Multiple genes for the variable regions are encoded in the human genome that contain three distinct types of segments. For example, the immunoglobulin heavy chain region contains 65 Variable (V) genes plus 27 Diversity (D) genes and 6 Joining (J) genes. The light chains also possess numerous V and J genes, but do not have D genes. By the mechanism of DNA rearrangement of these regional genes it is possible to generate an enormous antibody repertoire - 65 V genes x 27 D genes x 6 J Genes= 10530 combinations, with additional variability through light chains.
Most T cell receptors are composed of an alpha chain and a beta chain. The T cell receptor genes are similar to immunoglobulin genes in that they too contain multiple V, D and J genes in their beta chains (and V and J genes in their alpha chains) that are rearranged during the development of the lymphocyte to provide that cell with a unique antigen receptor.
In the developing B cell, the first recombination event to occur is between one D and one J gene segment of the heavy chain locus. Any DNA between these two genes is deleted. This D-J recombination is followed by the joining of one V gene, from a region upstream of the newly formed DJ complex, forming a rearranged VDJ gene. All other genes between V and D segments of the new VDJ gene are now deleted from the cell’s genome. Primary transcript (unspliced RNA) is generated containing the VDJ region of the heavy chain and both the constant mu and delta chains (Cμ and Cδ). (i.e. the primary transcript contains the segments: V-D-J-Cμ-Cδ). The primary RNA is processed to add a polyadenylation (poly-A) tail after the Cμ chain and to remove sequence between the VDJ segment and this constant gene segment. Translation of this mRNA leads to the production of the Ig μ heavy chain protein.
The kappa (κ) and lambda (λ) chains of the immunoglobulin light chain loci rearrange in a very similar way, except the light chains lack a D segment. In other words, the first step of recombination for the light chains involves the joining of the V and J chains to give a VJ complex before the addition of the constant chain gene during primary transcription. Translation of the spliced mRNA for either the kappa or lambda chains results in formation of the Ig κ or Ig λ light chain protein.
Assembly of the Ig μ heavy chain and one of the light chains results in the formation of membrane bound form of the immunoglobulin IgM that is expressed on the surface of the immature B cell.
During T cell development, the T cell receptor (TCR) chains undergo essentially the same sequence of ordered recombination events as that described for immunoglobulins. D-to-J recombination occurs first in the β chain of the TCR. This process can involve either the joining of the Dβ1 gene segment to one of six Jβ1 segments or the joining of the Dβ2 gene segment to one of six Jβ2 segments. DJ recombination is followed (as above) with Vβ-to-DβJβ rearrangements. All genes between the Vβ-Dβ-Jβ genes in the newly formed complex are deleted and the primary transcript is synthesized that incorporates the constant domain gene (Vβ-Dβ-Jβ-Cβ). mRNA transcription splices out any intervening sequence and allows translation of the full length protein for the TCR Cβ chain.
The rearrangement of the alpha (α) chain of the TCR follows β chain rearrangement, and resembles V-to-J rearrangement described for Ig light chains (see above). The assembly of the β- and α- chains results in formation of the αβ-TCR that is expressed on a majority of T cells.
Other enzymes of the VDJ recombinase are expressed in multiple cell types and are involved in DNA repair following the activity of RAG1 and RAG2. One of these enzymes is called the DNA-dependent protein kinase complex (DNA-PK) that repairs double-stranded DNA. DNA-PK binds to each end of the broken DNA and recruits several other proteins, including Artemis nuclease, XRCC4 (X-ray repair cross-complementing factor 4), DNA ligase IV, Cernunnos (also called XLF or XRCC4-like factor), and any of several DNA polymerases. DNA-PK complexes on each DNA end phosphorylate (add phosphate groups to) each other, resulting in activation of Artemis. Artemis then breaks the hairpin loop that was formed by the RAG proteins. XRCC4 and Cernunnos act in concert with DNA-PK to align the two DNA ends with each other, and also help to recruit terminal transferase, which adds nucleotides randomly to the ends. DNA polymerases λ and μ insert additional nucleotides as needed to make the two ends compatible for joining. Ligase IV finally links DNA strands on opposite ends of the break to each other, completing the joining process.
Because of the variability in the exact position of cleavage of the hairpin loop by Artemis, as well as the random nucleotide addition by terminal transferase, the final DNA sequence, and thus the sequence of the resulting antibody, is highly variable, even when the same two V, D, or J segments are joined. This great diversity allows VDJ recombination to generate antibodies even to microbes that neither the organism nor its ancestors have ever previously encountered.