexes assembled on 12/12 or 23/23 substrates and analyzed in parallel show little or no evidence of nicking, a result consistent with previous reports (17). ...
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.
... Hairpin formation occurs only in the context of the 12/23 paired complex; however, nicking can occur at a single RSS. [9][10][11] The assembly of the 12/23 paired complex appears to occur by a well-ordered series of macromolecular associations that is best represented by the capture model. 11 In this model the RAG proteins appear to first bind and nick the 12-RSS. ...
RAG1 and RAG2 proteins catalyze site-specific DNA cleavage reactions in V(D)J recombination, a process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS. For example, while RAG1 alone is capable of recognizing the conserved elements of the RSS, it is not clear if or how RAG2 may enhance sequence-specific associations with the RSS. To shed light on this issue, we examined the association of RAG1, with and without RAG2, with consensus RSS versus non-RSS substrates using fluorescence anisotropy and gel mobility shift assays. The results indicate that while RAG1 can recognize the RSS, the sequence-specific interaction under physiological conditions is masked by a high-affinity non-sequence-specific DNA binding mode. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence-specific DNA, resulting in a large differential in binding affinity for the RSS versus the non-RSS sites. We conclude that this represents a major means by which RAG2 contributes to the initial recognition of the RSS and that, therefore, association of RAG1 with RAG2 is required for effective interactions with the RSS in developing lymphocytes.
... n, and the same is true for the nicking of the 23-substrate alone. This indicates that even if a RAG complex binds to a 12-and a 23-RSS simultaneously, it would still nick them independently. Based on the results here, we can infer a kinetic scheme for the RAG-mediated nicking and hairpinning during the initiation of V(D)J recombination (Fig. 5).8, 9, 14, 46, 47). However, in all of those previous studies, it was unclear whether two 12-substrates might synapse to permit the nicking step or the 12-substrates were being nicked individually (and prior to any synapsis). The same applies to reactions involving only 23-substrates. Our immobilization and kinetics studies clearly indicate th ...
In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before
any catalytic steps (strand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning
the role of synapsis in nicking. Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in
any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not
affect their nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find
that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for
a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor
an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type
reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps
in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification
of the immune repertoire.
... The hairpin-formation reaction occurs within the paired complex (PC): a 12-and a 23-RSS are synapsed by the RAG proteins and HMG1/2 (Fig. 2). Nicking can occur asyn-Fugmann chronously at the isolated RSSs prior to formation of the PC; synchronous hairpin formation leading to two DNA double-strand breaks, however, is catalyzed exclusively within the PC (36)(37)(38). ...
RAG1 and RAG2 are the key components of the V(D)J recombinase machinery that catalyses the somatic gene rearrangements of antigen receptor genes during lymphocyte development. In the first step of V(D)J recombination--DNA cleavage--the RAG proteins act together as an endonuclease to excise the DNA between two individual gene segments. They are also thought to be involved in the subsequent DNA joining step. In vitro, the RAG proteins catalyze the integration of the excised DNA element into target DNA completing a process similar to bacterial transposition. In vivo, this reaction is suppressed by an unknown mechanism. The individual roles of RAG1 and RAG2 in V(D)J recombination and transposition reactions are discussed based on mutation analyses and structure predictions.
... The RAG Cleavage Efficiency of Human V H Targets Varies Markedly-The reaction time courses conformed to the expected kinetics for nicking and hairpin formation (41,44,45), and the products increased over the initial 50 min when incubated at 37°C (Fig. 2). Cleavage efficiency was determined as the percentage of the substrate that is converted to the nicked or hairpin product at the 30-min time point. ...
The human immunoglobulin heavy chain locus contains 39 functional human VH elements. All 39 VH elements (with their adjacent heptamer/nonamer signal) were tested for site-specific cleavage with purified human core RAG1
and RAG2, and HMG1 proteins in a 12/23-coupled cleavage reaction. Both nicking and hairpin formation were measured. The individual
VH cleavage efficiencies vary over nearly a 30-fold range. These measurements will be useful in considering the factors affecting
the generation of the immunoglobulin and T-cell receptor repertoires in the adult humans. Interestingly, when these cleavage
efficiencies are summed for each of the VH families, the six VHfamily efficiencies correspond closely to the observed profile of unselected VH family usage in the peripheral B cells of normal adult humans. This correspondence raises the possibility that the dominant
factor determining VH element utilization within the 1-megabase human genomic VH array is simply the individual RAG cleavage efficiencies.
... Overall, RAG-catalyzed DNA DSB yields coding sequences terminated by covalently sealed hairpins (coding ends) and blunt-ended breaks terminated at RSS heptamers (signal ends). The 12/23 rule is enforced at formation of the coding end hairpins, since transesterification preferentially occurs in the context of the synaptic complex, which consists of the RAG proteins bound to both a 12-RSS and a 23-RSS (8,9). In contrast, the nicking reaction may occur with RAG proteins bound to a single 12-or 23-RSS. ...
Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig V(H) gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability.
