VDJ recombination

VDJ recombination, also known as antigen receptor gene rearrangement or antigen-independent diversification, is a diversity generating assembly process affecting the variable domain of immunoglobulin and TCR genes.

▼: 12/23 rule : B cells : class-switch recombination : CSR : double strand breaks : E2A encoded proteins : hairpin : HMG-1, HMG-2 : lymphoid-specific components : nonlymphoid-restricted components : RAG1 & RAG2 : RSS : SAGA : SHM : somatic hypermutation : synaptic complex : transposon : VDJ genes :▼

The heavy (H) plus kappa (κ) or lambda (λ) chain combinations of BCRs (H-κ/λ) and the alpha (α) and beta (β) or gamma (γ) and delta (δ) chain combinations of TCRs (αβ or γδ) are encoded by roughly three hundred different gene segments, yet produce an estimated 5 x 10^7 to 10^9 surface receptors (B+T). The segments are scattered on human chromosomes 2, 14, and 22.

Segments:
BCR heavy chains: 51 VH, 27 DH, 6 JH, 9 CH gene segments on human chromosome 14 (93 VDJC). The CH segments are 1 µ (IgM), 1 δ (IgD), 4 γ (IgG), 1 ε (IgE), and 2 α (IgA)
BCR light chains 40 Vκ , 31 Vλ , 5 Jκ , 4 Jλ gene segments on human chromosome 14 (80 VJ)

TCR 50 Vα, 50 Jα, 20 Vβ, 13 Jβ , 2 Dβ gene segments (100 α, 35 β), and a smaller number of γδ gene segments. The κ segments are on human chromosome 2 and the λ segments are on human chromosome 22

Antigen receptor gene rearrangement of variable (V), diversity (D) and joining (J) gene segments generates this enormous repertoire of antigen receptors with different antibody specificities, providing the versatility that is essential to normal immune functioning. It has been estimated that around 10^9 distinct antibody molecules can be generated by VDJ recombination. The genes encoding the variable region domain for the heavy chain lack a complete exon, instead segments encoding the V region are split into arrays of gene segments. Light chain genes are also organised on different chromosomes, but they have no D gene segments. There are 51 functional VH genes and 41 Vk genes. D (diversity) and J (junctional) genes code for amino acids at the carboxyl end of V regions including CDR3.

Each heavy chain is derived from a V, D, J, and C region in a sequence of steps:
1. A D and J sequence are spliced
2. A V segment is spliced to the DJ segment, all intervening Vs and Js are deleted when the random V and J are joined. This brings V, D, and J gene segments together in a translational reading frame at the DNA level, generating a mRNA product: Leader, V, D, J, C, poly A

Each light chain begins with the V and J sequences combined, with a few thousand base pairs separating the J and the C regions. This is then transcribed into a primary transcript, polyadenylated, and the intervening sequence is spliced out. This generates the mRNA product: Leader, V, J, C, poly A.

Antigen-dependent immunoglobulin gene diversification, via somatic hypermutation (SHM), and class-switch recombination (CSR) occur in mature B cells during the humoral immune response. SHM generates point mutations and CSR generates different antibody isotypes by recombination 1, 2.

Highly conserved recombination signal sequences (RSS), comprising a heptamer and a nonamer motif with an intervening 12- or 23-bp spacer, enable VDJ recombination of the immunoglobulin and TCR loci involving RSS with different spacers following the 12/23 rule (3). Right - click to enlarge - simplified process of VDJ recombination in which V and D coding segments of immunoglobulin and T cell receptor genes are flanked by short recombination signal sequences (RSS), which are in opposite orientations at the 5' and 3' termini of the coding sequences. That is, RSS are located at the 3' end of each V segment, 3' and 5' ends of each D segment, and at the 3' end of each J segment. The RSS are recognized (1, 2)by a complex of the lymphocyte-specific recombination proteins, recombination activating genes, RAG1 and RAG2 (for recombination activating genes). These enzymes cleave (c) the DNA between the coding sequence and the RSS, creating double-strand breaks (DSB). The broken coding strands are then ligated (j) by nonhomologous end-joining to yield a rearranged gene segment (D-J, then DJ-V):
● a coding join (D-J or V-DJ for heavy chains; V-J for light chains), which is retained, and
● a signal join, formed from a loop of DNA from which has been deleted all the intervening DNA initially present between the 2 gene segments. The signal join segment is discarded.
(For more comprehensive diagrams and legends see here.) Џ AV animations click on thumbnails Џ

