The murine V_H region extends over about 2.5 megabases on chromosome 12 and includes roughly 100 functional segments (depending on mouse strain) plus additional V_H pseudogene segments (Table 6.1). All V_H elements are in the same transcriptional orientation as the D, J_H, and C_H regions.³ The V_H segments are classified into 16 distinct families based on sequence similarity; V_H elements within a family show more than 80% nucleotide sequence identity. Elements of individual V_H families are largely clustered together, though some interdigitation occurs (Fig. 6.4). The V_H families can be grouped into three “clans” based on sequence conservation primarily of their framework regions (framework region 1, codons 6 to 24, and framework region 3, codons 67 to 85, respectively), which form the more conserved structural backbone of the Ig variable region. Importantly, these clans are conserved between man, mouse, and frog, suggesting that their emergence in the repertoire preceded the amphibian-reptile divergence.⁴

About 50 kb downstream of the most 3′ V_H element resides the murine D cluster spanning about 80 kb, depending on
the mouse strain (see Table 6.1). Each D segment is flanked by 12 RSSs on both sides, so that the 12/23 rule ensures that all assembled V genes carry a D element between their V and J segments (which are both flanked by 23 RSSs that prevent direct V to J rearrangements).

The murine D elements are classified into four families: DSP2, DFL16, DST4, and DQ52. Although D regions could theoretically contribute to Ig diversity by being read in all three frames, the mouse has evolved mechanisms that strongly favor one of them.⁵ Four functional germline J_H sequences reside about 0.7 kb downstream of the most 3′ D region, DQ52.

The human V_H locus spans 1.1 Mb at the telomeric end of chromosome 14 (14q32.33) (see Table 6.1). The human germline V_H segments—numbering roughly 40 to 45—fall into seven families that, in contrast to the family clusters characteristic of the murine locus, are extensively interdigitated (see Fig. 6.4). Some human V_H sequences are polymorphic owing to V_H insertions or deletions in different allelic chromosomes. Twenty-four additional germline V_H sequences have been mapped to chromosome 15 and 16 and represent nonfunctional “orphans” that were apparently duplicated from the IgH locus on chromosome 14.⁶

Twenty-six human D elements are located in an ˜40 kb region about 20 kb downstream of VH6, the most 3′ of the V_H genes.⁷ This D cluster is comprised of four tandem duplications of a 9.5 kb segment containing a representative of each of six D families. The twenty-seventh D element—DHQ52— is the only one showing sequence similarity to a mouse segment (DQ52) and shares a homologous location just 5′ to J_H1. In contrast to mice, humans use all reading frames of D elements.⁷ One reading frame encodes primarily hydrophilic residues, one encodes hydrophobic residues, and one includes frequent stop codons. Some D elements contain stop codons that can be removed by nuclease trimming during VDJ assembly. As in mice, the human J_H cluster is immediately downstream of DHQ52.

Each B-lymphocyte initially produces IgM by expressing an assembled variable region linked to Cµ, but may use CSR (discussed later in this chapter) to replace Cµ with one of the several C_H regions lying downstream, thereby allowing expression of IgG, IgA, or IgE (Fig. 6.5A). Eight murine C_H genes span about 200 kb of DNA on chromosome 12; these genes were linked by contiguous clones in 1982.⁸ Several γ pseudogenes lie within the clustered γ functional genes⁹ (Fig. 6.5B). The coding sequences of all C_H genes are oriented in the same direction.

The human C_H genes were similarly cloned, and then eventually completely linked by the Human Genome Project. The human IgH locus contains a large duplication, with two copies of a γ-γ-ε-α unit separated by a γ pseudogene (see Fig. 6.5B). One of the duplicated ε sequences is also a pseudogene, and a third closely homologous ε-related sequence—a “processed” pseudogene—is present on chromosome 9.

The IgH locus has also been examined in several other species besides mouse and human, and several notable differences have been observed. Rabbits, for example, have 13 Cα sequences and only a single Cγ gene¹⁰; this unusual expansion of genes contributing to mucosal immunity may be related to the peculiar habit of coprophagy in these animals. In contrast to the multiplicity of rabbit Cα genes, pigs have only one Cα gene and eight Cγ genes. Camels are unusual in having H chains that function in the absence of L chains.¹¹

Igs are found either as secreted molecules in the serum or as membrane-bound receptors. The membrane-bound µ chains contain a C-terminal hydrophobic transmembrane domain consisting of 26 uncharged hydrophobic amino acids encoded by additional membrane exons, and these residues anchor the protein in the cell membrane lipid bilayer. The membrane (µ_m) and secreted (µ_s) forms are derived from the same gene by alternative splicing (Fig. 6.6). The same general gene structure has been found for other C_H genes, suggesting that differential splicing accounts for the two forms of all Ig isotypes.

