V(D)J recombination (variable–diversity–joining rearrangement) is the mechanism of
somatic recombination that occurs only in developing
lymphocyte
A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include T cells (for cell-mediated and cytotoxic adaptive immunity), B cells (for humoral, antibody-driven adaptive immunity), an ...
s during the early stages of T and B cell maturation. It results in the highly diverse repertoire of
antibodies/immunoglobulins and
T cell receptor
The T-cell receptor (TCR) is a protein complex, located on the surface of T cells (also called T lymphocytes). They are responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. ...
s (TCRs) found in
B cell
B cells, also known as B lymphocytes, are a type of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or inserted into the plasm ...
s and
T cell
T cells (also known as T lymphocytes) are an important part of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell ...
s, respectively. The process is a defining feature of the
adaptive immune system
The adaptive immune system (AIS), also known as the acquired immune system, or specific immune system is a subsystem of the immune system that is composed of specialized cells, organs, and processes that eliminate pathogens specifically. The ac ...
.
V(D)J recombination in mammals occurs in the primary lymphoid organs (
bone marrow
Bone marrow is a semi-solid biological tissue, tissue found within the Spongy bone, spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It i ...
for B cells and
thymus
The thymus (: thymuses or thymi) is a specialized primary lymphoid organ of the immune system. Within the thymus, T cells mature. T cells are critical to the adaptive immune system, where the body adapts to specific foreign invaders. The thymus ...
for T cells) and in a nearly random fashion rearranges variable (V), joining (J), and in some cases, diversity (D) gene segments. The process ultimately results in novel
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
sequences in the antigen-binding regions of immunoglobulins and TCRs that allow for the recognition of
antigen
In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response.
...
s from nearly all pathogens including
bacteria
Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
,
viruses
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Viruses are found in almo ...
,
parasites
Parasitism is a close relationship between species, where one organism, the parasite, lives (at least some of the time) on or inside another organism, the host, causing it some harm, and is adapted structurally to this way of life. The en ...
, and
worms
The World Register of Marine Species (WoRMS) is a taxonomic database that aims to provide an authoritative and comprehensive catalogue and list of names of marine organisms.
Content
The content of the registry is edited and maintained by scien ...
as well as "altered self cells" as seen in
cancer
Cancer is a group of diseases involving Cell growth#Disorders, abnormal cell growth with the potential to Invasion (cancer), invade or Metastasis, spread to other parts of the body. These contrast with benign tumors, which do not spread. Po ...
. The recognition can also be
allergic
Allergies, also known as allergic diseases, are various conditions caused by hypersensitivity of the immune system to typically harmless substances in the environment. These diseases include hay fever, food allergies, atopic dermatitis, alle ...
in nature (''e.g.'' to
pollen
Pollen is a powdery substance produced by most types of flowers of seed plants for the purpose of sexual reproduction. It consists of pollen grains (highly reduced Gametophyte#Heterospory, microgametophytes), which produce male gametes (sperm ...
or other
allergen
An allergen is an otherwise harmless substance that triggers an allergic reaction in sensitive individuals by stimulating an immune response.
In technical terms, an allergen is an antigen that is capable of stimulating a type-I hypersensitivi ...
s) or may match host tissues and lead to
autoimmunity
In immunology, autoimmunity is the system of immune responses of an organism against its own healthy cells, tissues and other normal body constituents. Any disease resulting from this type of immune response is termed an " autoimmune disease ...
.
In 1987,
Susumu Tonegawa was awarded the
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine () is awarded yearly by the Nobel Assembly at the Karolinska Institute for outstanding discoveries in physiology or medicine. The Nobel Prize is not a single prize, but five separate prizes that, acco ...
"for his discovery of the genetic principle for generation of antibody diversity".
Background
Human
antibody
An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as pathogenic bacteria, bacteria and viruses, includin ...
molecules (including
B cell receptors) are composed of heavy and light chains, each of which contains both ''constant'' (C) and ''variable'' (V) regions, genetically encoded on three
loci:
*The immunoglobulin heavy locus (
IGH@) on chromosome 14, containing the gene segments for the
immunoglobulin heavy chain
The immunoglobulin heavy chain (IgH) is the large polypeptide subunit of an antibody (immunoglobulin). In the human genome, the IgH gene loci are on chromosome 14.
A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two I ...
.
*The immunoglobulin kappa (κ) locus (
IGK@) on chromosome 2, containing the gene segments for one type (κ) of
immunoglobulin light chain
]
The immunoglobulin light chain is the small Peptide, polypeptide subunit of an antibody (immunoglobulin).
A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains.
In humans
There are two types of light ...
.
*The immunoglobulin lambda (λ) locus (
IGL@) on chromosome 22, containing the gene segments for another type (λ) of immunoglobulin light chain.
Each heavy chain or light chain gene contains multiple copies of three different types of gene segments for the variable regions of the antibody proteins. For example, the human immunoglobulin heavy chain region contains 2 Constant (Cμ and Cδ) gene segments and 44 Variable (V) gene segments, plus 27 Diversity (D) gene segments and 6 Joining (J) gene segments.
