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Summary Long DNA palindromes are implicated in chromosomal rearrangement, but their roles in the underlying molecular events remain a matter of conjecture. One notion is that palindromes induce DNA breaks after assuming a cruciform structure, the four-way DNA junction providing a target for cleavage by Holliday junction (HJ)-specific enzymes. Though compelling, few components of the "cruciform resolution" proposal are established. Here we address fundamental properties and genetic dependencies of palindromic DNA metabolism in eukaryotes. Plasmid-borne palindromes introduced into S. cerevisiae are site-specifically broken in vivo, and the breaks exhibit unique hallmarks of an HJ resolvase mechanism. In vivo resolution requires Mus81, for which the bacterial HJ resolvase RusA will substitute. These results provide confirmation of cruciform extrusion and resolution in the context of eukaryotic chromatin. Related observations are that, unchecked by a nuclease function provided by Mre11, episomal palindromes launch a self-perpetuating breakage-fusion-bridge-independent copy number increase termed "escape."

The vertebrate immune system can respond to an ever-changing spectrum of pathogens, and V(D)J recombination plays a central role in achieving this stunning versatility. V(D)J recombination first appeared in vertebrates about 450 million years ago and seemingly emerged without antecedent in evolution, an observation suggesting that the essentials of the recombination machinery were acquired through horizontal transfer and subsequent exaptation. This chapter examines the idea that the very apparatus is derived from part of a DNA transposon by comparing the biochemistry of transposases and the RAG recombinase. We hypothesize that the recombinase not only functions in lymphocytes but has since its acquisition also acted on germ cell genomic sequence, thereby influencing the structure and organization of immunoglobulin and T cell receptor loci. Lastly, we consider the nature of the immune system in the ancestral vertebrate, prior to the development of V(D)J recombination, and review current thinking on how V(D)J recombination fits into the evolution of adaptive immunity as a whole.

In mice, kappa light chains dominate over lambda in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested kappa and lambda RSS flanked by their natural 5' and 3' flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a kappa RSS and its flanks support six- to nine-fold higher levels of recombination than a lambda counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual kappa or lambda RSS (plus flanks) occurs with comparable frequencies, but that a pair of kappa RSS (plus flanks) support significantly higher levels of double cleavage than a pair of lambda RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same kappa or lambda RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 kappa RSS (plus natural flanks) as compared to their lambda counterparts. We conclude that the overuse of kappa compared to lambda segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of kappa RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.

DNA palindromes are associated with rearrangement in a variety of organisms. A unique opportunity to examine the impact of a long palindrome in mammals is afforded by the Line 78 strain of mice. Previously it was found that the transgene in Line 78 is likely to be palindromic and that the symmetry of the transgene was responsible for a high level of germ line instability. Here we prove that Line 78 mice harbor a true 15.4-kb palindrome, and through the establishment of cell lines from Line 78 mice we have shown that the palindrome rearranges at the impressive rate of about 0.5% per population doubling. The rearrangements observed to arise from rapid palindrome modification are consistent with a center-break mechanism where double-strand breaks, created through hairpin nicking of an extruded cruciform, are imprecisely rejoined, thus introducing deletions at the palindrome center. Significantly, palindrome rearrangements in somatic tissue culture cells almost completely mirrored the structures generated in vivo in the mouse germ line. The close correspondence between germ line and somatic events indicates the possibility that center-break modification of palindromes is an important mechanism for preventing mutation in both contexts. Permanent cell lines carrying a verified palindrome provide an essential tool for future mechanistic analyses into the consequences of palindromy in the mammalian genome.

International audience; DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

Estimates put the origin of V(D)J recombination at ∼450 million years ago (for reviews, see references 1, 2). It has been speculated that it all started with a chance occurrence, the integration of a mobile element into a gene encoding an Ig domain ([3][1]). The acquisition of the V(D)J

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.

Higher eukaryotes are proficient at remodeling palindromic DNA. As shown here, a fully palindromic 15.4 kb circular DNA can be introduced into rodent cells with the novel result that the molecule is reproducibly and site-specifically converted to a monomeric circle. The dimer-to-monomer conversion has not been described previously, and in particular is undetectable in Escherichia coli. Comparative DNA sequence analyses of the new junctions found within the monomer circles suggest that the resolution process involves formation of hairpin DNA structures followed by the introduction of single-strand nicks near their termini. By extension, hairpin nicking combined with non-homologous end-joining may be important as a general mechanism for the main-tenance of genomic stability in mammalian cells. It is suggested that the absence of a comparable strategy for coping with palindrome-induced structure in E.coli and other unicellular organisms underlies a fundamental difference in DNA sequence organiz-ation in the genomes of prokaryotes versus higher eukaryotes.