Early B cells make roughly similar quantities of both µm and µ_s, whereas maturation to the plasma cell stage is associated with strong predominance of µ_s production, facilitating high-level secretion of circulating Ig. The balance between the two ribonucleic acid (RNA) splice forms of µ has been interpreted as a competition between splicing of the CH4 and M1 exons versus the cleavage/polyadenylation at the upstream µ_s poly(A) site. These processes are mutually exclusive because CH4-M1 splice removes the µ_s poly(A) site, while cleavage at the µ_s poly(A) site removes the membrane exons.

Cis-regulatory elements (and corresponding transacting RNA binding proteins) control the balance between these
processes. They include a GU-rich element downstream of the µs poly(A) site,¹² the polyadenylation factor cleavage stimulator factor 64,¹³ and the U1A protein.¹⁴ These factors likely function downstream of B-lymphocyte-induced maturation protein-1 (BLIMP-1) whose expression in Ig-secreting plasma cells was also found to be critical for µ_s poly(A) site utilization.¹⁵ Cis-acting sequences affecting the ratio of alternative splice forms have been described for other isotypes besides Cµ, particularly Cα.¹⁶

Membrane Ig serves as the antigen-recognition component of the BCR that is critical for initiating the signal for lymphocyte activation following contact with antigen. Transduction is mediated by an associated protein dimer composed of the BCR components Igα and Igβ (CD79a and CD79b) whose cytoplasmic domains contain immunoreceptor tyrosine-based activation motifs similar to those found in the CD3 chains mediating TCR signaling. Additional signaling is mediated by conserved tyrosines in the cytoplasmic tails of the IgG and IgE H chains, which serve as a phosphorylation-dependent docking sites for the signaling adapter Grb2.¹⁷ Binding of Grb2 enhances BCR signaling and subsequent B-cell proliferation.

The murine Vκ locus spans about 3.2 mb on chromosome 6¹⁸ and contains 20 Vκ families, some of which are shared by human and mouse (see Table 6.1). Vκ sequences within a single family are largely clustered together. Some Vκ elements lie in the opposite orientation to that of the Jκ and Cκ elements, and these Vκ segments undergo VJ recombination by an inversion rather than deletion (Fig. 6.7). A few Vκ sequences have been localized to chromosome 16 and 19 and are considered orphan genes.

The human Vκ locus (see Table 6.1) lies on the short arm of chromosome 2 (2p11-2) spanning ˜2 mb of DNA.¹⁹ The locus includes a large inverted duplication, so that most Vκ sequences exist in pairs with one copy lying in the cluster proximal to Jκ (and in the same orientation) and a second copy (inverted) in the distal cluster. The average sequence similarity between duplicates is 98.9%, suggesting the duplication occurred less than 5 million years ago. This is consistent with the absence of such duplication in chimpanzees, which diverged from the human lineage approximately 6 million years ago. Interestingly, about 5% of human alleles also lack the distal duplication.

Outside the Igκ locus, at least 25 orphan Vκ segments have been identified in clusters on chromosome 1, 2, and 22. The orphan cluster located in the long arm of chromosome 2 was probably separated from the major locus—on the short arm of this chromosome—by a pericentric inversion (which must have occurred rather recently in evolution as it is absent from chimpanzee and gorilla).

In comparison to the H chain genes, the organization of the C region segments in the κ locus is relatively simple (see Table 6.1). A single Cκ gene with a single exon and with no reported alternative splice products is found in both mouse and human. While all five Jκ elements are functional in humans, the third J element in mice has not been observed in functional κ L chains.

Apart from the typical Vκ-Jκ rearrangements, an additional recombination event occurs uniquely in the κ locus. The event is mediated by V(D)J recombination utilizing a 23-RSS element—designated Recombining Sequence in the mouse²⁰ and Kappa Deleting Element in the human²¹—that is positioned in an intergenic region downstream of Cκ; the recombination results in the deletion of the Cκ exon. Hence, Cκ fragments are undetectable on Southern blots of DNA from λ-expressing human lymphoid cells,²² as in most B cells the Cκ genes are apparently deleted from both chromosomes before Igλ gene rearrangement begins.

In laboratory mouse strains, only about 5% of the B-lymphocytes utilize Igλ L chain, and the diversity of these L chains is meager due to the very small number of V region genes (Fig. 6.8). Complete sequence analysis²³ of the murine locus revealed two V-J-C clusters (Vλ2-Vλx-Jλ2Cλ2-Jλ4Cλ4 and Vλ1-Jλ3Cλ3-Jλ1Cλ1) separated by about 110 kb. Each Jλ is linked to its own Cλ region gene, but Jλ4 is nonfunctional. Recombination occurs largely within each cluster, although Vλ2Cλ1 products are occasionally observed. The ancestry of the Vλx element is uncertain, as it is rather dissimilar to the other Vλ segments; indeed, it resembles Vκ as much as Vλ. In contrast to the Igκ locus, the Vλ segments are flanked by 23-RSS and the Jλ gene segments by 12-RSS (see Fig 6.3).

