Recombination-activating gene

Last updated
recombination-activating gene 1
Identifiers
Symbol RAG1
NCBI gene 5896
HGNC 9831
OMIM 179615
RefSeq NM_000448
UniProt P15918
Other data
Locus Chr. 11 p13
Search for
Structures Swiss-model
Domains InterPro
recombination-activating gene 2
Identifiers
Symbol RAG2
NCBI gene 5897
HGNC 9832
OMIM 179616
RefSeq NM_000536
UniProt P55895
Other data
Locus Chr. 11 p13
Search for
Structures Swiss-model
Domains InterPro
Recombination-activating protein 2
Identifiers
SymbolRAG2
Pfam PF03089
InterPro IPR004321
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary
Recombination-activating protein 1
Identifiers
SymbolRAG1
Pfam PF12940
InterPro IPR004321
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary

The recombination-activating genes (RAGs) encode parts of a protein complex that plays important roles in the rearrangement and recombination of the genes encoding immunoglobulin and T cell receptor molecules. There are two recombination-activating genes RAG1 and RAG2, whose cellular expression is restricted to lymphocytes during their developmental stages. The enzymes encoded by these genes, RAG-1 and RAG-2, are essential to the generation of mature B cells and T cells, two types of lymphocyte that are crucial components of the adaptive immune system. [1]

Contents

Function

In the vertebrate immune system, each antibody is customized to attack one particular antigen (foreign proteins and carbohydrates) without attacking the body itself. The human genome has at most 30,000 genes, and yet it generates millions of different antibodies, which allows it to be able to respond to invasion from millions of different antigens. The immune system generates this diversity of antibodies by shuffling, cutting and recombining a few hundred genes (the VDJ genes) to create millions of permutations, in a process called V(D)J recombination. [1] RAG-1 and RAG-2 are proteins at the ends of VDJ genes that separate, shuffle, and rejoin the VDJ genes. This shuffling takes place inside B cells and T cells during their maturation.

RAG enzymes work as a multi-subunit complex to induce cleavage of a single double stranded DNA (dsDNA) molecule between the antigen receptor coding segment and a flanking recombination signal sequence (RSS). They do this in two steps. They initially introduce a ‘nick’ in the 5' (upstream) end of the RSS heptamer (a conserved region of 7 nucleotides) that is adjacent to the coding sequence, leaving behind a specific biochemical structure on this region of DNA: a 3'-hydroxyl (OH) group at the coding end and a 5'-phosphate (PO4) group at the RSS end. The next step couples these chemical groups, binding the OH-group (on the coding end) to the PO4-group (that is sitting between the RSS and the gene segment on the opposite strand). This produces a 5'-phosphorylated double-stranded break at the RSS and a covalently closed hairpin at the coding end. The RAG proteins remain at these junctions until other enzymes (notably, TDT) repair the DNA breaks.

The RAG proteins initiate V(D)J recombination, which is essential for the maturation of pre-B and pre-T cells. Activated mature B cells also possess two other remarkable, RAG-independent phenomena of manipulating their own DNA: so-called class-switch recombination (AKA isotype switching) and somatic hypermutation (AKA affinity maturation). [2] Current studies have indicated that RAG-1 and RAG-2 must work in a synergistic manner to activate VDJ recombination. RAG-1 was shown to inefficiently induce recombination activity of the VDJ genes when isolated and transfected into fibroblast samples. When RAG-1 was cotransfected with RAG-2, recombination frequency increased by a 1000-fold. [3] This finding has fostered the newly revised theory that RAG genes may not only assist in VDJ recombination, but rather, directly induce the recombinations of the VDJ genes.

Structure

As with many enzymes, RAG proteins are fairly large. For example, mouse RAG-1 contains 1040 amino acids and mouse RAG-2 contains 527 amino acids. The enzymatic activity of the RAG proteins is concentrated largely in a core region; Residues 384–1008 of RAG-1 and residues 1–387 of RAG-2 retain most of the DNA cleavage activity. The RAG-1 core contains three acidic residues (D600, D708, and E962) in what is called the DDE motif, the major active site for DNA cleavage. These residues are critical for nicking the DNA strand and for forming the DNA hairpin. Residues 384–454 of RAG-1 comprise a nonamer-binding region (NBR) that specifically binds the conserved nonamer (9 nucleotides) of the RSS and the central domain (amino acids 528–760) of RAG-1 binds specifically to the RSS heptamer. The core region of RAG-2 is predicted to form a six-bladed beta-propeller structure that appears less specific than RAG-1 for its target.