Unusual DNA structures promote genetic instability. One such example is hairpin DNA, which can form from palindromic sequences and triplet repeats, and is also a characteristic intermediate in V(D)J recombination. We previously found that a large 15.3-kb palindrome that was introduced as a transgene into the mouse germline was highly unstable. Although it could be transmitted, the transgene was found to be rearranged in up to 56% of the progeny, and rearrangement events often involved deletion at the center of symmetry. Here, the fine structure of centrally deleted palindromes was sampled by analysis of recombinant junctions isolated from testes DNA, providing further evidence for a model, previously proposed, that accounts for such deletions on the basis of a hairpin-tip nicking activity. In addition to central deletions, gene conversion events were also elevated in the transgenic palindrome. We have now analyzed instability in two mouse sublines in which (as a result of inversion) the transgenic palindrome had been shortened to 4.2 kb. In these sublines, the transgene was still subject to both rearrangement and gene conversion events at a high frequency, similar to the original 15.3-kb palindrome. Recombination was not limited to the sequences constituting the inverted repeat, but was seen to include sequences lying outside the palindrome. As discussed, the salient feature in all of these observations, a high level of genetic change associated with palindromic DNA, underscores the significance of hairpin DNA and hairpin-tip nicking in genome stability.

V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.

The fine-structures of V(D)J joining products present a cache of information about the recombination process. Testable hypotheses with predictive value are beginning to provide a surprisingly detailed picture of the joining operation. Here, one feature of V(D)J junctions, the appearance of short palindromic inserts, is discussed, and experimental evidence in support of a joining model involving a hairpin-terminated DNA intermediate will be summarized.

SummaryWhereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

Wolf–Hirschhorn syndrome (WHS) is a malformation syndrome associated with growth retardation, mental retardation, and immunodeficiency resulting from a hemizygous deletion of the short arm of chromosome 4, called the WHS critical region (WHSC). The WHSC1 gene is located in this region, and its loss is believed to be responsible for a number of WHS characteristics. We identified WHSC1 in a genetic screen for genes involved in responding to replication stress, linking Wolf–Hirschhorn syndrome to the DNA damage response (DDR). Here, we report that the WHSC1 protein is a member of the DDR pathway. WHSC1 localizes to sites of DNA damage and replication stress and is required for resistance to many DNA-damaging and replication stress-inducing agents. Through its SET domain, WHSC1 regulates the methylation status of the histone H4 K20 residue and is required for the recruitment of 53BP1 to sites of DNA damage. We propose that Wolf–Hirschhorn syndrome results from a defect in the DDR.

Recurrent chromosomal abnormalities, especially chromosomal translocations, are strongly associated with certain subtypes of leukemia, lymphoma and solid tumors. The appearance of particular translocations or associated genomic alterations can be important indicators of disease prognosis, and in some cases, certain translocations may indicate appropriate therapy protocols. To date, most of our knowledge about chromosomal translocations has derived from characterization of the highly selected recurrent translocations found in certain cancers. Until recently, mechanisms that promote or suppress chromosomal translocations, in particular, those responsible for their initiation, have not been addressed. For translocations to occur, two distinct chromosomal loci must be broken, brought together (synapsed) and joined. Here, we discuss recent findings on processes and pathways that influence the initiation of chromosomal translocations, including the generation fo DNA double strand breaks (DSBs) by general factors or in the context of the Lymphocyte-specific V(D)J and IgH class-switch recombination processes. We also discuss the role of spatial proximity of DSBs in the interphase nucleus with respect to how DSBs on different chromosomes are justaposed for joining. In addition, we discuss the DNA DSB response and its role in recognizing and tethering chromosomal DSBs to prevent translocations, as well as potential roles of the classical and alternative DSB end-joining pathways in suppressing or promoting translocations. Finally, we discuss the potential roles of long range regulatory elements, such as the 3'IgH enhancer complex, in promoting the expression of certain translocations that are frequent in lymphomas and, thereby, contributing to their frequent appearance in tumors.

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.