The human Vλ region was characterized by intensive cloning, sequencing, and mapping of Vλ elements and ultimately by the complete sequence analysis of 1 Mb covering the entire locus²⁴ (see Table 6.1). Within the Vλ cluster lies the human VpreB gene (discussed below), as well as several genes and pseudogenes unrelated to the Igλ system.

λ L chains are much more abundant in man than in mouse (about 40% of human L chains are λ versus about 5% in mouse). Four forms of human λ chains have been classified serologically, with differences residing in a small number of amino acids in the C region. The serologic classification of Kern+ corresponds to a glycine at position 152 versus a serine in Kern-. The Oz+ designation corresponds to a lysine at position 190 versus an arginine in the Oz- variant. Similarly, Mcg+ λ chains (versus Mcg-) contain asparagine 112 (versus alanine), threonine 114 (versus serine) and lysine 163 (versus threonine).

Four functional human Jλ-Cλ segments and three pseudogenes are clustered within an approximately 33 kb region of DNA (see Fig. 6.8) and the four major expressed human λ isotypes correspond to the functional JCλ1, JCλ2, JCλ3, and
JCλ7, with the latter encoding an isotype provisionally designated Mcp.²⁵ JCλ6 may be functional in some individuals, and the common allele—which has a 4 bp insertion leading to a deletion of the C-terminal third of the Cλ region— can nevertheless undergo Vλ-Jλ recombination, encoding a truncated protein that can associate with H chains. A variety of polymorphic variants of the human λ locus have been detected, apparently the result of gene duplication, as shown in Figure 6.8.²⁶ Lastly, three Cλ-related sequences have been discovered near the major Jλ-Cλ cluster. One of these, designated λ14.1, represents the human homolog of the murine “surrogate” L chain λ5 (see following discussion).

A model for the detailed mechanism of the V(D)J recombination event must account for the observed features of the recombination products and of their germline precursors. In the germline precursors, the RSSs with their heptamer, nonamer, and appropriate 12 or 23 bp spacers are necessary and sufficient to create efficient recombination targets; model substrates in which RSSs flank DNA sequences completely unrelated to Ig genes are competent to undergo recombination. The model shown in Figure 6.9 will serve as a framework for discussion of the recombination mechanism. The recombination is thought to begin with binding of the RAG1-RAG2 complex to the RSSs that flank the two gene segments to be recombined. Simultaneous DNA cleavage occurs precisely between the RSSs and the gene segments. The two ends of the RSSs (frequently named “signal ends”) are joined directly, forming “signal joints.” In contrast, the ends of the gene segments (also referred to as “coding ends”) are processed prior to joining and are ultimately ligated together, giving rise to “coding joints” and completing the recombination event.

In the recombination products, signal joints are typically direct ligation products of the signal ends: the RSSs are joined directly at the heptamers (“back-to-back”), and nucleotide additions or deletions at these junctions are quite rare. The properties of the coding joints, however, are more complex, as the joining reaction at these DNA ends is “imprecise.” The following features are frequently present:

To study broken DNA ends as intermediates in V(D)J recombination, several laboratories employed ligation-mediatedpolymerase chain reaction (LM-PCR) to detect signal ends. This technique involves ligating blunt double-stranded oligonucleotide linkers to blunt genomic DNA breaks, and then amplifying the ligation junctions between a primer in the ligated oligonucleotide and a primer based on known sequence from the ligated genomic DNA; amplification products can
then be cloned and sequenced. LM-PCR analyses of both TCR and Ig genes undergoing V(D)J recombination showed the signal ends to be blunt double strand breaks (dsbs), usually exactly at the heptamer border.³² Similar LM-PCR experiments failed to detect the coding ends unless they were pretreated with mung bean nuclease, a single-strandspecific endonuclease that recognizes the distortion of DNA at a hairpin structure. Sequences of these LM-PCR products from coding ends suggested that the hairpins are precisely at the end of the coding elements, usually without loss or gain of a single nucleotide.³³ By Southern blot analyses, coding ends were found to have two properties suggestive of a hairpin-like structure: 1) resistance to exonuclease treatment, and 2) doubling of the apparent length of restriction fragments under denaturing electrophoresis conditions.³⁴

Hairpin ends represent V(D)J recombination intermediates that, in wild-type cells, are opened at the hairpin tip (or a few nucleotides away from it) by the Artemis nuclease (discussed below). P nucleotides result from opening the loop at an asymmetric position (see Fig. 6.9); this model would explain why P nucleotides are never observed at coding ends that have been “nibbled” after opening of the hairpin. P nucleotide segments in the rare coding joints observed in SCID mice are unusually long and likely result from resolution of hairpins by nicking enzymes that, unlike Artemis, do not focus on the area near the tip of hairpin loops but instead nick in variable positions in the double-stranded hairpin “stem.”³⁴

If a V segment and a J segment are both oriented in the same direction, they can recombine by excising the DNA between the coding sequences and ligating the two coding ends. Ligation of the two signal ends produces a DNA circle that generally lacks replication origins and therefore fails to replicate as cells divide after V(D)J recombination. Such excision circles are therefore generally absent in mature B-lymphocytes that have already undergone several rounds of proliferation after completing the Ig gene assembly. By isolating circular DNA from cells actively undergoing Vκ-Jκ rearrangement, it is possible to isolate and characterize the circular molecules bearing signal joints.³⁵

As mentioned previously, some germline Vκ genes are oriented in the opposite direction from the Jκ-Cκ region. In these cases, VJ recombination occurs by an inversion of the DNA between the recombining V and J segments, leaving both the VκJκ coding joint and the signal joint (formed by ligating the RSSs) retained in the chromosome (see Fig. 6.7). This demonstrates that the enzymatic machinery “sees” only the DNA in the immediate vicinity of the recombination site and is insensitive to the topology of the DNA strands far from this site.