RAG1 and RAG2, the proteins that mediate VDJ recombination, are closely related to transposases, and it is believed that evolution of the vertebrate genome includes their entry as part of a Transib superfamily transposon.

Several proteins mediate VDJ recombination (4):
● Lymphoid-specific components of the recombination machinery, RAG-1, RAG-2 together constitute the recombinase. Terminal deoxynucleotidyl transferase (TdT) mediates the incorporation of nontemplate-dependent nucleotides.
● Nonlymphoid-restricted components include DNA-PKcs, Ku70, Ku80, XRCC4, ligase 4, Artemis, and possibly HMG1 and HMG2.

All these proteins are involved in repair of DNA double strand breaks in addition to their rôle in VDJ recombination. The nonlymphoid-specific components probably participate in the processing and joining steps of VDJ recombination. HMG1 and HMG2 are two additional nonlymphoid-specific components that have been implicated in VDJ recombination. Experiments have demonstrated that these proteins increase the in vitro efficiency of cleavage by RAGs (5, 6).

During the initial stages of antigen receptor gene rearrangement, RAG-1/RAG-2 form a complex with the RSS, which is partly stabilized by interactions between the nonamer binding domain of RAG-1 and the nonamer motif. Bridging of 12 and 23 RSS, in a synaptic complex, is critical for DNA cleavage and for it to be facilitated by the DNA bending proteins HMG1 and HMG2. Within the synaptic complex, RAG-1/RAG-2 efficiently introduce a nick at each RSS through a hydrolysis reaction at the heptamer/coding flank border, which generates a 3' hydroxyl end. A transesterification reaction, resembling the mechanism of transpositional recombination, next creates a double strand break as the free 3' hydroxyl of the nicked strand is used in a nucleophilic attack on the opposing strand generating a covalently sealed hairpin intermediate, known as the hairpin coding end (5, 6). [s]

E2A encoded proteins, including E12 and E47, regulate site-specific DNA recombination. The extreme N-terminal domain of E2A has been shown to recruit the co-activator protein complex, SAGA, which contains histone acetylase activity, so it is conceivable that the E2A proteins regulate recombination by promoting locus accessibility. Possible mechanisms by which E2A proteins regulate recombination include localized accessibility, looping, and direct recruitment of RAG proteins.[l]

The VDJ recombination mechanism in jawed vertebrates is catalyzed by the RAG1 and RAG2 proteins, which are believed to have emerged approximately 500 million years ago from transposon-encoded proteins. Although no transposase sequence similar to RAG1 or RAG2 has been found, the approximately 600-amino acid “core” region of RAG1 required for its catalytic activity is significantly similar to the transposase encoded by DNA transposons that belong to the Transib superfamily. It has been demonstrated that recombination signal sequences (RSSs) were derived from terminal inverted repeats of an ancient Transib transposon. Furthermore, the critical DDE catalytic triad of RAG1 is shared with the Transib transposase as part of conserved motifs.[r] These findings refute one of Behe's claims for irreducible complexity of complex biochemical features.

. chromosome 2, chromosome 14, chromosome 22 .

▲:12/23 rule ф antibodies ф antigen »» Basic mechanisms of evolution : B cells : class-switch recombination : CSR : double strand breaks ~ double strand breaks : E2A encoded proteins : hairpin : HMG-1, HMG-2 : lymphoid-specific components : nonlymphoid-restricted components »» point mutation : RAG1 & RAG2 »» Recombination : RSS : SAGA : SHM : somatic hypermutation ~ splicing : synaptic complex : transposon ~ transposons : VDJ genes :▲

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