Adeno-associated virus (AAV) is a small nonpathogenic, replication-defective DNA virus with a single-stranded DNA genome. Recombinant AAV (rAAV) is among the most promising gene delivery vectors for human gene therapy (15, 28). rAAV lacks machinery for virus genome insertion into host chromosomal DNA but does integrate into chromosomes at a low frequency (reviewed in reference 29). Murine leukemia virus preferentially integrates near transcription start sites (59), while human immunodeficiency virus favors transcribed regions for integration (49). As for rAAV, previous studies have shown that rAAV serotype 2 (rAAV2) vector integration occurs preferentially in genes and near gene regulatory sequences in mouse liver (36, 40) and in cultured human cells (32). A recent observation by Miller et al. has suggested that rAAV does not cause chromosomal breaks but integrates at preexisting chromosomal breakage sites (30). Although rAAV vector integration frequency is considered to be low, it is important to further understand the interactions between rAAV vector and host chromosomal DNA in various tissues in experimental animals. This is because a study has demonstrated increased incidence of liver cancer in rAAV2 vector-treated animals (5), the mechanisms for which remain elusive. In addition, the use of new serotype vectors with robust transduction efficiency, such as rAAV8, can increase vector genome loads in cells, which may pose an increased risk of undesirable genomic alterations in rAAV-transduced cells. Such robust serotype vectors now are widely used for many preclinical studies for gene therapy of various human diseases. Moreover, we have hypothesized that elucidation of the mechanisms of rAAV integration may help in understanding ongoing genomic instability in living animals, which is difficult to investigate with currently available strategies but is important for studies on carcinogenesis and aging. Furthermore, it has been shown that rAAV vectors serve as a powerful tool to study the mechanisms of fundamental biological processes in cells, such as DNA damage responses and DNA repair (11). To begin to further understand rAAV integration in various tissues of experimental animals, it is essential to establish a system by which rAAV integration sites can be identified on a large scale in nondividing cells with high efficiency and reliability without any cell manipulations. This minimizes possible technical biases and enables identification of rAAV integrations in nonhepatic tissues, for which selection is not easy to perform. However, currently available methods for high-throughput rAAV integration site analysis all rely on cell division either under a selective pressure (40) or without selection (32). Cell division is required for diluting out extrachromosomal rAAV vector genomes with high complexity, which are abundantly present in cells in a quiescent state and inhibit efficient isolation of rAAV integrants. In the present study, we have established a novel high-throughput method to identify rAAV integration sites in nondividing cells with high efficiency and high reliability independently of cell division, transgene expression, or selection. We have identified a thousand rAAV integration sites in quiescent somatic cells in mouse liver, skeletal muscle, and heart, and we discovered that DNA palindromes are a common target for rAAV integration. DNA palindromes are found prevalently in many organisms, including mammals (24, 58; S. M. Lewis, T. Zheng, S. Chen, T. Alleyne, J. Cheung, T. Chiang, and R. Richard, unpublished data). They have gained attention recently due to accumulating evidence that they have roles in promoting genomic instability in eukaryotes. This DNA motif has been shown to be involved in gene amplification (2, 7, 43, 46, 52-54, 60), nonrandom chromosomal translocations causing human diseases (6, 9, 12, 16-20, 55), genomic instability in animal models (1, 3, 4, 22, 23), retrovirus integration (14), and RAG protein-mediated transposition (45). Thus, the discovery that rAAV integrates preferentially at DNA palindromes not only provides further insights into the mechanisms of rAAV integration but also provides an unprecedented opportunity to study the biological impact and properties of naturally occurring DNA palindromes in tissues of living animals.

The nature of any long palindrome that might exist in the human genome is obscured by the instability of such sequences once cloned in Escherichia coli. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. With this approach we have investigated an intronic sequence in the human Neurofibromatosis 1 (NF1) locus that is represented by multiple conflicting versions in GenBank. We find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. A side-by-side comparison of the same plasmids in E.coli versus. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. The high quality sequence obtained from the yeast-based approach provides insight into the various mechanisms that destabilize a palindrome in E.coli, yeast and humans, into the diversification of a highly polymorphic site within the NF1 locus during primate evolution, and into the association between palindromy and chromosomal translocation.

