Nonhomologous end joining and V(D)J recombination require an additional factor.

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T(-)B(-) severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue in Saccharomyces cerevisiae functions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.

Cellular and biochemical impact of a mutation in DNA ligase IV conferring clinical radiosensitivity.

DNA ligase IV functions in DNA non-homologous end-joining, in V(D)J recombination, and during brain development. We previously reported a homozygous mutation (R278H) in DNA ligase IV in a developmentally normal leukemia patient who overresponded to radiotherapy. The impact of this hypomorphic mutation has been evaluated using cellular, biochemical, and structural approaches. Structural modeling using T7 DNA ligase predicts that the activity and conformational stability of the protein is likely to be impaired. We show that wild type DNA ligase IV-Xrcc4 is an efficient double-stranded ligase with distinct optimal requirements for adenylate complex formation versus rejoining. The mutation impairs the formation of an adenylate complex as well as reducing the rejoining activity. Additionally, it imparts temperature-sensitive activity to the protein consistent with the predictions of the structural modeling. At the cellular level, the mutation confers a unique V(D)J recombination phenotype affecting the fidelity of signal joint formation with little effect on the frequency of the reaction. These findings suggest that hypomorphic mutations in ligase IV may allow normal development but confer marked radiosensitivity.

DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.

Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA.

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.

V(D)J recombination: site-specific cleavage and repair.

V(D)J recombination is a site-specific gene rearrangement process that contributes to the diversity of antigen receptor repertoires. Two lymphoid-specific proteins, RAG1 and RAG2, initiate this process at two recombination signal sequences. Due to the recent development of an in vitro assay for V(D)J cleavage, the mechanism of cleavage has been elucidated clearly. The RAG complex recognizes a recombination signal sequence, makes a nick at the border between signal and coding sequence, and carries out a transesterification reaction, resulting in the production of a hairpin structure at the coding sequence and DNA double-strand breaks at the signal ends. RAG1 possesses the active site of the V(D)J recombinase although RAG2 is essential for signal binding and cleavage. After DNA cleavage by the RAG complex, the broken DNA ends are rejoined by the coordinated action of DNA double-strand break repair proteins as well as the RAG complex. The junctional variability resulting from imprecise joining of the coding sequences contributes additional diversity to the antigen receptors.

Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase.

The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking and hairpin formation) is severely impaired. Because both chemical steps are affected by the same mutations, a single active site appears responsible for both reactions. Two independent lines of evidence demonstrate that at least two of these acidic residues are directly involved in coordinating a divalent metal ion: The substitution of Cys for Asp allows rescue of some catalytic function, whereas an alanine substitution is no longer subject to iron-induced hydroxyl radical cleavage. Our results support a model in which the RAG1 protein contains the active site of the V(D)J recombinase and are interpreted in light of predictions about the structure of RAG1.

V(D)J recombination: on the cutting edge.

The vertebrate immune system has evolved an elegant mechanism for generating an enormous diversity in antigen receptor binding specificity from a limited amount of genetic information. Recent advances are rapidly increasing our understanding of this unusual site-specific DNA rearrangement that assembles the antigen receptor genes during lymphoid development.

Roles of the "dispensable" portions of RAG-1 and RAG-2 in V(D)J recombination.

V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispensable" regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.

Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly.

Immunoglobulin genes are assembled during lymphoid development by a series of site-specific rearrangements that are tightly regulated to ensure that functional antibodies are generated in B (but not T) cells and that a unique receptor is present on each cell. Because a common V(D)J recombinase comprising RAG1 and RAG2 proteins is used for both B- and T-cell antigen receptor assembly, lineage-specific rearrangement must be modulated through differential access to sites of recombination. We show here that the C-terminus of the RAG2 protein, although dispensable for the basic recombination reaction and for Ig heavy chain DH to JH joining, is essential for efficient VH to DJH rearrangement at the IgH locus. Thus, the RAG2 protein plays a dual role in V(D)J recombination, acting both in catalysis of the reaction and in governing access to particular loci.

