Research Article: V(D)J Recombination and the Evolution of the Adaptive Immune System

Date Published: October 13, 2003

Publisher: Public Library of Science

Author(s): Eleonora Market, F. Nina Papavasiliou

Abstract: In order for the immune system to generate its vast numbers of receptors, B- and T-cell receptor genes are created by recombining preexisting gene segments. This well- coordinated set of reactions is explained.

Partial Text: The immune system needs to be able to identify and ultimately destroy foreign invaders. To do so, it utilizes two major types of immune cells, T cells and B cells (or, collectively, lymphocytes). Lymphocytes display a large variety of cell surface receptors that can recognize and respond to an unlimited number of pathogens, a feature that is the hallmark of the “adaptive” immune system. To react to such a variety of invaders, the immune system needs to generate vast numbers of receptors. If the number of different types of receptors present on lymphocytes were encoded by individual genes, the entire human genome would have to be devoted to lymphocyte receptors. To establish the necessary level of diversity, B- and T-cell receptor (BCR and TCR, respectively) genes are created by recombining preexisting gene segments. Thus, different combinations of a finite set of gene segments give rise to receptors that can recognize unlimited numbers of foreign invaders. This is accomplished by a supremely well-coordinated set of reactions, starting with cleaving DNA within specific, well-conserved recombination signal sequences (RSSs). This highly regulated step is carried out by the lymphocyte-specific recombinationactivating genes (RAG1 and RAG2). The segments are then reassembled using a common cellular repair mechanism.

In the first part of the “cut-and-paste” reaction, breaks within both strands of the DNA helix (double-stranded breaks) are made within the RSS sites; in the second part, the newly created breaks are repaired by the cell’s general DNA repair pathway. In the initial phase, two lymphocyte-specific proteins that are encoded by the recombinationactivating genes (RAG1 and RAG2) work together to recognize and bind RSSs. The complex consisting of these two proteins, RAG1–RAG2 (henceforth RAG), cuts the DNA between the rearranging DNA segments and the adjacent RSS motifs (Figure 1B). The second step of the reaction glues together the ends of the chromosome containing the rearranging segments, which will ultimately code for the receptor and are called coding joints (CJs). The portion of DNA between the rearranged segments is shed from the genome, but it too gets glued together in a minicircle (a signal joint [SJ]). Typically, SJs are rapidly and precisely fused, but CJs are ligated more slowly, in part because their fusion is not precise—small insertions are present quite often, and even deletions can be detected. (For detailed reviews, see Fugmann et al. 2000; Gellert 2002). Both pasting reactions are necessary for creating the receptors as well as for preventing havoc within the genome.

RAG proteins carry out the first enzymatic step of the reaction—site-specific cleavage of DNA (van Gent et al. 1995). Artificial expression of RAGs in mammalian cells other than B- or T-lymphocytes suggests that RAG is the only lymphocyte-specific factor required for this recombination event to occur (Schatz and Baltimore 1988). Indeed, in mice whose RAG genes have been deleted (RAG−/−), V(D)J recombination is completely abolished, and these mice have neither mature B nor T cells (Mombaerts et al. 1992; Shinkai et al. 1992). A similar type of immunodeficiency, called Omenn syndrome, is seen in people with mutations in their RAG genes (Villa et al. 1998).

RSSs are found next to every variable (V), diversity (D), and joining (J) segment. They consist of three distinct elements: a heptamer and a nonamer sequence, separated by a spacer element—either 12 or 23 bp long (Figure 2) (Tonegawa 1983; Akira et al. 1987). Although the two RAG proteins work together in a protein complex, they do have unique functions. RAG1 binds both the 12- and 23-RSSs with equal affinities, while RAG2 does not bind either RSS sequence. This suggests that RAG1 forms the initial complex with DNA, which then recruits and is stabilized by RAG2 (Fugmann et al. 2000).

After RAG cuts the DNA at the RSS, the two sides of the break are different. The coding ends are closed to resemble hairpins, while the RSS ends are open and blunt. These blunt RSS ends are rejoined rapidly, forming SJs, but before the coding ends can be fused, the hairpins must be opened (Gellert 2002).

As mentioned, RAG−/− mice do not have mature B and T cells. This causes a severe combined immunodeficiency syndrome (SCID) characterized by a complete block in B- and T-cell development, but no other defects (Mombaerts et al. 1992; Shinkai et al. 1992). However, there are other molecular deficiencies that also have a SCID phenotype. One of these is mapped to the enzyme DNA protein kinase (DNA-PKcs), which is required for the proper joining of DNA ends (Bosma et al. 1988). Mice deficient in DNA-PKcs can initiate V(D)J recombination, but cannot form the CJs (Gao et al. 1998). These mice are also sensitive to processes that induce DNA double-stranded breaks, such as ionizing radiation (Gao et al. 1998). Hence, the repair pathway responsible for fixing DNA breaks caused by radiation also creates CJs. Indeed, along with DNA-PKcs, other proteins of this nonhomologous end-joining repair pathway are important for the completion of V(D)J recombination (such as Ku70 and Ku80, Artemis, XRCC4, and DNA ligase IV) (Bassing et al. 2002).

From the discovery of the RAG genes on, investigators have suspected that V(D)J recombination may be the result of the landing of a transposable genetic element (a “jumping gene” or transposon) into the vertebrate genome. The clues were many. Firstly, the compact organization of the RAG locus resembles a transposable element (Schatz et al. 1989). Secondly, RAGs cut the DNA after binding RSSs throughout the BCR and TCR loci (Gellert 2002). RSSs resemble the ends of other transposable elements. Biochemically, the reaction shares characteristics with enzymes found in other transposable elements (Gellert 2002). Finally, these genes appear abruptly in evolution: they are present in the jawed vertebrates (like the shark), but not in more ancient organisms (Schluter et al. 1999).

V(D)J recombination is absolutely crucial for the adaptive immune response. In its absence, our immune system is compromised. When it is not properly controlled, it gives rise to chromosomal translocations and B-and T-cell cancers. The elucidation of all steps of the reaction and attempts to understand exactly how these steps are regulated to avoid disastrous side effects are areas of study that have occupied researchers in the past and will continue to do so in the future.

Source:

http://doi.org/10.1371/journal.pbio.0000016

 

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