The Ig and T cell receptor (TCR) loci have an exceptionally dynamic evolutionary history, but the mechanisms responsible remain a subject of speculation. Ig and TCR genes are unique in vertebrates in that they are assembled from V, D, and J segments by site-specific recombination in developing lymphocytes. Here we examine the extent to which the V(D)J recombination in germline cells may have been responsible for remodeling Ig and TCR loci in mammals by asking whether gene segments have evolved as a unit, or whether, instead, recombination signal sequences (RSSs) and coding sequences have different phylogenies. Four distinct types of RSS have been defined in the human Ig heavy-chain variable region (Vh) locus, namely H1, H2, H3, and H5, and no other RSS type has been detected in other mammalian species. There is a well-supported discrepancy between the evolutionary history of the RSSs as compared with the Vhcoding sequences: the RSS type H2 of one Vhgene segment has clearly become replaced by a RSS type H3 during mammalian evolution, between 115 and 65 million years ago. Two general models might explain the RSS swap: the first involves an unequal crossing over, and the second implicates germline activation of V(D)J recombination. The Vh-H2/RSS-H3 recombination product has likely been selected during the evolution of mammals because it provides better V(D)J recombination efficiency.

Unintended DNA rearrangements in a differentiating lymphocyte can have severe, oncogenic consequences, but the mechanisms for avoiding pathogenic outcomes in V(D)J recombination are not well understood. The first level at which fidelity is instituted is in discrimination by the recombination proteins between authentic and inauthentic recombination signal sequences. Nevertheless, this discrimination is not absolute and cannot fully eliminate targeting errors. To learn more about the basis of specificity during V(D)J recombination, we have investigated whether it is possible for the recombination machinery to detect an inaccurately targeted sequence subsequent to cleavage. These studies indicate that even postcleavage steps in V(D)J recombination are sequence specific and that noncanonical sequences will not efficiently support the resolution of recombination intermediates in vivo. Accordingly, interventions after a mistargeting event conceivably occur at a late stage in the joining process and the likelihood may well be crucial to enforcing fidelity during antigen receptor gene rearrangement.

To study molecular events involved in B lymphocyte development and V(D)J rearrangement, we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here, we show that B lineage cells generatedin vitrofrom ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first, they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second, they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third, they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover, the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably, EAB cells possess functional V(D)J recombinase activity. In particular, the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation.

Despite the importance of hairpin opening in antigen receptor gene assembly, the molecular machinery that mediates this reaction has not been defined. Here, we show that RAG1 plus RAG2 can open DNA hairpins. Hairpin opening by RAGs is not sequence specific, but in Mg 2+ , hairpin opening occurs only in the context of a regulated cleavage complex. The chemical mechanism of hairpin opening by RAGs resembles RSS cleavage and 3′ end processing by HIV integrase and Mu transposase in that these reactions can proceed through alcoholysis. Mutations in either RAG1 or RAG2 that interfere with RSS cleavage also interfere with hairpin opening, suggesting that RAGs have a single active site that catalyzes several distinct DNA cleavage reactions.

Genetic instability is promoted by unusual sequence arrangements and DNA structures. Hairpin DNA structures can form from palindromes and from triplet repeats, and they are also intermediates in V(D)J recombination. We have measured the genetic stability of a large palindrome which has the potential to form a one-stranded hairpin or a two-stranded cruciform structure and have analyzed recombinants at the molecular level. A palindrome of 15.3 kb introduced as a transgene was found to be transmitted at a normal Mendelian ratio in mice, in striking contrast to the profound instability of large palindromes in prokaryotic systems. In a significant number of progeny mice, however, the palindromic transgene is rearranged; between 15 and 56% of progeny contain rearrangements. Rearrangements within the palindromic repeat occur both by illegitimate and homologous, reciprocal recombination. Gene conversion within the transgene locus, as quantitated by a novel sperm fluorescence assay, is also elevated. Illegitimate events often take the form of an asymmetric deletion that eliminates the central symmetry of the palindrome. Such asymmetric transgene deletions, including those that maintain one complete half of the palindromic repeat, are stabilized so that they cannot undergo further illegitimate rearrangements, and they also exhibit reduced levels of gene conversion. By contrast, transgene rearrangements that maintain the central symmetry continue to be unstable. Based on the observed events, we propose that one mechanism promoting the instability of the palindrome may involve breaks generated at the hairpin structure by a hairpin-nicking activity, as previously detected in somatic cells. Because mammalian cells are capable of efficiently repairing chromosome breaks through nonhomologous processes, the resealing of such breaks introduces a stabilizing asymmetry at the center of the palindrome. We propose that the ability of mammalian cells to eliminate the perfect symmetry in a palindromic sequence may be an important DNA repair pathway, with implications regarding the metabolism of palindromic repeats, the mutability of quasipalindromic triplet repeats, and the early steps in gene amplification events.