... Once the RAG proteins have created the double-strand breaks, the appropriate joining of both the coding and signal ends require factors that mediate nonhomologous DNA end joining [4]. There is evidence that the RAG proteins may prefer to assemble first on the 12-RSS to form the single RSS complex [5][6][7][8], followed by recruitment of the 23-RSS to form the paired complex [5,8,9]. The DNA-bending proteins, HMGB1 or HMGB2, facilitate formation of the latter complex [10]. ...
Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity. An understanding of the physicochemical properties of the RAG proteins, their association, and their interaction with the RSS is not yet well developed.
Here, we further our investigations into the self-association properties of RAG1 by demonstrating that despite the presence of multiple RAG1 oligomers, only the dimeric form maintains the ability to interact with RAG2 and the RSS. However, facile aggregation of the dimeric form at physiological temperature may render this protein inactive in the absence of RAG2. Upon addition of RAG2 at 37 degrees C, the preferentially stabilized V(D)J recombinase:RSS complex contains a single dimer of RAG1.
Together these results confirm that the functional form of RAG1 in V(D)J recombination is in the dimeric state, and that its stability under physiological conditions likely requires complex formation with RAG2. Additionally, in future structural and functional studies of RAG1, it will be important to take into account the temperature-dependent self-association properties of RAG1 described in this study.
It has been proposed that the modern immune system has evolved from a transposon in an ancient vertebrate. While much is known about the mechanism by which bacterial transposable elements catalyze double-strand breaks at their ends, less is known about how eukaryotic transposable elements carry out these reactions. We have examined the mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage. We show that the nontransferred strand is cleaved initially, unlike prokaryotic transposons which cleave the transferred strand first. First strand cleavage is not tightly coupled to second strand cleavage and can occur independently of synapsis, as happens in V(D)J recombination but not in transposition of prokaryotic transposons. Unlike V(D)J recombination, however, second strand cleavage of mariner does not occur via a hairpin intermediate.
During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the "nonamer binding region," which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease.
V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences.
V(D)J recombination is initiated by the synapsis and cleavage of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins. Our understanding of these processes has been greatly aided by the development of in vitro biochemical assays of RAG binding and cleavage activity. Accumulating evidence suggests that synaptic complex assembly occurs in a step-wise manner and that the RAG proteins catalyze RSS cleavage by mechanisms similar to those used by bacterial transposases. In this chapter we will review the molecular mechanisms of RAG synaptic complex assembly and 12/23-regulated RSS cleavage, focusing on recent advances that shed new light on these processes.
The products of recombination-activating genes RAG1 and RAG2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2.
During V(D)J recombination, site specific DNA excision is dictated by the binding of RAG1/2 proteins to the conserved recombination signal sequence (RSS) within the genome. The interaction between RAG1/2 and RSS is thought to involve a large DNA distortion that is permissive for DNA cleavage. In this study, using atomic force microscopy imaging (AFM), we analyzed individual RAG-RSS complexes, in which the bending angle of RAG-associated RSS substrates could be visualized and quantified. We provided the quantitative measurement on the conformations of specific RAG-12RSS complexes. Previous data indicating the necessity of RAG2 for recombination implies a structural role in the RAG-RSS complex. Surprisingly, however, no significant difference was observed in conformational bending with AFM between RAG1-12RSS and RAG1/2-12RSS. RAG1 was found sufficient to induce DNA bending, and the addition of RAG2 did not change the bending profile. In addition, a prenicked 12RSS bound by RAG1/2 proteins displayed a conformation similar to the one observed with the intact 12RSS, implying that no greater DNA bending occurs after the nicking step in the signal complex. Taken together, the quantitative AFM results on the components of the recombinase emphasize a tightly held complex with a bend angle value near 60 degrees , which may be a prerequisite step for the site-specific nicking by the V(D)J recombinase.
A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies.
This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination,
could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation.
It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel
system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include
two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate
the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind
and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals
only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site
hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions,
thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.
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.
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.
The RAG1 and RAG2 proteins together initiate V(D)J recombination by performing cleavage of chromosomal DNA adjacent to antigen receptor gene segments. Like the adaptive immune system itself, RAG1 and RAG2 are found only in jawed vertebrates. The hypothesis that RAG1 and RAG2 arose in evolution as components of a transposable element has received dramatic support from our recent finding that the RAG proteins are a fully functional transposase in vitro. This result strongly suggests that antigen receptor genes acquired their unusual structure as a consequence of the insertion of a transposable element into an ancestral receptor gene by RAG1 and RAG2 approx 450 million years ago.
V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly
affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the coding end sequence effect
can be qualitatively and quantitatively recapitulated in vitro with purified RAG proteins. We find that coding end sequence
specifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin
formation step is not affected by the coding end sequence. Furthermore, the coding end sequence effect can be ablated by prenicking
the substrate, indicating that the coding end effect is specific to the nicking step. In reactions in which both 12- and 23-substrates
are present, a suboptimal coding end sequence on one signal can slow down hairpin formation at the partner signal, a result
consistent with models in which coordination between the signals occurs at the hairpin formation step. The coding end sequence
effect on nicking and the coupling of the 12- and 23-substrates explains how hairpin formation can be rate limiting for some
12/23 pairs, whereas nicking can be rate limiting when low-efficiency coding end sequences are involved.