In addition to the canonical coding and signal joints, several “nonstandard” recombination joints have been documented, that, though not contributing to physiologic Ig gene assembly, represent tell-tale signs of a recombination event.³⁶ In the first phase of V(D)J recombination, the DNA is cut at both gene segment-RSS boundaries that participate in the reaction, thereby generating four DNA ends. In principle, there are three possible topologies in which these DNA ends can be rejoined:

“Hybrid” and “open and shut” joints have been observed in transfected plasmids bearing artificial recombination substrates³⁶ as well as in endogenous Ig loci in vivo.³⁷

As discussed previously, imprecise joining of gene segments causes about two-thirds of all recombination products to be out-of-frame. Thus, a B-lymphocyte could end up with nonproductively rearranged Igκ genes on both alleles. However, germline Vκ segments lying upstream of an initial Vκ-Jκ recombination junction can recombine with Jκ segments lying downstream of the junction, producing a “secondary” recombination event, as shown in Figure 6.10A.
Such secondary recombination also occurs in cells that have assembled a productive Vκ-Jκ joint if the encoded antigen binding domain recognizes an autoantigen. This type of secondary recombination, known as “receptor editing,” is considered in more detail later in this chapter.

In the IgH locus, secondary D-J rearrangements sometimes occur, but only until V_H-DJ_H recombination removes all unused upstream D_H segments (Fig. 6.10B). VDJ rearrangement eliminates all the 12-RSSs from the IgH locus that could pair with the 23-RSSs flanking the upstream V_H elements. Sometimes, these V_H segments do, however, recombine with an established VDJ unit, displacing most of the originally assembled V_H element,³⁸ a process sometimes called V_H replacement. Such events are mediated by cryptic RSSs (mainly a heptamer sequence) that is present near the 3′ end of about 70% of all V_H genes (see Fig. 6.10B). Such internal cryptic RSSs are not generally found in L chain genes. As discussed previously for the L chain, secondary recombination represents a rescue mechanism for cells with nonproductive rearrangements on both H chain chromosomes, and for cells whose encoded antibody recognizes an autoantigen.

A major advance in the investigation of V(D)J recombination was the identification of two genes whose products are critical for this process in the B and T cell lineages. In the pioneering experiments, Schatz and Baltimore⁴⁰ stably transfected fibroblasts with a construct containing a selectable marker whose expression was dependent on V(D)J recombination; as expected, no measurable recombination occurred in this nonlymphoid cell. However, when either human or murine genomic DNA was transfected into these fibroblasts, a small fraction of recipient cells stably expressed recombinase activity, activating the selectable marker. This suggested that a single transfected genomic DNA fragment was able confer recombinase activity in a fibroblast. (Presumably the fibroblast contained endogenous copies of the same genes, but their expression was repressed by mechanisms that could not repress the transfected genes.) This active fragment was cloned and turned out to contain two closely linked genes, designated RAG1 and RAG2, respectively. Both RAG1 and RAG2 are essential for recombination; therefore, these genes would not have been discovered by this transfection technique if they had not been closely linked in the genome. The genes are notable for having no introns splitting up their open reading frame in most species, and for their opposite transcriptional orientation in all species examined.

A crucial role for the RAG genes in V(D)J recombination was supported by the conservation of these genes in all jawed vertebrate species analyzed thus far, from shark through man. RAG1 and RAG2 are expressed together in developing B and T cells, specifically at the stages at which V(D)J recombinase activity is required for the assembly of Ig and TCR genes. Moreover, mouse strains in which either gene has been eliminated by homologous recombination (gene “knockouts”) have no mature B or T cells, as the result of their inability to initiate V(D)J recombination.^41,42 Similarly, a subset of human patients with SCID syndrome characterized by the complete absence of T- or B-lymphocytes have been found to have null mutations in RAG genes.⁴³ Patients with hypomorphic alleles often have a complex set of features (oligoclonal T cells, hepatosplenomegaly, eosinophilia, decreased serum Ig but elevated IgE) known as the Omenn syndrome, which can also be caused by defects in other genes involved in V(D)J recombination. Interestingly, the same RAG mutation in different patients can cause either Omenn syndrome or SCID, depending on unknown factors.⁴⁴

RAG1 shows intrinsic binding affinity for the RSS nonamer sequence via its nonamer binding domain even in the absence of RAG2. Exhaustive mutational analysis has revealed that RAG1 contains the catalytic center of the RAG complex, composed of three amino acids critical for all enzymatic activity: D600, D708, and E962.^45,46 RAG2, on the other hand, serves as a regulatory cofactor; it has no intrinsic binding affinity for RSSs, but once bound to RAG1 improves the strength and specificity of RAG1 RSS contacts.^47,48 It is also enhances RAG activity on chromosomal substrates and it restricts V(D)J recombination to the G0/G1 stage of the cell cycle (both features are discussed below).