Two lines of evidence point to a hairpin DNA intermediate in V(D)J joining (V, variable; D, diversity; J, joining) [Lieber, M.R. (1991) FASEB J. 4, 2934-2944]. One is the presence of P nucleotide insertions (short inverted-repeat sequence) in V(D)J junctions [Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y. & Tonegawa, S. (1989) Cell 59, 859-870]; a second is the detection of site-specifically broken DNA molecules with covalently closed (hairpin) termini in thymus DNA [Roth, D. B., Menetski, J. P., Nakajima, P., Bosma, M. J. & Gellert, M. (1989) Cell 70, 983-991]. However, P nucleotide insertions could be generated in ways not involving a hairpin structure, and because physical evidence for hairpin-ended DNA fragments has been obtained only with mutant mice, there is some uncertainty regarding the role of hairpin molecules in the normal V(D)J joining pathway. To determine whether mammalian cells are capable of metabolizing this odd type of DNA terminus and whether, in doing so, junctions with P insertions are in fact created, a linear DNA molecule with a hairpin closure at each end was transfected into several murine cell lines. The hairpin-ended molecules were recircularized, and the junctions exhibited P insertions at a high frequency. This result directly links the presence of P insertions to a hairpin precursor, providing strong evidence for the notion that a hairpin DNA intermediate exists in V(D)J recombination. A comparison of hairpin end joining in various cells, including those derived from mice with the severe combined immunodeficiency (scid) mutation, is presented.

Publisher Summary 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.

Antigen receptor genes acquire junctional inserts upon assembly from their component, germ line-encoded V, D, and J segments. Inserts are generally of random sequence, but a small number of V-D, D-J, or V-J junctions are exceptional. In such junctions, one or two added base pairs inversely repeat the sequence of the abutting germ line DNA. (For example, a gene segment ending AG might acquire an insert beginning with the residues CT upon joining). It has been proposed that the nonrandom residues, termed "P nucleotides," are a consequence of an obligatory end-modification step in V(D)J recombination. P insertion in normal, unselected V(D)J joining products, however, has not been rigorously established. Here, we use an experimentally manipulable system, isolated from immune selection of any kind, to examine the fine structure of V(D)J junctions formed in wild-type lymphoid cells. Our results, according to statistical tests, show the following, (i) The frequency of P insertion is influenced by the DNA sequence of the joined ends. (ii) P inserts may be longer than two residues in length. (iii) P inserts are associated with coding ends only. Additionally, a systematic survey of published P nucleotide data shows no evidence for variation in P insertion as a function of genetic locus and ontogeny. Together, these analyses establish the generality of the P nucleotide pattern within inserts but do not fully support previous conjectures as to their origin and centrality in the joining reaction.

We describe novel products of V(D)J recombination in which signal sequences become joined to coding elements, in contrast to the standard reaction whose products are junctions of two signal sequences or two coding elements. In this variant reaction, the recombination machinery evidently recognizes signal sequences and introduces strand breaks at the normal positions, but then connects the elements in unusual combinations. The lack of fixed directionality indicates that recombination sites are not uniquely aligned when strand exchange occurs. The discovery of these variant junctions suggests a model for the evolution of the antigen receptor loci.

A cell line transformed by Abelson murine leukemia virus, called PD, is capable of carrying out kappa-gene rearrangement while growing in culture. Subclones of PD have diverse kappa-gene structures, and some derivatives show evidence of continued joining activity after as many as three subclonings. Analysis of PD sublineages has shown that a rearranged chromosome can undergo secondary kappa-gene rearrangements, producing either a new rearrangement or a deletion of C kappa. Although the PD line actively rearranges its kappa genes, its rearranged heavy-chain genes show little variation, and there is no rearrangement of lambda genes. In PD subclones, DNA fragments representing the reciprocal product of kappa-gene rearrangement are often evident, and they may undergo either further rearrangement or deletion. The implications of multiple rearrangements on a single chromosome and of the maintenance of reciprocal fragments are considered in the context of a model that postulates that the V kappa and J kappa segments are not all organized in the DNA in the same transcriptional direction, leading to inversions rather than deletions during joining.