Cryo-electron microscopy structures of the synaptic RAG complexes reveal a closed dimer conformation with generation of new intermolecular interactions between two RAG1-RAG2 monomers upon DNA binding, compared to the Apo-RAG complex which constitutes as an open conformation. [4] Both RAG1 molecules in the closed dimer are involved in the cooperative binding of the 12-RSS and 23-RSS intermediates with base specific interactions in the heptamer of the signal end. The first base of the heptamer in the signal end is flipped out to avoid the clash in the active center. Each coding end of the nicked-RSS intermediate is stabilized exclusively by one RAG1-RAG2 monomer with non-specific protein-DNA interactions. The coding end is highly distorted with one base flipped out from the DNA duplex in the active center, which facilitates the hairpin formation by a potential two-metal ion catalytic mechanism. The 12-RSS and 23-RSS intermediates are highly bent and asymmetrically bound to the synaptic RAG complex with the nonamer binding domain dimer tilts towards the nonamer of the 12-RSS but away from the nonamer of the 23-RSS, which emphasizes the 12/23 rule. Two HMGB1 molecules bind at each side of 12-RSS and 23-RSS to stabilize the highly bent RSSs. These structures elaborate the molecular mechanisms for DNA recognition, catalysis and the unique synapsis underlying the 12/23 rule, provide new insights into the RAG-associated human diseases, and represent a most complete set of complexes in the catalytic pathways of any DDE family recombinases, transposases or integrases.

Evolution

Based on core sequence homology, it is believed that RAG1 evolved from a transposase from the Transib superfamily. [5] No Transib family members include an N-terminal sequence found in RAG1 suggesting the N-terminal of RAG1 came from a separate element. The N-terminal region of RAG1 has been found in the transposable element N-RAG-TP in the sea slug, Aplysia californica , which contains the entire RAG1 N-terminal. [6] It is likely that the full RAG1 structure was derived from the recombination between a Transib and the N-RAG-TP transposon. [7]

A transposon with RAG2 arranged next to RAG1 has been identified in the purple sea urchin. [8] Active Transib transposons with both RAG1 and RAG2 ("ProtoRAG") has been discovered in B. belcheri (Chinese lancelet) and Psectrotarsia flava (a moth). [9] [10] The terminal inverted repeats (TIR) in lancelet ProtoRAG have a heptamer-spacer-nonamer structure similar to that of RSS, but the moth ProtoRAG lacks a nonamer. The nonamer-binding regions and the nonamer sequences of lancelet ProtoRAG and animal RAG are different enough to not recognize each other. [9] The structure of the lancelet protoRAG has been solved ( PDB: 6b40 ​), providing some understanding on what changes lead to the domestication of RAG genes. [11]

Although the transposon origins of these genes are well-established, there is still no consensus on when the ancestral RAG1/2 locus became present in the vertebrate genome. Because agnathans (a class of jawless fish) lack a core RAG1 element, it was traditionally assumed that RAG1 invaded after the agnathan/gnathostome split 1001 to 590 million years ago (MYA). [12] However, the core sequence of RAG1 has been identified in the echinoderm Strongylocentrotus purpuratus (purple sea urchin), [13] the amphioxi Branchiostoma floridae (Florida lancelet). [14] Sequences with homology to RAG1 have also been identified in Lytechinus veriegatus (green sea urchin), Patiria minata (sea star), [8] the mollusk Aplysia californica, [15] and protostomes including oysters, mussels, ribbon worms, and the non-bilaterian cnidarians. [16] These findings indicate that the Transib family transposon invaded multiple times in non-vertebrate species, and invaded the ancestral jawed vertebrate genome about 500 MYA. [8] It is hypothesized that the absence of RAG-like genes in jawless vertebrates and urochordates [16] is due to horizontal gene transfer or gene loss in certain phylogenetic groups due to conventional vertical transmission. [13] Recent analysis has shown the RAG phylogeny to be gradual and directional, suggesting an evolutionary path that relies on vertical transmission. [16] This hypothesis suggests that the RAG1/2-like pair may have been present in its current form in most metazoan lineages and was lost in the jawless vertebrate and urochordate lineages. [7] There is no evidence that the V(D)J recombination system arose earlier than the vertebrate lineage. [7] It is currently hypothesized that the invasion of RAG1/2 is the most important evolutionary event in terms of shaping the gnathostome adaptive immune system vs. the agnathan variable lymphocyte receptor system.