Attempts to determine the molecular role of the RAG proteins in cell-free recombination assays were initially hampered by poor solubility of the proteins, but functional analyses of truncated RAG genes (using RAG expression vectors cotransfected into fibroblasts along with recombination substrate plasmids) revealed that surprisingly large segments of both proteins could be deleted without eliminating recombinase activity, and some of the remaining core regions were
soluble and could be handled relatively easily in experiments. This work allowed the demonstration that in a cell-free in vitro system, core regions of the two RAG proteins together are capable of carrying out cleavage of substrate DNAs as well as hairpin formation on the coding end.⁴⁹

The RAG-mediated cleavage occurs in two steps: first a nick is introduced on the top strand between a gene segment and the adjacent heptamer (see Fig. 6.9), then the 3′-hydroxyl group participates as the nucleophile in a direct transesterification reaction to attack the phosphodiester bond adjacent to the heptamer on the bottom strand (see Fig. 6.9), yielding a DNA hairpin structure on the coding end and a new 3′-hydroxyl group on the 3′ end of the bottom heptamer strand.⁵⁰ After DNA cleavage, the RAG proteins remain in a complex with the DNA ends and facilitate aspects of the joining phase. Mutant forms of RAG1 or RAG2 have been reported that are competent for cleavage but show impairment in coding or signal joint formation.⁵¹

While nicking can occur asynchronously at the 12-RSS and 23-RSS, hairpin formation is “coupled” and occurs synchronously at both RSSs. In vitro, coupled cleavage requires only the RAG proteins, HMG1/2 (discussed below) and Mg²⁺ as the divalent metal ion in the reaction buffer. In vivo, DNA dsb formation at an individual RSS is dangerous as it could give rise to translocations, and it is thought that Mg²⁺ promotes an optimal molecular “architecture” for controlled V(D)J recombination. In vivo experiments indeed suggest that RAG proteins may bind to and introduce a nick at a single 12-RSS, but do not complete DNA cleavage until a matching 23-RSS is captured into the RAG-RSS complex.⁵²

In addition to the “classical” activities of RAG proteins on DNA segments containing RSSs, these proteins can also catalyze DNA strand cleavage on “nonstandard” substrates.

Although the “core” RAG proteins have been useful for elucidating the molecular mechanism of the cleavage step of V(D)J recombination in biochemical studies, it is clear that the “noncore” portions of each protein confer important functions, as expected from their sequence conservation across species. Broadly speaking, the “noncore” regions ensure regulated and efficient recombination on the physiological substrates (i.e., imperfect RSSs deviating from the perfect consensus heptamer and nonamer) in the context of chromatin. The functions of the “noncore” regions have largely been inferred by comparing V(D)J recombination products from cells expressing core RAG proteins versus full-length versions, and more recently by in vitro studies using full-length RAG proteins that are now available for such analyses.

The C-terminal region of RAG2 has multiple functions and is important for achieving normal numbers of B- and T-lymphocytes in vivo,⁶³ for the formation of precise signal joints during IgH recombination,⁶⁴ and for protecting against RAG-mediated DNA transposition.^51,65 These functions are thought to be conferred at least in part, by a plant homeo domain (PHD) zinc finger fold that is formed by amino acids 414 to 487 in murine RAG2. This PHD domain binds specifically to the tails of histone H3 that are trimethylated at lysine 4 (H3K4Me3),^66,67,68 a histone modification that is associated with “open” chromatin and that is uniquely present on “accessible” RSSs in Ig loci (discussed below). In vitro studies suggest that the binding of the RAG2 PHD domain to histone tails causes a conformational change that increases the catalytic activity of the RAG complex.⁶⁹

Furthermore, the RAG2 C terminus regulates RAG2 protein levels—and hence V(D)J recombinase activity—across the cell cycle to prevent dsbs during DNA synthesis or mitosis, when such breaks could lead to chromosomal deletions.³² RAG1 protein and messenger RNA (mRNA) transcript levels of both RAG genes vary little across the cell cycle, but phosphorylation of RAG2 at Thr490 by the cyclin-dependent
kinase cdk2 mediates its destruction via ubiquitination and proteasomal degradation during S phase.⁷⁰ Mice expressing RAG2 with a T490A mutation (which cannot be phosphorylated) showed RAG2 protein and dsbs throughout the cell cycle, demonstrating the importance of the RAG2 degradation signal in cell-cycle control of V(D)J recombination.^71,72

