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V(D)J recombination
Encyclopedia
VJ recombination, also known as somatic recombination, is a mechanism of genetic recombination
in the early stages of immunoglobulin
(Ig) and T cell receptor
s (TCR) production of the immune system
. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene
segments of vertebrate
s, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigen
s from bacteria
, virus
es, parasites, dysfunctional cells such as tumor
cells, and pollen
s.
molecules (and B cell receptors) comprise heavy and light chains with both constant (C) and variable (V) regions that are encoded by genes on three loci
.
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; roughly 10^8 combinations are possible, although some are removed due to self reactivity.
Most T cell receptor
s 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.
Failure of the cell to create a successful product that does not self-react leads to cell apoptosis
.
, 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 polyadenylated
(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.
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.
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 seven 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 cell
s.
(RSSs) that are recognized by a group of enzymes known collectively as the VDJ recombinase. RSSs are composed of seven conserved nucleotides (a heptamer) that reside next to the gene encoding sequence followed by a spacer (containing either 12 or 23 unconserved nucleotides) followed by a conserved nonamer (9 base pairs). The RSSs are present on the 3’ side (downstream) of a V region and the 5’ side (upstream) of the J region. These are the sides that will be involved in the joining. Only a pair of dissimilar spacer RSSs are efficiently recombined (i.e. one with a spacer of 12 nucleotides will be recombined with one that has a spacer containing 23 nucleotides). This is known as the 12/23 rule of recombination (or "one turn/two turn" rule).
-1 and -2 (RAG1 and RAG2). These enzymes associate with each other to recognize the RSS sequences and induce DNA cleavage at the RSS sequences. This cleavage only takes place on one strand of DNA, which leads to a nucleotide attack and creation of a hairpin loop.
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. RAG1 is homologous to transposase
. 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 the enzyme, terminal deoxynucleotidyl transferase (TdT)
, which adds nucleotides randomly to the ends, providing junctional diversity
. 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 deoxynucleotidyl transferase (TdT), 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.
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
in the early stages of immunoglobulin
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
(Ig) and T cell receptor
T cell receptor
The T cell receptor or TCR is a molecule found on the surface of T lymphocytes that is responsible for recognizing antigens bound to major histocompatibility complex molecules...
s (TCR) production of the immune system
Immune system
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own...
. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
segments of vertebrate
Vertebrate
Vertebrates are animals that are members of the subphylum Vertebrata . Vertebrates are the largest group of chordates, with currently about 58,000 species described. Vertebrates include the jawless fishes, bony fishes, sharks and rays, amphibians, reptiles, mammals, and birds...
s, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
s from bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es, parasites, dysfunctional cells such as tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
cells, and pollen
Pollen
Pollen is a fine to coarse powder containing the microgametophytes of seed plants, which produce the male gametes . Pollen grains have a hard coat that protects the sperm cells during the process of their movement from the stamens to the pistil of flowering plants or from the male cone to the...
s.
Background
Human antibodyAntibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
molecules (and B cell receptors) comprise heavy and light chains with both constant (C) and variable (V) regions that are encoded by genes on three loci
Locus (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
.
- Immunoglobulin heavy locus (IGH@) on chromosome 14, containing genes for the immunoglobulin heavy chain
- Immunoglobulin kappa (κ) locus (IGK@) on chromosome 2, containing genes for the immunoglobulin light chainImmunoglobulin light chain]The immunoglobulin light chain is the small polypeptide subunit of an antibody .A typical antibody is composed of two immunoglobulin heavy chains and two Ig light chains.-In humans:...
- Immunoglobulin lambda (λ) locus (IGL@) on chromosome 22, containing genes for the immunoglobulin light chainImmunoglobulin light chain]The immunoglobulin light chain is the small polypeptide subunit of an antibody .A typical antibody is composed of two immunoglobulin heavy chains and two Ig light chains.-In humans:...
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; roughly 10^8 combinations are possible, although some are removed due to self reactivity.
Most T cell receptor
T cell receptor
The T cell receptor or TCR is a molecule found on the surface of T lymphocytes that is responsible for recognizing antigens bound to major histocompatibility complex molecules...
s are composed of an alpha chain
Alpha chain
The alpha chain of a T cell receptor for antigens is one of the membrane-bound heterodimers. The other heterodimer is the beta chain. Each chain contains one variable region and one constant region. The V and C regions are homologous to immunoglobulin V and C regions...