During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.
V(D)J recombination proceeds through a series of protein:DNA complexes mediated in part by the RAG1 and RAG2 proteins. These proteins are responsible for sequence-specific DNA recognition and DNA cleavage, and they appear to perform multiple postcleavage roles in the reaction as well. Here we review the interaction of the RAG proteins with DNA, the chemistry of the cleavage reaction, and the higher order complexes in which these events take place. We also discuss postcleavage functions of the RAG proteins, including recent evidence indicating that they initiate the process of coding end processing by nicking hairpin DNA termini. Finally, we discuss the evolutionary and functional implications of the finding that RAG1 and RAG2 constitute a transposase, and we consider RAG protein biochemistry in the context of several bacterial transposition systems. This suggests a model of the RAG protein active site in which two divalent metal ions serve alternating and opposite roles as activators of attacking hydroxyl groups and stabilizers of oxyanion leaving groups.
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.
Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3' ends of linear transposon or donor DNA are joined to the 5' phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.
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.
It has been proposed that the modern immune system has evolved from a transposon in an ancient vertebrate. While much is known about the mechanism by which bacterial transposable elements catalyze double-strand breaks at their ends, less is known about how eukaryotic transposable elements carry out these reactions. We have examined the mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage. We show that the nontransferred strand is cleaved initially, unlike prokaryotic transposons which cleave the transferred strand first. First strand cleavage is not tightly coupled to second strand cleavage and can occur independently of synapsis, as happens in V(D)J recombination but not in transposition of prokaryotic transposons. Unlike V(D)J recombination, however, second strand cleavage of mariner does not occur via a hairpin intermediate.
Chromosome breakage--a dangerous event that has triggered the evolution of several double-strand break repair pathways--has been co-opted by the immune system as an integral part of B- and T-cell development. This is a daring strategy, as improper repair can be deadly for the cell, if not for the whole organism. Even more daring, however, is the choice of a promiscuous transposase as the nuclease responsible for chromosome breakage, as the possibility of transposition brings an entirely new set of risks. What mechanisms constrain the dangerous potential of the recombinase and preserve genomic integrity during immune-system development?
The complete cleavage phase of V(D)J recombination includes four phases: binding of the active RAG complexes to the 12- or 23-signals, nicking of the signals, synapsis of the two signals, and hairpin formation at both signals concurrently. We have done time courses for the complete cleavage phase of the V(D)J recombination reaction and quantitated the amount of active RAG enzyme. We have also formulated a kinetic model for the binding, nicking, synapsis, and hairpin formation phases. We have utilized free solution enzymatic measurements for the binding and nicking phases as we do mathematical simulations of the kinetic model. This permits iteration of rate constants for the synapsis and hairpin formation phases until the model fits the observed overall cleavage time course. This process yields a rate constant for the hairpin formation that is 0.004 min(-1), which corresponds to an average catalytic cycle time of 250 min. This value is exceedingly close to a measured value of this constant that relied on wash-out of an inhibitory cofactor. The agreement indicates that this is likely to be the rate of the hairpin step over a wide range of range of conditions and irrespective of the DNA sequence of the V, D or J coding end located adjacent to the signal. These findings indicate that, under optimal in vitro conditions, the core RAG proteins carry out nicking at a rate which is nearly 150-fold faster than hairpin formation. The physiologic implications of this and other kinetic inferences of these time courses are discussed.
The lymphoid-specific factors, recombination-activating gene 1 (RAG1) and RAG2, initiate V(D)J recombination by introducing DNA double-stand breaks at specific sites in the genome. In addition to this critical endonuclease activity, the RAG proteins catalyze other chemical reactions that can affect the outcome of V(D)J recombination, one of which is transposition. While the transposition activity of the RAG proteins is thought to have been critical for the evolution of modern antigen-receptor loci, it has also been proposed to contribute to chromosomal translocations and lymphoid malignancy. A major challenge has been to determine how the transposition activity of the RAG proteins is regulated in vivo. Although a variety of mechanisms have been suggested by recent studies, a clear resolution of this issue remains elusive.
V(D)J recombination is a form of site-specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell-specific proteins, recombination activating genes RAG-1 and RAG-2, and ubiquitously expressed architectural DNA-binding proteins and DNA-repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein-DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence-specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein-DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post-cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein-DNA complexes that support them, and factors and forces that control them.