The N-terminal noncore region of RAG1 is required in vivo for optimal RAG1 activity and for the formation of precise signal joints in D-J recombination.⁶⁴ This region of RAG1 contains a RING finger domain that seems to be required for ubiquitination of several proteins, including histone H3.⁷³

Apart from the obvious importance of the RAG proteins in understanding the initial steps of V(D)J recombination, knowledge of these proteins and their genes has allowed two major technical advances that have opened the way to many additional experiments. First, various nonlymphoid cell lines with known defects in various DNA repair genes have been transfected with the RAG genes to identify genes involved V(D)J recombination (these factors are described below). Second, availability of the RAG1 and RAG2 knockout mice has been instrumental in a large number of immunology studies. These mice completely lack functional B cells or T cells, and are not “leaky” like SCID mice, which develop some functional B and T cells, especially as the animals age. Thus the RAG-deficient mice can be used to study the importance of the “innate” immune system (i.e., responses that occur in the absence of antigen-specific lymphocytes) in particular immune responses. They can also be used as recipients for various lymphocyte populations to explore the roles of different cell types. They can also be used as recipients for various lymphocyte populations to explore the roles of different cell types. They can be transfected with transgenes encoding specific Ig genes to study the roles of specific antibodies in B cell development and in immune responses. Finally, they can be used in “RAG complementation” experiments designed to assess the phenotype —in lymphocytes—of various other gene knockouts.⁷⁴ In RAG complementation, embryonic stem cells in which the gene of interest has been knocked out by homologous recombination are injected into homozygous RAG2 knockout (RAG2-/-) blastocysts. This procedure yields chimeric mice in which all B and T cells derive from the embryonic stem cells deleted for the gene of interest, as these are the only source of intact RAG genes to support lymphocyte development. Such animals can be made more easily than a knockout mouse line, and can be used to study the effect of gene deletion in lymphocytes independent of effects the deletion may have in other cells. In particular, for cases where the gene knockout causes embryonic lethality due to effects on nonlymphoid cells, RAG complementation allows the selective knockout in lymphocytes to be studied in the background normal gene expression in nonlymphoid cells.

The search for RAG cofactors that stimulate cleavage activity in biochemical assays led to the identification of HMG1.⁷⁵ HMG1 (and the closely related HMG2) are abundant and ubiquitous proteins that bind DNA in a non-sequence-specific manner and to cause a local bend in DNA. The two RAG proteins can form a stable signal complex with a 12-RSS, but efficient complex formation with a 23-RSS requires the addition of either HMG1 or HMG2.⁷⁶ HMG1/2 apparently stabilizes the bending of the 23-RSS that is induced by the RAG proteins themselves.⁷⁷

The RAG proteins are the essential lymphocyte-specific factors in the DNA cleavage phase of V(D)J recombination, but DNA repair factors that are part of a DNA repair pathway known as nonhomologous end joining (NHEJ) are essential for the joining phase. NHEJ is the major pathway for repair of dsbs (such as those induced by ionizing radiation or reactive oxygen species) during the G0-G1 phases of the cell cycle. (In the S and G2 phases, the additional chromatid genome copy enables breaks to be repaired by homologous recombination.) The six classical core components of NHEJ are Ku70, Ku80, DNA-PKcs, XRCC4, DNA Ligase IV, Artemis, and Cernnunos/XLF, but additional proteins play a role in some models of NHEJ.

The DNA-PK Complex. The first gene for an NHEJ component to be recognized as participating in V(D)J recombination was the SCID gene. This gene was originally identified as being mutated in the scid mouse strain that is immunodeficient due to a marked impairment in V(D)J recombination of both Ig and TCR genes. Lymphocytes from scid mice are able to perform the RAG-mediated cleavage reaction, and can also form signal joints, but are markedly defective in coding joint formation. Subsequently, it was found that the scid mutation also impairs NHEJ, causing radiosensitivity.

The gene mutated in the scid mouse strain encodes DNAPKcs, a large protein (460 kD) with a kinase domain near its C terminus that is related to phosophoinositide-3-kinase (PI3K). This kinase is DNA-dependent and represents the catalytic subunit (hence “cs”) of a heterotrimer known as the DNA-PK complex. The other components are Ku70 and Ku80 (also referred to as Ku86), which were originally identified as the autoantigens recognized by a patient antiserum (Ku was the coded name of the patient, and the numbers refer to the approximate size of the proteins, 70 kD and 80 to 86 kD, respectively). Together, these two very abundant proteins form a heterodimer that binds to the ends of double-stranded DNA independent of the nucleotide sequence of the DNA. The DNA-Ku complex can then recruit DNAPKcs and activate autophosphorylation of this protein.⁷⁸ In vitro activation of DNA-PKcs was found to be efficient when DNA ends either were at high concentration or, if at low concentration, were on DNA fragments long enough to circularize readily. In contrast, when the DNA-PKcs was located on the ends of DNA fragments too short to circularize (and too dilute for efficient intermolecular interactions with other DNA ends), the DNA-PKcs activation was much reduced. These observations suggest that kinase activation can occur only after two DNA ends are brought together by DNA-PKcs in “synapsis.”^79,80 Further phosphorylation of
DNA-PKcs inactivates the protein and may prepare it for removal once DNA ends have been sealed.