The structure of immunoglobulin-related gene was analyzed in individual Abelson murine leukemia virus (A-MuLV)-transformed lymphoid cell lines. Essentially all of these lines, whether immunoglobulin-containing or null, had DNA rearrangements in the vicinity of the JH regions on both chromosomes as well as deletions of at least 5 kb of DNA 5' to JH. None of these lines, however, except rare light chain producers, had detectable rearrangement at either their kappa or lambda light chain loci. In contrast to A-MuLV-transformants derived from bone marrow. Those from early fetal liver frequently contained more than two and sometimes 12 or more distinct, rearranged JH-containing fragments. Cellular subclones derived from these lines had a subset, usually two, of the fragments found in the parent line. Therefore, heavy chain gene rearrangement appears to precede that of light chain gene rearrangement and is still continuing in certain cultured A-MuLV transformants.

Immunoglobulin K genes are constructed during lymphocyte differentiation by the joining of two DNA elements, VK and JK, to form both a VKJK coding unit and a reciprocal recombination product. The two products formed in single VK-to-JK joining events can be directly isolated through the use of a retrovirally introduced recombination substrate. The structural analysis of a number of recombinants and the derivation of secondary recombination products define some of the basic features of the mechanism of immunoglobulin gene assembly.

VK to JK recombination occurred within a DNA construct introduced into cells in the form of a defective retrovirus. Analysis of one recombinant demonstrated that VK–JK joining can occur via inversion with a net loss of a few base pairs.

Two aerobic, gramnegative, red-pigmented, rod-shaped bacteria were compared morphologically and physiologically with Serratia species, which they resembled superficially. The pigment produced by the marine isolates was shown to be similar to prodigiosin, the red pigment of S. marcescens . The isolates had a single polar flagellum, were oxidative, and did not produce acetoin from glucose or reduce nitrates, which made them distinct from both S. marcescens and S. marinorubra . The latter conformed well to the descriptions of S. marcescens in Bergey's Manual . The marine isolates displayed an absolute growth requirement for sea water or its equivalent. The growth requirement for sea water was replaced by sea-water levels of sodium, potassium, and magnesium chloride. Pigment was produced only when this salt mixture was further supplemented with calcium chloride. Neither sea water nor a high salt level was required for growth or prodigiosin synthesis by the Serratia species examined.

SummaryGenome-wide analysis of thymic lymphomas from Tp53−/− mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification.

It may be significant that the 12/23 rule distinguishes V(D)J recombination from both TPN and CSSR. The crucial and integral nature of the 12/23 rule is underscored by several observations. One is that the rearranging loci analyzed in modern examples of ancient vertebrate radiations always contain both versions of the V(D)J joining signal (reviewed inLewis 1994xLewis, S.M. Adv. Immunol. 1994; 56: 27–150Crossref | PubMedSee all ReferencesLewis 1994). Another is that during in vivo recombination, the 12/23 rule is enforced early, at the cleavage step (Steen et al. 1996xSteen, S.B, Gomelsky, L, and Roth, D.B. Genes to Cells. 1996; 1: 543–553Crossref | PubMedSee all ReferencesSteen et al. 1996). This constraint has been reconstructed in vitro with purified RAG1 and 2 alone (van Gent et al. 1996bxvan Gent, D.C, Ramsden, D.A, and Gellert, M. Cell. 1996; 85: 107–113Abstract | Full Text | Full Text PDF | PubMed | Scopus (225)See all Referencesvan Gent et al. 1996b).Why don't any of the analyzed CSSR or transposition systems have a 12/23 rule, or its equivalent? A teleological view is that the 12/23 rule is found in V(D)J recombination because V(D)J recombination is required to accurately manipulate hundreds, not just two or three, potential recombination sites. The 12/23 rule has the appearance, at least, of a particularly economical solution to the problem of avoiding unprofitable combinations in the case of a recombination system that is confronted with multi-site arrays.Perhaps at this juncture, a hunt for origins would best be served by looking among multi-site recombination systems for one that more clearly shares basic attributes with V(D)J recombination. Conceivably such a system could be transpositional, where a transposase is required to handle numerous recombination targets created by high-copy transposon integration. Alternatively, relatives of known CSSR systems (as for example Min; Figure 3Figure 3; Iida et al. 1990xIida, S, Sandmeier, H, Huber, P, Giestand-Nauer, R, Schneitz, K, and Arber, W. Mol. Microbiol. 1990; 4: 991–997Crossref | PubMed | Scopus (11)See all ReferencesIida et al. 1990) that serve to rearrange a multi-site array might bear closer scrutiny. There is also the possibility that further afield, recombination systems that enable RNA to wander, “integrons” to move, or DNA to exit en masse from a genome may provide key insights (see, for example,5xHall, R.M and Collis, C.M. Mol. Microbiol. 1995; 15: 593–600Crossref | PubMed | Scopus (474)See all References, 9xKlobutcher, L.A and Herrick, G. Nucl. Acids Res. 1995; 23: 2006–2013Crossref | PubMed | Scopus (80)See all References, 13xPyle, A.M. Nature. 1996; 381: 280–281Crossref | PubMed | Scopus (7)See all References). In the meantime, there is no question that the comparative molecular analyses carried out to date have been extremely informative (2xDifilippantonio, M.J, McMahan, C.J, Eastman, Q.M, Spanopoulou, E, and Schatz, D.G. Cell. 1996; 87: 253–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (165)See all References, 16xSpanopoulou, E, Zaitseva, F, Wang, F.-H, Santagata, S, Baltimore, D, and Panaoytou, G. Cell. 1996; 87: 263–276Abstract | Full Text | Full Text PDF | PubMed | Scopus (189)See all References, 19xvan Gent, D.C, Mizuuchi, D, and Gellert, M. Science. 1996; 271: 1592–1594Crossref | PubMedSee all References). At the present pace, it would not be surprising to see the V(D)J recombination system soon begin to rival the more classical site- directed recombination systems both in terms of the rich detail with which it is understood, and as a paradigm for revealing fundamental features of protein–DNA interactions.