V(D)J recombination generates functional immunoglobulin and T-cell receptor genes in developing lymphocytes. The recombination-activating gene 1 (RAG1) and RAG2 proteins catalyze site-specific DNA cleavage in this recombination process. Biochemical studies have identified catalytically active regions of each protein, referred to as the core regions. Here, we review our progress in the identification and characterization, in biophysical and biochemical terms, of topologically independent domains within both the non-core and core regions of RAG1. Previous characterizations of a structural domain identified in the non-core region of RAG1 from residues 265-380, referred to as the zinc-binding dimerization domain, are discussed. This domain contains two zinc-binding motifs, a RING finger and a C2H2 zinc finger. Core RAG1 also consists of multiple domains, each of which functions individually in one or more of the essential macromolecular interactions formed by the intact core protein. Two structural domains referred to as the central and the C-terminal domains that include residues 528-760 and 761-979 of RAG1, respectively, have been identified. The interactions of the central and C-terminal domains in core RAG1 with the recombination signal sequence (RSS) have contributed additional insight to a developing model for the RAG1-RSS complex.
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.
RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain
of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central
domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore,
the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory
effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of
the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We
propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions
that may lead to genomic instability in B and T lymphocytes.
In V(D)J recombination, the RAG1 and RAG2 protein complex cleaves the recombination signal sequences (RSSs), generating a
hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23
bp. Here we report that in the synaptic complex, recombination-activating gene (RAG) proteins interact with the 7-mer and
unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking
of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported
to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction
and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding
DNA distortion and hairpin formation in the context of the 12/23 rule.
We have developed a cell-free system in which the bacterial transposon Tn7 inserts at high frequency into its preferred target site in the Escherichia coli chromosome, attTn7; Tn7 transposition in vitro requires ATP and Tn7-encoded proteins. Tn7 transposes via a cut and paste mechanism in which the element is excised from the donor DNA by staggered double-strand breaks and then inserted into attTn7 by the joining of 3' transposon ends to 5' target ends. Neither recombination intermediates nor products are observed in the absence of any protein component or DNA substrate. Thus, we suggest that Tn7 transposition occurs in a nucleoprotein complex containing several proteins and the substrate DNAs and that recognition of attTn7 within this complex provokes strand cleavages at the Tn7 ends.
Immunoglobulin and T-cell receptor genes are assembled during lymphocyte development by a novel, highly regulated series of gene rearrangement reactions known as V(D)J recombination. All rearranging loci are flanked by conserved heptamer-nonamer recombination signal sequences. Gene rearrangement results in the imprecise fusion of coding sequences and the precise fusion of signal sequences. DNA molecules with double-stranded breaks near signal sequences have been detected in cells undergoing V(D)J recombination of the TCR delta locus. We have devised a ligation-mediated PCR assay that detects broken-ended molecules in purified genomic DNA. Using this assay we found that DNA breaks occurring precisely at the signal sequence-coding sequence junction are a general feature of V(D)J recombination, appearing in association with each type of rearranging immunoglobulin gene segment. We show that a significant fraction of these broken ends are blunt and 5'-phosphorylated. In addition, detection of these broken-ended signal sequences is dependent on the activity of RAG-1 and RAG-2, and is restricted to the G0/G1 phase of the cell cycle. The pattern of broken-ended molecules detected in cells at various stages of development reflects the activity of the V(D)J recombinase at different loci during B- and T-cell development.
The bacterial transposon Tn7 uses a cut and paste mechanism to translocate between non-homologous insertion sites. In the first step of recombination, double-strand breaks at each transposon end disconnect the element from the donor backbone; in the second step, the now exposed 3' transposon ends join to the target DNA. To dissect the chemical steps in these reactions, we have used mutant transposons altered at and near their extreme termini. We find that the initiating double-strand breaks result from a collaboration of two distinct DNA strand processing activities, one mediating cleavages at the 3' ends of Tn7, which can be blocked by changes at the transposon tips, and another mediating cleavages at the 5' ends. The joining of exposed 3'transposon ends to the target DNA can be blocked by changing the transposon tips. Our results suggest that the target joining step occurs through two usually concerted, but actually separable, reactions in which individual 3' transposon ends are joined to separate strands of the target DNA. Thus Tn7 transposition involves several distinct DNA processing reactions: strand cleavage and strand transfer reactions at the 3' ends of the transposon, and separate strand cleavage reactions at the 5' ends of the transposon.
Cleavage of V(D)J recombination signals by purified RAG1 and RAG2 proteins permits the dissection of DNA structure and sequence requirements. The two recognition elements of a signal (nonamer and heptamer) are used differently, and their cooperation depends on correct helical phasing. The nonamer is most important for initial binding, while efficient nicking and hairpin formation require the heptamer sequence. Both nicking and hairpin formation are remarkably tolerant of variations in DNA structure. Certain flanking sequences inhibit hairpin formation, but this can be bypassed by base unpairing, and even a completely single-stranded signal sequence is well utilized. We suggest that DNA unpairing around the signal-coding border is essential for the initiation of V(D)J combination.