The light chain genes possess either a single (Cκ) or four (Cλ) Constant gene segments with numerous V and J gene segments but do not have D gene segments.
DNA rearrangement causes one copy of each type of gene segment to go in any given lymphocyte, generating an enormous antibody repertoire; roughly 3×10
11 combinations are possible, although some are removed due to self reactivity.
Most
T cell receptor
The T-cell receptor (TCR) is a protein complex, located on the surface of T cells (also called T lymphocytes). They are responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. ...
s are composed of a variable
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 gene segments in their beta chains (and V and J gene segments in their alpha chains) that are rearranged during the development of the lymphocyte to provide that cell with a unique antigen receptor. The T cell receptor in this sense is the topological equivalent to an antigen-binding fragment of the antibody, both being part of the immunoglobulin superfamily.
An autoimmune response is prevented by eliminating cells that self-react. For T cells, this occurs in the thymus by testing the cell against an array of self antigens expressed through the function of the
autoimmune regulator
The autoimmune regulator (AIRE) is a protein that in humans is encoded by the ''AIRE'' gene. It is a 13Kilo-base pair, kbp gene on chromosome 21q22.3 that encodes 545 amino acids. AIRE is a transcription factor expressed in the Thymus#Medulla, me ...
(AIRE). The immunoglobulin lambda light chain locus contains protein-coding genes that can be lost with its rearrangement. This is based on a physiological mechanism and is not pathogenetic for leukemias or lymphomas. A cell persists if it creates a successful product that does not self-react, otherwise it is pruned via
apoptosis
Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...
.
Immunoglobulins
Heavy chain
In the developing
B cell
B cells, also known as B lymphocytes, are a type of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or inserted into the plasm ...
, 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 gene segments is deleted. This D-J recombination is followed by the joining of one V gene segment, from a region upstream of the newly formed DJ complex, forming a rearranged VDJ gene segment. All other gene segments between V and D segments 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 (C
μ and C
δ). (i.e. the primary transcript contains the segments: V-D-J-C
μ-C
δ). The primary RNA is processed to add a
polyadenylated (poly-A) tail after the C
μ chain and to remove sequence between the VDJ segment and this constant gene segment.
Translation
Translation is the communication of the semantics, meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The English la ...
of this mRNA leads to the production of the
IgM heavy chain protein.
Light chain
The kappa (κ) and lambda (λ) chains of the immunoglobulin light chain loci rearrange in a very similar way, except that 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.
T cell receptors
During
thymocyte
A thymocyte is an immune cell present in the thymus, before it undergoes transformation into a T cell. Thymocytes are produced as stem cells in the bone marrow and reach the thymus via the blood.
Thymopoiesis describes the process which turns thy ...
development, the
T cell receptor
The T-cell receptor (TCR) is a protein complex, located on the surface of T cells (also called T lymphocytes). They are responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. ...
(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 D
β1 gene segment to one of six J
β1 segments or the joining of the D
β2 gene segment to one of six J
β2 segments.
DJ recombination is followed (as above) with V
β-to-D
βJ
β rearrangements. All gene segments between the V
β-D
β-J
β gene segments in the newly formed complex are deleted and the primary transcript is synthesized that incorporates the constant domain gene (V
β-D
β-J
β-C
β). mRNA transcription splices out any intervening sequence and allows translation of the full length protein for the TCR β-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 cell
T cells (also known as T lymphocytes) are an important part of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell ...
s.
Mechanism
Key enzymes and components
The process of V(D)J recombination is mediated by VDJ recombinase, which is a diverse collection of enzymes. The key enzymes involved are
recombination activating genes 1 and 2 (RAG),
terminal deoxynucleotidyl transferase
Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphom ...
(TdT), and
Artemis
In ancient Greek religion and Greek mythology, mythology, Artemis (; ) is the goddess of the hunting, hunt, the wilderness, wild animals, transitions, nature, vegetation, childbirth, Kourotrophos, care of children, and chastity. In later tim ...
nuclease, a member of the ubiquitous
non-homologous end joining
Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. It is called "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair ...
(NHEJ) pathway for DNA repair. Several other enzymes are known to be involved in the process and include
DNA-dependent protein kinase (DNA-PK),
X-ray repair cross-complementing protein 4 (XRCC4),
DNA ligase IV,
non-homologous end-joining factor 1 (NHEJ1; also known as Cernunnos or XRCC4-like factor
LF, the recently discovered Paralog of XRCC4 and XLF (PAXX), and DNA polymerases λ and μ. Some enzymes involved are specific to lymphocytes (''e.g.'', RAG, TdT), while others are found in other cell types and even ubiquitously (''e.g.'', NHEJ components).