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

Division of Biology California Institute of Technology Pasadena, California 91125 f Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 Much of the variety in binding specificities of the immune system is generated when subgenic DNA elements are assembled into antigen receptor genes during T and B cell differentiation. This process has attracted interest both because of its important role in the differentiation of the immune system and because it is the only site-specific recombination system known to operate in vertebrates. The natural substrates for immunoglobulin and T cell receptor gene rearrangement are complex loci (seven have been described in mice) composed of multiple cop- ies of V, D in some cases, and J segments. A complete an- tigen receptor gene is created by fusion of one V to one J, or one V to one D to one J. The basic operation (which we call V-J joining whether it involves joining V to J, V to D, or D to J), is represented in the left column of the figure. Two coding segments (shown as boxes) are detached from their joining signal sequences (shown as triangles) and then attached to one another to form a contiguous coding sequence. The junction is called a coding joint. A recipro- cal fusion of the signal sequences leads to a signal joint. (General principles are reviewed in Tonegawa, 1983.) The details of this process can be probed using recom- bination substrates that are constructed to contain a de- fined pair (or pairs) of gene segments. These substrates are designed either to become integrated into the genome of the recipient cells or to remain extrachromosomal. In ei- ther case, when introduced into recombinationally active cell lines, the gene segments are targeted by the recombi- nation machinery and become rearranged in the same fashion as their endogenous counterparts. The ease of experimentation afforded by this methodology has led to a rapid increase in available information about V-J recom- bination. Here we will review some of the key features of the process and present a current model of how it may work. One clearcut result is that the joining signals alone are the necessary and sufficient targets for recombination. Joining signals are simple DNA sequence motifs consist- ing of conserved heptamer and nonamer sequences separated by a nonconserved spacer of 12 or 23 bp. With- out joining signals, no recombination occurs. On the other hand, if V, D, or J coding elements are replaced by other DNA, the new sequences abutting the joining signals are still recombined site specifically (Lewis et al., 1985; Akira et al., 1987; Hesse et al., 1987). Thus, of the four elements

We have studied the VDJ recombination reaction in early B and T cells from mice homozygous for the severe combined immune deficiency (scid) mutation. Schuler et al. (1986) previously found abnormal deletions by Southern blot analysis at rearranged IgH and TCR β alleles in transformed B and T lymphocytes from seid mice. We have recently completed a study (Lieber et al. 1988a) in which we transfected scid lymphoid cell lines with VDJ recombination substrates that remain extrachromosomal. These substrates allow large numbers of recombinant reaction products to be collected (Hesse et al. 1987; Lieber et al. 1987), thereby permitting detailed analysis of the VDJ recombination reaction in scid lymphoid cells.