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
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
receptor.
Failure of the cell to create a successful product that does not self-react leads to cell apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
.
In immunoglobulins
Heavy chain
In the developing B cellB cell
B cells are lymphocytes that play a large role in the humoral immune response . The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells and eventually develop into memory B cells after activation by antigen interaction...
, 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
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
(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 polyadenylated
Polyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
(poly-A) tail after the Cμ chain and to remove sequence between the VDJ segment and this constant gene segment. Translation
Translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of...
of this mRNA leads to the production of the Ig μ 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 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
IGM
IGM as an acronym or abbreviation can refer to:* Immunoglobulin M , the primary antibody against A and B antigens on red blood cells* International Grandmaster, a chess ranking* intergalactic medium* Intragroup medium - see: Intracluster medium...
that is expressed on the surface of the immature B cell.
In T cell receptors
During thymocyteThymocyte
Thymocytes are hematopoietic progenitor cells present in the thymus. Thymopoiesis is the process in the thymus by which thymocytes differentiate into mature T lymphocytes. The primary function of thymocytes is the generation of T lymphocytes . The thymus provides an inductive environment, which...
development, the T cell receptor
T cell receptor
The T cell receptor or TCR is a molecule found on the surface of T lymphocytes that is responsible for recognizing antigens bound to major histocompatibility complex 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 Dβ1 gene segment to one of six Jβ1 segments or the joining of the Dβ2 gene segment to one of seven 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 cell
T cell
T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells , by the presence of a T cell receptor on the cell surface. They are...
s.
Recombination signal sequences
The regional genes (V, D, J) are flanked by Recombination Signal SequencesRecombination Signal Sequences
The regional genes , used to generate T-cell receptors and Immunoglobulin molecules, are flanked by Recombination Signal Sequences that are recognized by a group of enzymes known collectively as the VDJ recombinase...
(RSSs) that are recognized by a group of enzymes known collectively as the VDJ recombinase. RSSs are composed of seven conserved nucleotides (a heptamer) that reside next to the gene encoding sequence followed by a spacer (containing either 12 or 23 unconserved nucleotides) followed by a conserved nonamer (9 base pairs). The RSSs are present on the 3’ side (downstream) of a V region and the 5’ side (upstream) of the J region. These are the sides that will be involved in the joining. Only a pair of dissimilar spacer RSSs are efficiently recombined (i.e. one with a spacer of 12 nucleotides will be recombined with one that has a spacer containing 23 nucleotides). This is known as the 12/23 rule of recombination (or "one turn/two turn" rule).
VDJ recombinase
VDJ recombinase refers to a collection of enzymes some of which are lymphocyte specific, and some that are expressed in many cell types. The initial steps of VDJ recombination are carried out by critical lymphocyte specific enzymes, called recombination activating geneRecombination activating gene
The recombination activating genes encode enzymes that play an important role in the rearrangement and recombination of the genes of immunoglobulin and T cell receptor molecules during the process of VDJ recombination...
-1 and -2 (RAG1 and RAG2). These enzymes associate with each other to recognize the RSS sequences and induce DNA cleavage at the RSS sequences. This cleavage only takes place on one strand of DNA, which leads to a nucleotide attack and creation of a hairpin loop.
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. RAG1 is homologous to transposase
Transposase
Transposase is an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism....
. 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 the enzyme, terminal deoxynucleotidyl transferase (TdT)
Terminal Deoxynucleotidyl Transferase
Terminal deoxynucleotidyl transferase , also known as DNA nucleotidylexotransferase or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells...
, which adds nucleotides randomly to the ends, providing junctional diversity
Junctional diversity
Junctional diversity describes the DNA sequence variations introduced by the improper joining of gene segments during the process of VJ recombination...
. 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 deoxynucleotidyl transferase (TdT), 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.
Further reading
- V(D)J Recombination. Series: Advances in Experimental Medicine and Biology, Vol. 650 Ferrier, Pierre (Ed.) Landes Bioscience 2009, XII, 199 p. ISBN: 978-1-4419-0295-5