Structure of receptor genes
There are 6 main clusters in the human genome encoding the chains of antigen-recognition receptors of B and T lymphocytes (immunoglobulins Ig and T-cell receptors TCR). The genes are localized on different chromosomes and, in germline configuration, consist of disconnected regions.
Coding segments are divided into:
- V segments (Variable). Form the main part of the variable domain (up to the CDR3 region). Their number reaches up to 129, but many are pseudogenes and are not expressed.
- D segments (Diversity). Responsible for additional diversity. Present only in immunoglobulin heavy H-chain clusters, as well as $\beta$- and $\delta$-chains of TCR.
- J segments (Joining). Short joining regions.
- C genes (Constant). Encode constant domains and determine the molecule isotype.
The mature gene configuration capable of encoding a full-length receptor is formed exclusively through somatic recombination, when germline regions join together.
Mechanism and the 12/23 rule
To enable enzymes to recognize DNA cleavage sites, recombination signal sequences (RSS) are located next to the coding segments. Each RSS consists of strictly conserved heptamer (7 nucleotides) and nonamer (9 nucleotides) motifs separated by a spacer exactly 12 or 23 base pairs long.
Segment assembly obeys the strict "12/23 rule": a segment with a 12-bp spacer can only join with a region having a 23-bp spacer.
The process itself is carried out by a multicomponent V(D)J recombination complex:
- Approximation and cleavage. Recombinases RAG-1 and RAG-2 bind to RSS, form a dimer, and create a DNA loop. Strand cleavage occurs precisely at the boundary of the coding segments.
- Removal of excess. The intermediate DNA along with signal sequences forms a circle (REC) and is permanently deleted from the genome.
- Hairpin formation and insertions. The ends of the cleaved coding regions fold into closed "hairpins". Then an endonuclease cleaves them laterally, and the strands unfold, forming palindromic P-insertions. Immediately afterwards, the enzyme TdT adds random nucleotides in a template-independent manner, forming N-insertions (up to 20 nucleotides).
- Repair. DNA ligase IV, with the participation of the Ku70/Ku80 complex, ligates the assembled segments.
Allelic exclusion
Recombination proceeds independently in each lymphocyte and begins strictly on one chromosome. This mechanism is called allelic exclusion.
If rearrangement is successful, RAG gene expression is blocked, and the gene on the paired chromosome remains in an untouched (germline) state. This is critically important to ensure cell monospecificity: a single lymphocyte must bear receptors of only one specificity.
However, the process itself is associated with a high error rate. About 2/3 of attempts lead to a reading frame shift. In case of failure on the first chromosome, the cell activates rearrangement on the second. If both attempts fail, the lymphocyte undergoes apoptosis.
Sources of hypervariability
Rearrangement occurs in the region where the third hypervariable region of the receptor (CDR3) is formed, the variability of which is colossal. Diversity is achieved through a combination of factors:
- Combinatorics: random selection of specific V, D, and J segments.
- Joining inaccuracy: enzymes can introduce breaks with a shift of several nucleotides.
- P- and N-insertions: addition of new, originally non-encoded bases.
- Reading frame shifts: the D segment can be read in three different frames.
Theoretically, this mechanism allows the generation of up to $10^{19}$ receptor variants. In practice, realizing this potential is impossible due to the limited number of cells in the body (the actual number of T-cell clones is about $2.5 \times 10^7$). However, this diversity is more than enough to recognize any antigens existing in nature.
Briefly: answers to topical questions
What enzymes and protein complexes are involved in V(D)J recombination?
V(D)J recombination involves a specialized recombination complex consisting of six main components. These include:
- Recombinase dimer (RAG-1/RAG-2) — recognizes signal sequences and cleaves DNA strands.
- DNA-dependent protein kinase — participates in hairpin resolution and repair.
- DNA ligase IV — ensures the rejoining of DNA strands.
- Terminal deoxynucleotidyl transferase (TdT) — catalyzes template-independent synthesis of oligodeoxynucleotides (N-insertions).
- HMG 1/2 heterodimer — facilitates the approximation of DNA regions.
- Ku70/Ku80 heterodimer — participates in the repair process.
The endonuclease enzyme also participates in hairpin resolution.
On which specific chromosomes are individual immunoglobulin and TCR gene clusters localized?
Immunoglobulin (IG) and T-cell receptor (TR) gene clusters are localized on four different human chromosomes. The distribution of gene loci is as follows:
| Chromosome | Immunoglobulin genes (IG) | T-cell receptor genes (TR) |
|---|---|---|
| Chromosome 2 | κ chain (IGK) | — |
| Chromosome 7 | — | β chain (TRB), γ chain (TRG) |
| Chromosome 14 | Heavy H-chain (IGH) | α chain (TRA), δ chain (TRD) |
| Chromosome 22 | λ chain (IGL) | — |
At which stages of antigen-independent B-lymphocyte differentiation does the rearrangement of heavy and light chain genes occur?
Rearrangement of immunoglobulin heavy and light chain genes occurs at strictly defined stages of B-lymphocyte development.
- Heavy chain genes (H-chains) rearrange first at the pro-B-II stage. This process includes two steps: first, D-J recombination occurs, followed by V-DJ recombination to form a mature gene.
- Light chain genes (L-chains) rearrange at the pre-B-II stage. In this process, V and J segments join directly (due to the absence of a D segment), with κ-chain genes involving first, and λ-chain genes upon failure.
What primary immunodeficiencies are caused by defects in the RAG1 and RAG2 genes?
Defects in the RAG1 and RAG2 genes lead to Omenn syndrome (Omenn syndrome) in case of incomplete RAG blockade. Gene function is partially preserved, allowing the V(D)J recombination necessary to generate the antigen-specific repertoire.
The 12/23 rule can be compared to railway assembly: a fragment with a "12"-type attachment connects only to a "23"-type groove. Identical ends (12+12 or 23+23) do not join. This protects the cell from incorrectly fusing two identical segments (e.g., V and V).
Class and exam questions
Which receptor chains possess D segments?
D segments are present only in immunoglobulin heavy H-chain clusters, as well as in T-cell receptor β- and δ-chain clusters. The remaining chains do not have them and assemble via V-J joining.
What are N-insertions and where do they come from?
These are entirely novel nucleotides, not encoded in the genome, which the enzyme terminal deoxynucleotidyl transferase (TdT) randomly attaches to the ends of the cleaved DNA. They enormously increase receptor variability.
What happens to the excised piece of DNA?
Unused genetic segments and signal sequences fold into a recombination excision circle (REC). It is physically excised and irrevocably lost by the cell.
What else to study on this topic
- Palindromic structure of heptamers, nonamers, and P-nucleotides
- Structure and functions of RAG-1 and RAG-2 recombinases in the DNA synapse
- Mechanism of T-lymphocyte lineage commitment (αβ or γδ)
- The phenomenon of peripheral receptor editing
- Strict sequence of chain rearrangement (heavy chains first, then light chains)
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