Ku genes are highly conserved through evolution, and homologs are even found encoded in the genome of some bacteria, consistent with a function in general NHEJ not restricted to V(D)J recombination. While mice with a targeted deletion of DNA-PKcs resemble the original scid mutation (i.e., defective coding but functional signal joint formation^81,82), Ku70 and Ku80 mutant cell lines are defective in both signal and coding joint formation, and Ku70- and Ku80-deficient mice exhibit a complete block in B- and T-cell development due to their inability to undergo V(D)J recombination.^83,84,85

DNA Ligase IV and XRCC4. An important role of activated Ku-DNA-PKcs complex is to recruit the additional components of NHEJ. One such component is DNA ligase IV, which is recruited to the Ku complex and activated by the protein XRCC4.^86,87 The evidence suggests that DNA ligase IV is the essential ligase that joins DNA ends in V(D)J recombination and NHEJ. Human patients with ligase IV deficiency (characterized by hypomorphic alleles) have a severe phenotype including chromosomal instability, developmental and growth retardation, radiosensitivity, and immunodeficiency with a T-B-NK+ phenotype.⁸⁸ The rare D_H-J_H junctions detected show extensive nucleotide deletion consistent with delayed ligation and prolonged exonuclease digestion.⁸⁹ In mice, disruption of either the XRCC4 or the DNA ligase IV gene causes embryonic lethality associated with neuronal apoptosis. Crossing these mice with p53 mutants does not improve V(D)J recombination, but rescues the mice from embryonic lethality, suggesting that neuronal cells may be unusually susceptible to p53-triggered apoptosis induced by normal low-level DNA damage during brain development; a similar mechanism may explain the severe human phenotype.⁹⁰ DNA ligase IV is the only NHEJ component absolutely required to join compatible sticky DNA ends in vitro, though XRCC4 can stimulate this activity significantly.⁸⁷

Cernunnos/XRCC4-like Factor. The next NHEJ component was independently discovered by two laboratories. One group used yeast two-hybrid screening to search for proteins interacting with XRCC4.⁹¹ The other group searched for the gene causing a syndrome of T+ B lymphocytopenia, increased radiosensitivity, and microcephaly in a Turkish family; these investigators used functional cDNA rescue of a patient’s cell line from a radiomimetic drug to identify the gene.⁹² The protein identified by both groups is a 299 amino acid nuclear protein, which was named Cernunnos or XLF. The protein has a predicted secondary structure similar to that of XRCC4, to which it binds in cells⁹³ as expected from its isolation via two-hybrid screen. When Cernunnos/XLF-deficient fibroblasts were transfected with RAG genes and a recombination substrate, imprecise signal joining was observed, similar to the defect in patients with hypomorphic DNA ligase IV mutations. These experiments all suggest a role for Cernunnos/XLF linked to the function of XRCC4 and ligase IV.

Artemis. The coding ends generated by RAG cleavage cannot be directly ligated because of their hairpin structure, and therefore V(D)J recombination requires a single-strand endonuclease activity to cleave the hairpins. This activity is conferred by the protein named Artemis, which was discovered through positional cloning of the genetic defect in a group of human SCID patients with defects in V(D)J recombination and increased radiation sensitivity.⁹⁴ Patients with homozygous null mutations of Artemis survive (no embryonic lethality) and show sensitivity to γ irradiation as well as defects in coding joints, while signal joint formation is normal. Hypomorphic Artemis mutations can cause features of the Omenn syndrome similar to those observed with hypomorphic RAG gene mutations.⁹⁵ Purified recombinant Artemis protein has an intrinsic exonuclease activity in vitro; however, when complexed with DNA-PKcs in the presence of DNA ends, it gains a single-strand endonuclease activity and, in an ATP-dependent step, becomes phosphorylated at multiple sites in the C-terminal region of the protein.^96,97 The Artemis endonuclease can cleave synthetic and RAG-generated hairpin ends as well as other singlestranded DNA near a transition to double-strand DNA.⁹⁸

DNA Polymerase X Family Members. If a hairpin opening leaves blunt ends or complementary sticky ends (like the ends generated by many restriction enzymes), in vitro joining experiments suggest that these ends can be joined by ligase IV without any additional processing.⁹⁹ However, as Artemis probably opens most hairpins noncomplementary DNA overhangs, further processing of DNA ends generally occurs before ligation completes the recombination. This processing may include further nuclease digestion (by Artemis or exonucleases) and apparently also involves variable DNA extension by three DNA polymerases—polymerase λ, polymerase µ, and terminal deoxynucleotidyl transferase (TdT)—all of which are members of the polymerase X family. Interestingly, all three proteins contain a Brca1-C-terminus domain, which is thought to confer binding to Ku.¹⁰⁰