Selective pressure

It is still unclear what forces led to the development of a RAG1/2-mediated immune system exclusively in jawed vertebrates and not in any invertebrate species that also acquired the RAG1/2-containing transposon. Current hypotheses include two whole-genome duplication events in vertebrates, [17] which would provide the genetic raw material for the development of the adaptive immune system, and the development of endothelial tissue, greater metabolic activity, and a decreased blood volume-to-body weight ratio, all of which are more specialized in vertebrates than invertebrates and facilitate adaptive immune responses. [18]

See also

Related Research Articles

<span class="mw-page-title-main">Transposable element</span> Semiparasitic DNA sequence

A transposable element is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transposition often results in duplication of the same genetic material. In the human genome, L1 and Alu elements are two examples. Barbara McClintock's discovery of them earned her a Nobel Prize in 1983. Its importance in personalized medicine is becoming increasingly relevant, as well as gaining more attention in data analytics given the difficulty of analysis in very high dimensional spaces.

<span class="mw-page-title-main">Retrotransposon</span> Type of genetic component

Retrotransposons are a type of genetic component that copy and paste themselves into different genomic locations (transposon) by converting RNA back into DNA through the reverse transcription process using an RNA transposition intermediate.

A transposase is any of a class of enzymes capable of binding to the end of a transposon and catalysing its movement to another part of a genome, typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the Tn3 transposon. The existence of transposons was postulated in the late 1940s by Barbara McClintock, who was studying the inheritance of maize, but the actual molecular basis for transposition was described by later groups. McClintock discovered that some segments of chromosomes changed their position, jumping between different loci or from one chromosome to another. The repositioning of these transposons allowed other genes for pigment to be expressed. Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause antibiotic resistance. Transposition is also important in creating genetic diversity within species and generating adaptability to changing living conditions.

<span class="mw-page-title-main">Omenn syndrome</span> Medical condition

Omenn syndrome is an autosomal recessive severe combined immunodeficiency. It is associated with hypomorphic missense mutations in immunologically relevant genes of T-cells such as recombination activating genes, Interleukin-7 receptor-α (IL7Rα), DCLRE1C-Artemis, RMRP-CHH, DNA-Ligase IV, common gamma chain, WHN-FOXN1, ZAP-70 and complete DiGeorge syndrome. It is fatal without treatment.

V(D)J recombination is the mechanism of somatic recombination that occurs only in developing lymphocytes during the early stages of T and B cell maturation. It results in the highly diverse repertoire of antibodies/immunoglobulins and T cell receptors (TCRs) found in B cells and T cells, respectively. The process is a defining feature of the adaptive immune system.

<span class="mw-page-title-main">Mobile genetic elements</span> DNA sequence whose position in the genome is variable

Mobile genetic elements (MGEs) sometimes called selfish genetic elements are a type of genetic material that can move around within a genome, or that can be transferred from one species or replicon to another. MGEs are found in all organisms. In humans, approximately 50% of the genome is thought to be MGEs. MGEs play a distinct role in evolution. Gene duplication events can also happen through the mechanism of MGEs. MGEs can also cause mutations in protein coding regions, which alters the protein functions. These mechanisms can also rearrange genes in the host genome generating variation. These mechanism can increase fitness by gaining new or additional functions. An example of MGEs in evolutionary context are that virulence factors and antibiotic resistance genes of MGEs can be transported to share genetic code with neighboring bacteria. However, MGEs can also decrease fitness by introducing disease-causing alleles or mutations. The set of MGEs in an organism is called a mobilome, which is composed of a large number of plasmids, transposons and viruses.

<span class="mw-page-title-main">RAG1</span> Protein-coding gene in the species Homo sapiens

Recombination activating gene 1 also known as RAG-1 is a protein that in humans is encoded by the RAG1 gene.

<span class="mw-page-title-main">RAG2</span> Protein-coding gene in the species Homo sapiens

Recombination activating gene 2protein is a lymphocyte-specific protein encoded by RAG2 gene on human chromosome 11. Together with RAG1 protein, RAG2 forms a V(D)J recombinase, a protein complex required for the process of V(D)J recombination during which the variable regions of immunoglobulin and T cell receptor genes are assembled in developing B and T lymphocytes. Therefore, RAG2 is essential for generation of mature B and T lymphocytes.

Trans-regulatory elements (TRE) are DNA sequences encoding upstream regulators, which may modify or regulate the expression of distant genes. Trans-acting factors interact with cis-regulatory elements to regulate gene expression. TRE mediates expression profiles of a large number of genes via trans-acting factors. While TRE mutations affect gene expression, it is also one of the main driving factors for evolutionary divergence in gene expression.