Distinct roles of RAG1 and RAG2 in binding the V(D)J recombination signal sequences.

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

Functional analysis of coordinated cleavage in V(D)J recombination.

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

Accessibility of nucleosomal DNA to V(D)J cleavage is modulated by RSS positioning and HMG1.

B and T cell receptor gene assembly by V(D)J recombination is tightly regulated during lymphoid development. The mechanisms involved in this regulation are poorly understood. Here we show that nucleosomal DNA is refractory to V(D)J cleavage. However, the presence of HMG1, a chromatin-associated nonhistone DNA-binding protein, stimulates V(D)J cleavage of nucleosomal templates. This HMG1 stimulation is differentially affected by the rotational or translational positioning of the recombination signal sequence on the histone octamer, with cleavage of the 12 bp spacer RSS showing sensitivity to rotational position and the 23 bp spacer RSS affected by its displacement from the dyad. These results suggest that V(D)J recombination can be modulated by controlling substrate accessibility and cleavage at the level of an individual nucleosome.

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

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

Regions of RAG1 protein critical for V(D)J recombination.

The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutation for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conversed sequence. We show here that this "dispensable" region of RAG1 is not necessary for coding joint formation or recombination of an integrated substrate, and this portion is not functionally redundant with the "dispensable" region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.

Cutting apart V(D)J recombination.

The past year has seen major advances in our understanding of the recombination mechanism by which antibody and T cell receptor genes are assembled during lymphoid development. The initial cleavage events can be carried out in vitro by purified RAG1 and RAG/ protein. In addition, a number of genes involved in later steps of the reaction have been cloned, opening the way for an in-depth biochemical analysis of this critical developmental process.

Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps.

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.

DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect.

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.

Rch1, a protein that specifically interacts with the RAG-1 recombination-activating protein.

RAG1 and RAG2 are lymphoid-specific genes that together induce V(D)J recombinase activity in a variety of nonlymphoid cell types. While no other lymphoid-specific factors are required to induce recombination, other factors with more widespread expression patterns have been implicated in the reaction. However, none of these factors have been cloned, and their relationship to the RAG proteins is unclear. Using the yeast two-hybrid assay, we have identified RCH1, a gene encoding a protein of molecular weight 58,000 that interacts specifically with RAG-1. The predicted Rch1 protein sequence is 47% identical to yeast SRP1, a protein associated with the nuclear envelope. A truncated form of Rch1, which retains the ability to interact with RAG-1, reduces V(D)J recombination activity in HeLa cells.

Analysis of regions of RAG-2 important for V(D)J recombination.

The recombinase activating genes RAG-1 and RAG-2 operate together to activate V(D)J recombination, and thus play an essential role in the generation of immune system diversity. As a first step in understanding the function of the RAG-2 protein, we have tested a series of deletion and insertion mutations for their ability to induce V(D)J joining of a variety of model substrates. Mutants were assayed for their ability to induce deletional and inversional V(D)J joining, thereby testing their proficiency at forming both signal and coding joints, and, in some cases, for their ability to carry out recombination of both extrachromosomal and integrated recombination substrates. All these reactions were affected similarly by any one mutation. Although the RAG-2 protein shows extensive evolutionary conservation across its length, we found that the carboxy-terminal portion of RAG-2, including an acidic region, is dispensable for all forms of recombination tested. In contrast, all mutations we created in the N-terminal region severely decreased recombination. Thus, the core active region required for V(D)J recombination is confined to the first three-quarters of the RAG-2 protein.

Activation of V(D)J recombination by RAG1 and RAG2.

V(D)J recombination is normally limited to lymphoid cells, but expression of the RAG1 and RAG2 genes allows other cell types to carry out this reaction. The products of these recently discovered genes may form part of the recombination machinery, and are a focal point for lymphoid development.