Recombination-activating gene 1 (RAG1), as well as RAG2, are the only lymphoid-specific genes required for V(D)J recombination. RAG1 protein contains a C3HC4 zinc-binding motif (zinc ring finger) that binds two zinc ions. We have found that RAG1 contains additional zinc-binding motifs in the form of two separate C2H2 zinc finger sequences. One of the zinc fingers, in combination with the C3HC4 subdomain, forms a highly specific dimerization domain. A combination of biophysical techniques has been used to determine the energetics of association, the overall shape of the dimerization domain, and the relative orientation of the monomeric subunits within the dimer. These results provide direct evidence that a C3HC4 motif is involved in a protein-protein interaction, in this case via homodimer formation. In addition, the observation that the dimerization domain includes multi-class zinc binding motifs, namely both a zinc finger and a C3HC4 subdomain, has important implications for other C3HC4-containing proteins. The position of this dimerization domain in the N-terminal third of the RAG1 sequence of 1040 amino acid residues may have a significant influence on the activities associated with the C-terminal domains of the protein.
Recent studies have demonstrated that DNA cleavage during V(D)J recombination is mediated by the RAG1 and RAG2 proteins. These proteins must therefore bind to the recombination signals, but the specific binding interaction has been difficult to study in vitro. Here, we use an in vivo one-hybrid DNA binding assay to demonstrate that RAG1, in the absence of RAG2, can mediate signal recognition via the nonamer, with the heptamer acting to enhance its binding. A region of RAG1 with sequence similarity to bacterial invertases is essential for DNA binding. Localization of RAG2 to the signal is dependent upon the presence of RAG1 and is substantially more efficient with a 12 bp spacer signal than with a 23 bp spacer signal.
The RAG1 and RAG2 proteins initiate V(D)J recombination by making specific double-strand DNA breaks at recombination signal sequences. We show here that RAG1 and RAG2 bind specifically to this sequence, forming a stable protein-DNA complex. The complex requires the conserved heptamer and nonamer motifs of the recombination signal as well as both the RAG1 and RAG2 proteins. This complex is able to either nick or form hairpins at the V(D)J signal sequence, depending on the divalent cation present. A complex trapped using Ca2+ is subsequently active when transferred to Mg2+ or Mn2+. After cleavage, the complex is destabilized and the RAG proteins dissociate. We term this early precursor in the V(D)J recombination reaction a "stable cleavage complex."
During V(D)J recombination, RAG1 and RAG2 cleave DNA adjacent to highly conserved recombination signals, but nothing is known about the protein-DNA complexes that exist after cleavage. Using a properly regulated in vitro V(D)J cleavage system, together with nuclease sensitivity, mobility shift, and immunoprecipitation experiments, we provide evidence that a stable complex is formed postcleavage between synapsed recombination signals. This complex includes the proteins RAG1, RAG2, HMG-1 or the closely related HMG-2 protein, and the components of the DNA-dependent protein kinase. The existence of such a stable complex explains a number of in vivo observations and suggests that remodeling of postcleavage synaptic complexes is an important step in the resolution of signal ends in V(D)J recombination.
Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by
a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination
requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23
rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products
of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1
and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the
existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.
V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 bp between the conserved heptamer and nonamer elements. The RAG1 and RAG2 proteins initiate the reaction by making double-strand DNA breaks at both signals, and must thus be able to operate on these two different spatial arrangements. We show that the DNA-bending proteins HMG1 and HMG2 stimulate cleavage and RAG protein binding at the 23 bp spacer signal. These findings suggest that DNA bending is important for bridging the longer spacer, and explain how a similar array of RAG proteins could accommodate a signal with either a 12 or a 23 bp spacer. An additional effect of HMG proteins is to stimulate coupled cleavage greatly when both signal sequences are present, suggesting that these proteins also aid the formation of a synaptic complex.
In the murine T cell receptor delta locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination.
Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.
Open and shut junctions are rare (V(D)J joining products in which site-specific recognition, cleavage and re-ligation of joining signals has been uncoupled from recombination. Here, we investigate the relationship of opening and shutting to recombination in two ways. First, we have tested a series of substrates containing one or two joining signals in an in vivo assay. Opening and shutting can be readily observed in substrates that have only one consensus joining signal. Thus, unlike recombination, the majority of open and shut events do not require interactions between two canonical joining signals. Next we examined two-signal substrates to investigate the effect of signal proximity on the frequency of dual open and shut events. These experiments indicate that at least some of the time opening and shutting can be a two-signal transaction. Together these results point to two mechanistically related, but distinct origins for open and shut joining events. In one case, cutting and closing may occur without interaction between two signals. In the other, we suggest that interaction of a canonical signal with 'cryptic' signal-like elements whose sequence is extensively diverged from canonical signals, may bias the V(D)J recombination machinery towards opening and shutting rather than recombination. Open and shut operations could in this way provide a means whereby mistakes in target recognition by the V(D)J recombination machinery produce a non-recombinant outcome, avoiding deleterious chromosomal rearrangements in lymphoid tissues.
Two conserved DNA sequences serve as joining signals in the assembly of immunoglobulins and T-cell receptors from V-, (D)-, and J-coding segments during lymphoid differentiation. We have examined V(D)J recombination as a function of joining signal sequence. Plasmid substrates with mutations in one or both of the heptamer-spacer-nonamer sequences were tested for recombination in a pre-B-cell line active in V(D)J recombination. No signal variant recombines more efficiently than the consensus forms of the joining signals. We find the heptamer sequence to be the most important; specifically, the three bases closest to the recombination crossover site are critical. The nonamer is not as rigidly defined, and it is not important to maintain the five consecutive As that distinguish the consensus nonamer sequence. Both types of signals display very similar sequence requirements and have in common an intolerance for changes in spacer length greater than 1 bp. Although the two signal types share sequence motifs, we find no evidence of a role in recombination for homology between the signals, suggesting that they serve primarily as protein recognition and binding sites.