To maintain the specificity of recombination, V(D)J recombinase recognizes and binds to
recombination signal sequences (RSSs) flanking the variable (V), diversity (D), and joining (J) genes segments. RSSs are composed of three elements: a heptamer of seven conserved nucleotides, a spacer region of 12 or 23 basepairs in length, and a nonamer of nine conserved nucleotides. While the majority of RSSs vary in sequence, the consensus heptamer and nonamer sequences are CACAGTG and ACAAAAACC, respectively; and although the sequence of the spacer region is poorly conserved, the length is highly conserved. The length of the spacer region corresponds to approximately one (12 basepairs) or two turns (23 basepairs) of the DNA helix. Following what is known as the 12/23 Rule, gene segments to be recombined are usually adjacent to RSSs of different spacer lengths (''i.e.'', one has a "12RSS" and one has a "23RSS"). This is an important feature in the regulation of V(D)J recombination.
Process
V(D)J recombination begins when V(D)J recombinase (through the activity of RAG1) binds a RSS flanking a coding gene segment (V, D, or J) and creates a single-strand nick in the DNA between the first base of the RSS (just before the heptamer) and the coding segment. This is essentially energetically neutral (no need for
ATP hydrolysis) and results in the formation of a free 3'
hydroxyl group
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
and a 5'
phosphate group
Phosphates are the naturally occurring form of the element phosphorus.
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosp ...
on the same strand. The reactive hydroxyl group is positioned by the recombinase to attack the
phosphodiester bond
In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...
of opposite strand, forming two DNA ends: a
hairpin (stem-loop) on the coding segment and a
blunt end on the signal segment. The current model is that DNA nicking and hairpin formation occurs on both strands simultaneously (or nearly so) in a complex known as a ''recombination center''.
The blunt signal ends are flush ligated together to form a circular piece of DNA containing all of the intervening sequences between the coding segments known as a signal joint (although circular in nature, this is not to be confused with a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...
). While originally thought to be lost during successive cell divisions, there is evidence that signal joints may re-enter the genome and lead to pathologies by activating
oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. s or interrupting
tumor suppressor gene
A tumor suppressor gene (TSG), or anti-oncogene, is a gene that regulates a cell (biology), cell during cell division and replication. If the cell grows uncontrollably, it will result in cancer. When a tumor suppressor gene is mutated, it results ...
function(s)
ef
The coding ends are processed further prior to their ligation by several events that ultimately lead to junctional diversity. Processing begins when DNA-PK binds to each broken DNA end and recruits several other proteins including Artemis, XRCC4, DNA ligase IV, Cernunnos, and several DNA polymerases. DNA-PK forms a complex that leads to its
autophosphorylation, resulting in activation of Artemis. The coding end hairpins are opened by the activity of Artemis. If they are opened at the center, a blunt DNA end will result; however in many cases, the opening is "off-center" and results in extra bases remaining on one strand (an overhang). These are known as palindromic (P) nucleotides due to the
palindromic nature of the sequence produced when DNA repair enzymes resolve the overhang. The process of hairpin opening by Artemis is a crucial step of V(D)J recombination and is defective in the
severe combined immunodeficiency (scid) mouse model.
Next, XRCC4, Cernunnos, and DNA-PK align the DNA ends and recruit terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that adds non-templated (N) nucleotides to the coding end. The addition is mostly random, but TdT does exhibit a preference for G/C nucleotides. As with all known DNA polymerases, the TdT adds nucleotides to one strand in a 5' to 3' direction.
Lastly, exonucleases can remove bases from the coding ends (including any P or N nucleotides that may have formed). DNA polymerases λ and μ then insert additional nucleotides as needed to make the two ends compatible for joining. This is a stochastic process, therefore any combination of the addition of P and N nucleotides and exonucleolytic removal can occur (or none at all). Finally, the processed coding ends are ligated together by DNA ligase IV.
All of these processing events result in a
paratope
In immunology, a paratope, also known as an antigen-binding site, is the part of an antibody which recognizes and binds to an antigen. It is a small region at the tip of the antibody's antigen-binding fragment and contains parts of the antibody' ...
that is highly variable, even when the same gene segments are recombined. V(D)J recombination allows for the generation of immunoglobulins and T cell receptors to antigens that neither the organism nor its ancestor(s) need to have previously encountered, allowing for an adaptive immune response to novel pathogens that develop or to those that frequently change (''e.g.'',
seasonal influenza
Flu season is an annually recurring time period characterized by the prevalence of an outbreak of influenza (flu). The season occurs during the cold half of the year in each hemispheres of Earth, hemisphere. It takes approximately two days to ...
). However, a major caveat to this process is that the DNA sequence must remain
in-frame in order to maintain the correct amino acid sequence in the final protein product. If the resulting sequence is out-of-frame, the development of the cell will be arrested, and the cell will not survive to maturity. V(D)J recombination is therefore a very costly process that must be (and is) strictly regulated and controlled.
See also
*
B cell receptor
*
T cell receptor
The T-cell receptor (TCR) is a protein complex, located on the surface of T cells (also called T lymphocytes). They are responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. ...
*
Basel Institute for Immunology
*
Charles M. Steinberg
*
NKT cell
*
Recombination-activating gene
References
Further reading
*
* V(D)J Recombination. Series: Advances in Experimental Medicine and Biology, Vol. 650 Ferrier, Pierre (Ed.) Landes Bioscience 2009, XII, 199 p.
{{Immune system
Immune system
Lymphocytes
Immunology