<span class="mw-page-title-main">HMGB2</span> Protein-coding gene in the species Homo sapiens

High-mobility group protein B2 also known as high-mobility group protein 2 (HMG-2) is a protein that in humans is encoded by the HMGB2 gene.

<span class="mw-page-title-main">GALNAC4S-6ST</span> Protein-coding gene in the species Homo sapiens

Carbohydrate sulfotransferase 15 is an enzyme that in humans is encoded by the CHST15 gene. It belongs to the N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase enzyme class.

<span class="mw-page-title-main">Junctional diversity</span> DNA sequence variations introduced in recombination

Junctional diversity describes the DNA sequence variations introduced by the improper joining of gene segments during the process of V(D)J recombination. This process of V(D)J recombination has vital roles for the vertebrate immune system, as it is able to generate a huge repertoire of different T-cell receptor (TCR) and immunoglobulin molecules required for pathogen antigen recognition by T-cells and B cells, respectively.

<span class="mw-page-title-main">KLHDC3</span> Protein-coding gene in the species Homo sapiens

Kelch domain-containing protein 3 is a protein that in humans is encoded by the KLHDC3 gene.

Recombination signal sequences are conserved sequences of noncoding DNA that are recognized by the RAG1/RAG2 enzyme complex during V(D)J recombination in immature B cells and T cells. Recombination signal sequences guide the enzyme complex to the V, D, and J gene segments that will undergo recombination during the formation of the heavy and light-chain variable regions in T-cell receptors and immunoglobulin molecules.

Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.

The Artemis complex is a protein complex that functions in V(D)J recombination, the somatic recombination process which generates diversity in T cell receptors and immunoglobulins. Mutations in the Artemis complex results in hypersensitivity to DNA double-strand break-inducing agents, such as radiation; and so people with mutations in the Artemis complex may develop radiosensitive severe combined immune deficiency (RS-SCID).

Antibody structure is made up of two heavy-chains and two light-chains. These chains are held together by disulfide bonds. The arrangement or processes that put together different parts of this antibody molecule play important role in antibody diversity and production of different subclasses or classes of antibodies. The organization and processes take place during the development and differentiation of B cells. That is, the controlled gene expression during transcription and translation coupled with the rearrangements of immunoglobulin gene segments result in the generation of antibody repertoire during development and maturation of B cells.

<span class="mw-page-title-main">Conservative transposition</span>

Transposition is the process by which a specific genetic sequence, known as a transposon, is moved from one location of the genome to another. Simple, or conservative transposition, is a non-replicative mode of transposition. That is, in conservative transposition the transposon is completely removed from the genome and reintegrated into a new, non-homologous locus, the same genetic sequence is conserved throughout the entire process. The site in which the transposon is reintegrated into the genome is called the target site. A target site can be in the same chromosome as the transposon or within a different chromosome. Conservative transposition uses the "cut-and-paste" mechanism driven by the catalytic activity of the enzyme transposase. Transposase acts like DNA scissors; it is an enzyme that cuts through double-stranded DNA to remove the transposon, then transfers and pastes it into a target site.

DNA transposons are DNA sequences, sometimes referred to "jumping genes", that can move and integrate to different locations within the genome. They are class II transposable elements (TEs) that move through a DNA intermediate, as opposed to class I TEs, retrotransposons, that move through an RNA intermediate. DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate. DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes. In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other genes associated with virulence. After replicating and propagating in a host, all transposon copies become inactivated and are lost unless the transposon passes to a genome by starting a new life cycle with horizontal transfer. It is important to note that DNA transposons do not randomly insert themselves into the genome, but rather show preference for specific sites.

Transib is a superfamily of interspersed repeats DNA transposons. It was named after the Trans-Siberian Express. It is similar to EnSpm/CACTA.