Sequences encoding immunoglobulin variable domains are known to be assembled from variable (V), diversity (D), and joining (J) segments by site-specific recombination. We present a sensitive and rapid assay for V-(D)-J recombination that uses plasmid DNA transiently introduced into transformed pre-B cells, and demonstrates that the recombination is independent of any unique chromosomal context. Sequences sufficient to constitute recombination sites are contained within the 84 and 42 bp flanking, respectively, the murine J kappa 1 and V kappa L8 segments, which include the known heptamer-nonamer V-(D)-J joining signals. Deletion and inversion occur at comparable frequencies. Thus, V-(D)-J recombination may be relatively insensitive to the topological arrangement of sites, and events at the two novel junctions produced by the reaction may be coupled.
The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.
A recently described pre-B cell line can be induced at high temperature to actively rearrange its immunoglobulin light-chain loci. We used this cell line to determine the fate of double-strand breaks generated by V(D)J rearrangement. After induction, 30%-40% of K loci had broken JK1 signal ends. JK1-coding ends were detectable, but 10- to 100-fold less frequent. Both covalently closed (hairpin) and open, blunt, processed coding ends were observed. Coding junctions involving JK1 accumulated with similar kinetics as JK1 signal ends, arguing that coding ends can be resolved quickly and efficiently to coding junctions, whereas signal ends remain mostly unjoined. Signal ends are then joined rapidly when cells are returned to the low temperature. These results support the model that broken signal ends and hairpin coding ends are authentic intermediates in V(D)J recombination. It appears that hairpin coding ends are rapidly opened, processed, and resolved to coding junctions, whereas joining of signal ends is clearly uncoupled from the joining of coding ends and can be much slower. Efficient formation of signal junctions may require cell cycle progression, or down-regulation of the recombination machinery.
We previously identified possible intermediates in V(D)J recombination at the TCR delta locus and characterized molecules with signal ends and with covalently sealed (hairpin) coding ends in thymocytes of scid mice by Southern blotting. Here, we use a sensitive ligation-mediated PCR assay to demonstrate that all coding ends detected in scid thymocytes are covalently sealed. Neither coding nor signal ends exhibit loss or addition of nucleotides. These data imply that hairpin formation is coupled to the initial cleavage at the signal/coding border, and that the cleavage step in V(D)J recombination is conservative. In scid/+ or wild-type thymocytes, hairpin coding ends are at least 1000-fold less abundant than signal ends. These results provide insight into the mechanism of V(D)J recombination.
The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 A resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a beta barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.
The RAG-1 protein plays an essential role in V(D)j recombination, but its exact function has not yet been defined. Here we report that a particular mutation in RAG-1 affects recombination by altering the specificity of target sequence usage. Recombination mediated by wild-type RAG-1 is tolerant of a wide range of coding sequences adjacent to the recombination signal. With the mutant RAG-1, recombination is much more demanding; efficient recombination is only found when particular dinucleotides are adjacent to the signal sequence heptamer. The mutant is also more sensitive than wild-type RAG-1 to certain alterations within the signal sequence. We suggest that the RAG-1 protein may interact physically with the target DNA at the coding-signal sequence border.
Severe combined immunodeficient (SCID) mice are deficient in a recombination process utilized in both DNA double-strand break repair and in V(D)J recombination. The phenotype of these mice involves both cellular hypersensitivity to ionizing radiation and a lack of B and T cell immunity. The catalytic subunit of DNA-dependent protein kinase, p350, was identified as a strong candidate for the murine gene SCID. Both p350 and a gene complementing the SCID defect colocalize to human chromosome 8q11. Chromosomal fragments expressing p350 complement the SCID phenotype, and p350 protein levels are greatly reduced in cells derived from SCID mice compared to cells from wild-type mice.
Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.
A tetramer of Mu transposase (MuA) cleaves the phage Mu DNA and joins these ends to a target DNA to catalyze transposition. Substitution mutations at Asp-269 or Glu-392 within MuA destroy both the DNA cleavage and joining activities without blocking tetramer assembly, indicating that the mutations specifically affect catalysis. Although inactive under standard reaction conditions (10 mM Mg2+), the mutant proteins are partially resuscitated by 10-20 mM Mn2+, concentrations 5- to 10-fold higher than optimal for wild-type MuA. Amino acid sequence alignment and the similar effects of mutations suggests that Asp-269 and Glu-392 of MuA may be analogs of the first Asp and final Glu of a conserved triad of acidic amino acids present in many transposases and the retroviral integrases (the D-D-35-E motif). The higher Mn2+ optima observed with MuA derivatives altered at these positions supports a role for the conserved acidic amino acids in coordinating divalent metal ions in the active sites of transposases.
Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and
V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar
to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these
mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role
for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling
pathway induced by DNA damage.
During Tn10 transposition, the transposon is fully excised from the donor site by double strand cleavages at the two ends of the element prior to integration at a new target site. Results presented here demonstrate that an interaction between the two transposon ends is required for double strand cleavage at either end. Furthermore, despite this essential interaction of ends, subsequent cleavages at the two ends can occur at observably distinct times prior to occurrence of strand transfer at either end. Moreover, the time between cleavages at the two ends is exaggerated by the presence of an appropriate mutation at one end of the element. Biological rationales for this constellation of mechanistic features are suggested. Additional results demonstrate that mutations at the three terminal basepairs of Tn10 confer defects subsequent to interaction of ends, in confirmation of inferences from genetic analysis. More specifically, mutations in bp 1-3 confer strong defects during conversion of the full excision intermediate to a complete strand transfer product; mutations in bp 1 and 2 also confer more subtle defects subsequent to interaction of ends but prior to full excision. Such defects might reflect roles for these basepairs in the chemical steps of transposition per se, the positioning of terminal residues for those chemical steps, and/or the coupling of cleavage(s) to subsequent conformational changes.
The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.
This chapter attempts an interdisciplinary perspective to consider that that both molecular biologists and immunologists have learned about the V (D) J recombination process, as instructed by cross-species (and cross-locus) comparisons. Immune recognition in vertebrates is based on the antigen receptors manufactured by B and T cells. The antigen-binding polypeptides within these multiunit conglomerates are encoded at the immunoglobulin (Ig) and T cell receptor (TCR) loci. Through the process called “V (D) J joining,” antigen receptor genes are constructed from multiply reiterated DNA segments in B and T cells. By this means, an enormous number of binding specificities can be generated in lymphoid cells from a relatively minimal amount of germline information. V (D) J rearrangement can culminate in the production of alternative or “nonstandard” junction products. Demonstrated nonstandard products can account for all theoretically possible signal end-to-coding end assortments. A picture emerges of V (D) J joining as an orderly process that is the sum of disorderly parts. Variability comes into play at a number of levels—namely, variable crossover sites, variable joining signals, variable strand exchange, variable degrees of reciprocity, and so forth. It is argued that the main tactic employed by other site-directed recombination systems is not in evidence for V (D) J joining.
The variable domains of immunoglobulins and T cell receptors are assembled through the somatic, site specific recombination
of multiple germline segments (V, D, and J segments) or V(D)J rearrangement. The recombination signal sequence (RSS) is necessary
and sufficient for cell type specific targeting of the V(D)J rearrangement machinery to these germline segments. Previously,
the RSS has been described as possessing both a conserved heptamer and a conserved nonamer motif. The heptamer and nonamer
motifs are separated by a ‘spacer’ that was not thought to possess significant sequence conservation, however the length of
the spacer could be either 12 +/− 1 bp or 23 +/− 1 bp long. In this report we have assembled and analyzed an extensive data
base of published RSS. We have derived, through extensive consensus comparison, a more detailed description of the RSS than
has previously been reported. Our analysis indicates that RSS spacers possess significant conservation of sequence, and that
the conserved sequence in 12 bp spacers is similar to the conserved sequence in the first half of 23 bp spacers.
V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.
Formation of double-strand breaks at recombination signal sequences is an early step in V(D)J recombination. Here we show that purified RAG1 and RAG2 proteins are sufficient to carry out this reaction. The cleavage reaction can be divided into two distinct steps. First, a nick is introduced at the 5' end of the signal sequence. The other strand is then broken, resulting in a hairpin structure at the coding end and a blunt, 5'-phosphorylated signal end. The hairpin is made as a direct consequence of the cleavage mechanism. Nicking and hairpin formation each require the presence of a signal sequence and both RAG proteins.
Nonreplicative transposition by Tn10/IS10 involves three chemical steps at each transposon end: cleavage of the two strands plus joining of one strand to target DNA. These steps occur within a synaptic complex comprising two transposon ends and monomers of IS10 transposase. We report four transposase mutations that individually abolish each of the three chemical steps without affecting the synaptic complex. We conclude that a single constellation of residues, the "active site," directly catalyzes each of the three steps. Analyses of reactions containing mixtures of wild-type and catalysis-defective transposases indicate that a single transposase monomer at each end catalyzes the cleavage of two strands and that strand transfer is carried out by the same monomers that previously catalyzed cleavage. These and other data suggest that one active site unit carries out all three reactions in succession at one transposon end.
In the first step of V(D)J recombination, the RAG1 and RAG2 proteins cleave DNA between a signal sequence and the adjacent coding sequence, generating a blunt signal end and a coding end with a closed hairpin structure. These hairpins are intermediates leading to the formation of assembled antigen receptor genes. It is shown here that the hairpins are formed by a chemical mechanism of direct trans-esterification, very similar to the early steps of transpositional recombination and retroviral integration. A minor variation in the reaction is sufficient to divert the process from transposition to hairpin formation.