Terminal Deoxynucleotidyl Transferase and N Regions. TdT, the primary source of untemplated “N region” additions in VDJ junctions, is an enzyme uniquely expressed in the thymus and bone marrow; in the B lineage, it is expressed almost exclusively in pro-B cells. It catalyzes the nontemplated addition of nucleotides to the 3′ end of DNA strands. Though no template determines the nucleotides added, the enzyme adds dG residues preferentially, consistent with N region sequences observed in VDJ joints. Both TdT expression and N nucleotide addition are characteristically absent from fetal lymphocytes.¹⁰¹ N region addition is common in H chain genes (recombined in pro-B cells) but rare in murine L chain genes (recombined in pre-B cells), though perhaps somewhat less rare in human.¹⁰² This is consistent with the observation that in mice the expression of a µ H chain may downregulate TdT expression,¹⁰³ contributing to the reduced level during the stage of L chain recombination.

Lymphocytes with engineered defects in their TdT genes produced rearranged Ig V regions with almost no N additions. Conversely, when TdT expression was engineered in cells undergoing κ or λ L chain rearrangement, the level of
N nucleotide addition to these coding joints was dramatically increased. Furthermore, mice engineered to undergo premature Vκ-Jκ joining in pro-B cells show an increased frequency of N region nucleotides in their recombined Vκ genes.¹⁰⁴ These results suggest that the low frequency of N region sequences in normal κ or λ recombinations is caused by the reduced levels of TdT at this stage of B-cell development (see following discussion).

The absence of N region addition in TdT mutant mice, as well as in normal fetal lymphocytes, is associated with an increase in the frequency of recombination junctions with microhomologies. These are short stretches of nucleotides that are present close to the end of both germline gene segments involved in the recombination event. These junctions suggest a joining intermediate in which the complementary single-stranded regions from the two coding ends hybridize to each other, much as “sticky ends” generated by restriction endonucleases can facilitate ligation of DNA fragments. This alternative joining pathway may restrict the diversity of neonatal antibodies; the resulting antibodies are possibly enriched in specificities for commonly encountered pathogens, or have broadened specificity, as has been reported for TCRs lacking N regions.¹⁰⁵ Decreased N region nucleotides and a high incidence of homology-mediated recombination have also been found in the rare coding joints formed in Ku80-/- mice, consistent with a role for Ku in recruiting TdT or supporting its action.¹⁰⁶

Polymerase µ and Polymerase λ. Polymerase µ and polymerase λ are ubiquitously expressed polymerases. Both readily fill in single-strand gaps in DNA and apparently participate in V(D)J recombination by filling in single-strand 3′ overhangs generated by asymmetric hairpin opening. Without this filling in, such overhangs might be resected by nucleases. Indeed, when in vitro NHEJ reconstitution experiments are performed using purified proteins and DNA fragments with overhanging ends, the omission of polymerase µ or polymerase λ increases the deletional trimming at junctions.¹⁰⁰ Similar excessive deletions at VDJ junctions are observed in mice lacking polymerase µ or polymerase λ. Remarkably, however, polymerase µ knockout mice show abnormalities only in their L chains,¹⁰⁷ whereas the deletions in polymerase λ knockouts are restricted to their H chains.¹⁰⁸ This selectivity may be explained by corresponding changes in the relative mRNA levels for these two polymerases at different stages of B-cell development.

DNA Damage Response Factors. In eukaryotic cells, DNA breaks initiate signals that halt cell division, induce DNA repair, and in some cases trigger apoptosis. Several proteins apart from NHEJ components can be detected at DNA breaks induced by V(D)J recombination or irradiation, including γ-H2AX, a phosphorylated form of the histone H2AX; ATM, the product of the gene mutated in the disease ataxia telangiectasia; Nbs1 (or nibrin), the product of the gene mutated in Nijmegen breakage syndrome; and 53BP1, p53 binding protein 1. The importance of these proteins in V(D)J recombination is not clear because defects in all three are compatible with near normal V(D)J recombination. Possibly, they participate in backup mechanisms to prevent aberrant V(D)J recombination and thus translocations.

Pax5/B-Cell-Specific Activator Protein. Pax5 (also known as B-cell-specific activator protein; BSAP) is a transcription factor required for normal B-cell development. Pax5-deficient mice are able to complete DJH recombination, but V_H to DJ_H recombination is impaired except for certain V_H genes located proximal to the D regions. Interestingly, 94% of human and mouse V_H coding genes were found to have potential Pax5 binding sites. Surprisingly, Pax5 was found to coimmunoprecipitate with RAG proteins, to potentiate in vitro cleavage of a V_H gene RSS, and to enhance V_H to DJ_H recombination in RAG-transfected fibroblasts; the latter enhancement required intact Pax5 binding sites in the V_H sequence.¹⁰⁹

In general, inactive genes tend to be located in the periphery of nuclei, while active genes are recruited to a more central nuclear location.¹²⁴

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