References

  1. 1 2 Jones JM, Gellert M (Aug 2004). "The taming of a transposon: V(D)J recombination and the immune system". Immunological Reviews. 200: 233–48. doi:10.1111/j.0105-2896.2004.00168.x. PMID   15242409. S2CID   12080467.
  2. ↑ Notarangelo LD, Kim MS, Walter JE, Lee YN (Mar 2016). "Human RAG mutations: biochemistry and clinical implications". Nature Reviews. Immunology. 16 (4): 234–46. doi:10.1038/nri.2016.28. PMC   5757527 . PMID   26996199.
  3. ↑ Oettinger MA, Schatz DG, Gorka C, Baltimore D (Jun 1990). "RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination". Science. 248 (4962): 1517–23. Bibcode:1990Sci...248.1517O. doi:10.1126/science.2360047. PMID   2360047.
  4. ↑ Ru H, Chambers MG, Fu TM, Tong AB, Liao M, Wu H (November 2015). "Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures". Cell. 163 (5): 1138–1152. doi:10.1016/j.cell.2015.10.055. PMC   4690471 . PMID   26548953.
  5. ↑ Kapitonov VV, Jurka J (June 2005). "RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons". PLOS Biology. 3 (6): e181. doi: 10.1371/journal.pbio.0030181 . PMC   1131882 . PMID   15898832.
  6. ↑ Panchin Y, Moroz LL (May 2008). "Molluscan mobile elements similar to the vertebrate Recombination-Activating Genes". Biochemical and Biophysical Research Communications. 369 (3): 818–823. doi:10.1016/j.bbrc.2008.02.097. PMC   2719772 . PMID   18313399.
  7. 1 2 3 Yakovenko I, Agronin J, Smith LC, Oren M (2021). "Guardian of the Genome: An Alternative RAG/Transib Co-Evolution Hypothesis for the Origin of V(D)J Recombination". Frontiers in Immunology. 12: 709165. doi: 10.3389/fimmu.2021.709165 . PMC   8355894 . PMID   34394111.
  8. 1 2 3 Kapitonov VV, Koonin EV (2015-04-28). "Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon". Biology Direct. 10 (1): 20. doi: 10.1186/s13062-015-0055-8 . PMC   4411706 . PMID   25928409.
  9. 1 2 Huang S, Tao X, Yuan S, Zhang Y, Li P, Beilinson HA, Zhang Y, Yu W, Pontarotti P, Escriva H, Le Petillon Y, Liu X, Chen S, Schatz DG, Xu A (June 2016). "Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination". Cell. 166 (1): 102–14. doi:10.1016/j.cell.2016.05.032. PMC   5017859 . PMID   27293192.
  10. ↑ Morales Poole JR, Huang SF, Xu A, Bayet J, Pontarotti P (June 2017). "The RAG transposon is active through the deuterostome evolution and domesticated in jawed vertebrates". Immunogenetics. 69 (6): 391–400. bioRxiv   10.1101/100735 . doi:10.1007/s00251-017-0979-5. PMID   28451741. S2CID   11192471.
  11. ↑ Zhang Y, Cheng TC, Huang G, Lu Q, Surleac MD, Mandell JD, Pontarotti P, Petrescu AJ, Xu A, Xiong Y, Schatz DG (May 2019). "Transposon molecular domestication and the evolution of the RAG recombinase". Nature. 569 (7754): 79–84. Bibcode:2019Natur.569...79Z. doi:10.1038/s41586-019-1093-7. PMC   6494689 . PMID   30971819.
  12. ↑ Kasahara M, Suzuki T, Pasquier LD (Feb 2004). "On the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates". Trends in Immunology. 25 (2): 105–11. doi:10.1016/j.it.2003.11.005. PMID   15102370.
  13. 1 2 Fugmann SD, Messier C, Novack LA, Cameron RA, Rast JP (Mar 2006). "An ancient evolutionary origin of the Rag1/2 gene locus". Proceedings of the National Academy of Sciences of the United States of America. 103 (10): 3728–33. Bibcode:2006PNAS..103.3728F. doi:10.1073/Pnas.0509720103. PMC   1450146 . PMID   16505374.
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  15. ↑ Panchin Y, Moroz LL (May 2008). "Molluscan mobile elements similar to the vertebrate Recombination-Activating Genes". Biochemical and Biophysical Research Communications. 369 (3): 818–23. doi:10.1016/j.bbrc.2008.02.097. PMC   2719772 . PMID   18313399.
  16. 1 2 3 Martin EC, Vicari C, Tsakou-Ngouafo L, Pontarotti P, Petrescu AJ, Schatz DG (2020-05-06). "Identification of RAG-like transposons in protostomes suggests their ancient bilaterian origin". Mobile DNA. 11 (1): 17. doi: 10.1186/s13100-020-00214-y . PMC   7204232 . PMID   32399063.
  17. ↑ Kasahara M (Oct 2007). "The 2R hypothesis: an update". Current Opinion in Immunology. Hematopoietic cell death/Immunogenetics/Transplantation. 19 (5): 547–52. doi:10.1016/j.coi.2007.07.009. PMID   17707623.
  18. ↑ van Niekerk G, Davis T, Engelbrecht AM (2015-09-04). "Was the evolutionary road towards adaptive immunity paved with endothelium?". Biology Direct. 10 (1): 47. doi: 10.1186/s13062-015-0079-0 . PMC   4560925 . PMID   26341882.

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