V(D)J recombination, the process that assembles antigen-receptor genes, is directed by signal sequences flanking the DNA segments to be joined. Signals consist of a conserved heptamer and nonamer separated by a spacer of either 12 or 23 base pairs. Recombination occurs almost exclusively between two signals with spacers of different lengths. This restriction, called the '12/23 rule', governs the organization and pattern of rearrangement of antigen-receptor loci. In vitro work demonstrating the direct roles of the Rag proteins in the initiation of V(D)J recombination did not recreate the 12/23 rule. Instead, double-strand breaks were formed efficiently at isolated signals. Here we show that extracts made from a lymphoid cell line that expresses truncated forms of the Rag1 and Rag2 proteins have a signal-cutting activity that obeys the 12/23 rule. Cleavage at the two signals is concerted and requires their synapsis, and mutations of one signal prevent cleavage at both.
Central to the Mu transpositional recombination are the two chemical steps; donor DNA cleavage and strand transfer. These reactions occur within the Mu transpososome that contains two Mu DNA end segments bound to a tetramer of MuA, the transposase. To investigate which MuA monomer catalyzes which chemical reaction, we made transpososomes containing wild-type and active site mutant MuA. By pre-loading the MuA variants onto Mu end DNA fragments of different length prior to transpososome assembly, we could track the catalysis by MuA bound to each Mu end segment. The donor DNA end that underwent the chemical reaction was identified. Both the donor DNA cleavage and strand transfer were catalyzed in trans by the MuA monomers bound to the partner Mu end. This arrangement explains why the transpososome assembly is a prerequisite for the chemical steps.
V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 AND RAG2 proteins was previously shown to demand only a single signal sequence. Here, we established conditions where 12- and 23-spacer signal sequences are both necessary for cleavage. Coupled cutting at both sites requires only the RAG1 and RAG2 proteins, but depends on the metal ion. In Mn2+, a single signal sequence supports efficient double strand cleavage, but cutting in Mg2+ requires two signal sequences and is best with the canonical 12/23 pair. Thus, the RAG proteins determine both aspects of the specificity of V(D)J recombination, the recognition of a single signal sequence and the correct 12/23 coupling in a pair of signals.
Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining.
The process of V(D)J recombination is the defining characteristic of lymphocyte development. This site-specific recombination reaction assembles the genes that encode immunoglobulin (lg) and T cell receptor proteins, and in many species is the source of much of the diversity in these gene products. The reaction is complex and almost certainly requires the coordinated activity of a large number of proteins. Most components of the V(D)J recombination enzymatic machinery (hereafter referred to as the V(D)J recombinase) are ubiquitously expressed, with only three lymphocyte-specific factors thus far identified: the products of the recombination activating genes (RAG-1 and RAG-2), and terminal deoxynucleotidyl transferase (TdT). TdT is not required for V(D)J recombination, but when present it functions to add nongermline-encoded nucleotides (N regions) to coding junctions and thereby greatly increases the diversity of the products of the reaction. The rag-1 and rag-2 proteins are central to the process of V(D)J recombination, and their biochemical properties and protein-protein interactions are the focus of this chapter.
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.
The V(D)J recombinase subunits Rag-1 and Rag-2 mediate assembly of antigen receptor gene segments. We studied the mechanisms of DNA recognition by Rag-1/Rag-2 using surface plasmon resonance. The critical step for signal recognition is binding of Rag-1 to the nonamer. This is achieved by a region of Rag-1 homologous to the DNA-binding domain of the Hin family of bacterial invertases and to homeodomain proteins. Strikingly, the Hin homeodomain can functionally substitute for the Rag-1 homologous region. Rag-1 also interacts with the heptamer but with low affinity. Rag-2 shows no direct binding to DNA. Once the Rag-1/Rag-2 complex is engaged on the DNA, subsequent cleavage is directed by the heptamer sequence. This order of events remarkably parallels mechanisms that mediate transposition in bacteria and nematodes.
V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both.
We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo.
These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo.
Diversity of vertebrate antigen receptors is accomplished in large part by a somatic gene rearrangement process known as V(D)J recombination. The first step of the reaction appears to be the creation of a double strand break immediately between the recombination signal sequence (RSS) and the coding gene segment to generate a signal end and a coding end. Signal ends have been shown, both in vitro and in vivo, to be precise and blunt, while coding ends generated in vitro are covalently sealed hairpins. It has been difficult to document the existence of coding ends in vivo in normal lymphoid precursors, presumably because of their low abundance. To date, they have been identified in vivo only in a transformed pre-B cell line and in cells from the mutant scid mouse, where they largely conform to the hairpin structure found in vitro. Here, we identify T cell receptor J alpha gene coding ends in normal murine thymocytes. We demonstrate that these ends are processed, not blunt, and that most are not hairpin terminated, in sharp contrast to previous in vivo and in vitro observations. These results provide the first direct demonstration of this important intermediate of V(D)J recombination in normal lymphoid precursors and have implications for the mechanism of coding joint formation in vivo.
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