Summary: Recombination activating protein 2
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Recombination activating gene Edit Wikipedia article
|recombination activating gene 1|
|Locus||Chr. 11 p13|
|recombination activating gene 2|
|Locus||Chr. 11 p13|
|Recombination activating protein 2|
|Recombination activating protein 1|
The recombination activating genes (RAGs) 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. There are two recombination activating gene products known as RAG-1 and RAG-2, whose cellular expression is restricted to lymphocytes during their developmental stages. RAG-1 and RAG-2 are essential to the generation of mature B and T lymphocytes, two cell types that are crucial components of the adaptive immune system.
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 VDJ recombination. 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 ânickâ 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 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).
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 largely concentrated in a core region; residues 384â1008 of RAG-1 and residues 1â387 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 384â454 of RAG-1 comprise a nonamer-binding region (NBR) that specifically binds the conserved nonomer (9 nucleotides) of the RSS and the central domain (amino acids 528â760) 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.
Based on core sequence homology, it is believed that the RAG-1 protein evolved from a transposon of the Transib superfamily. Although the transposon origins of these genes are well established, there is still no consensus on when the ancestral RAG1/2 became present in the vertebrate genome. Because agnathans lack a core RAG1 element, it was traditionally assumed that RAG1 invaded after the agnathan/gnathastome split 1001 to 590 million years ago (MYA). However, recently the core sequence of RAG1 has been identified in the echinoderm Strongylocentrotus purpuratus (purple star fish)  and in the amphioxi Brachiostoma floridae (Florida lancelet). These findings indicate RAG1 may have invaded much earlier in evolutionary history that previously thought. Based on current evidence it is not clear whether RAG1 invaded in a very early ancestor of all deuterostomia (approx. 896 MYA) and was later lost in gnathostomes, or if RAG1 may have possibly invaded the genome multiple times  It should also be noted that RAG1/2 is only found in gnathostomes, and not in agnathans. 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.
- Jones, Jessica M.; Gellert, Martin (2004). "The taming of a transposon: V(D)J recombination and the immune system". Immunological Reviews 200: 233â48. doi:10.1111/j.0105-2896.2004.00168.x. PMID 15242409.
- Kapitonov, V.V; Jurka, Jerzy (Jun 2005). "RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons". PLoS Biology 3 (6): 998â1011. doi:10.1371/journal.pbio.0030181. PMC 1131882. PMID 15898832.
- "On the origins of the adaptive immune system: Novel insights from invertebrates and cold-blooded vertebrates". Trends in Immunology 25 (2): 105â111. Feb 2004. doi:10.1016/j.it.2003.11.005. PMID 15102370.
- Fugmann, S. D.; Messier, C; Novack, LA; Cameron, RA; Rast, JP (7). "An ancient evolution origin of the Rag1/2 gene locus". Proceedings of the National Academy of Sciences of the United States of America 103 (10): 3728â3733. doi:10.1073/Pnas.0509720103. PMC 1450146. PMID 16505374.
- Holland, L. Z.; Albalat, R.; Azumi, K.; Benito-Gutierrez, E.; Blow, M. J.; Bronner-Fraser, M.; Brunet, F.; Butts, T. et al. (Aug 2008). "The amphioxus genome illuminates vertebrate origins and cephalochordate biology". Genome Research 18 (8): 1380â1380. doi:10.1101/gr.073676.107. PMC 2493399. PMID 18562680.
- Flajnik, MF; Kasahara, Masanori (Jan 2010). "Origin and evolution of the adaptive immune system: genetic events and selective pressures". Nature Reviews Genetics 11 (1): 47â59. doi:10.1038/nrg2703. PMID 19997068.
- Janeway CA, Jr. et al. (2005). Immunobiology. (6th ed.). Garland Science. ISBN 0-443-07310-4.
- Abbas AK and Lichtman AH (2003). Cellular and Molecular Immunology (5th ed.). Saunders, Philadelphia. ISBN 0-7216-0008-5.
- Sadofsky, Moshe J. (August 2004). "Recombination-activating gene proteins: more regulation, please". Immunol. Rev. 200: 83â9. doi:10.1111/j.0105-2896.2004.00164.x. PMID 15242398.
- De, Pallabi; Rodgers, Karla K. (August 2004). "Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1". Immunol. Rev. 200: 70â82. doi:10.1111/j.0105-2896.2004.00154.x. PMID 15242397.
- Kapitonov, Vladimir V.; Jurka, Jerzy (May 2005). "RAG1 Core and V(D)J Recombination Signal Sequences Were Derived from Transib Transposons". PLoS Biol. 3 (6): e181. doi:10.1371/journal.pbio.0030181. PMC 1131882. PMID 15898832.
- Travis, John (November 1998). "The Accidental Immune System; Long ago, a wandering piece of DNAâperhaps from a microbeâcreated a key strategy". Science News 154 (19): 302. doi:10.2307/4010948. A simple explanation of recombination activating gene for the general reader.
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V-D-J recombination is the combinatorial process by which the huge range of immunoglobulin and T cell binding specificity is generated from a limited amount of genetic material. This process is synergistically activated by RAG1 and RAG2 in developing lymphocytes. Defects in RAG2 in humans are a cause of severe combined immunodeficiency B cell negative and Omenn syndrome.
Internal database links
|Similarity to PfamA using HHSearch:||Kelch_3|
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR004321
The variable portion of the genes encoding immunoglobulins and T cell receptors are assembled from component V, D, and J DNA segments by a site-specific recombination reaction termed V(D)J recombination. V(D)J recombination is targeted to specific sites on the chromosome by recombination signal sequences (RSSs) that flank antigen receptor gene segments. The RSS consists of a conserved heptamer (consensus, 5'-CACAGTG-3') and nonamer (consensus, 5'-ACAAAAACC-3') separated by a spacer of either 12 or 23 bp. Efficient recombination occurs between a 12-RSS and a 23-RSS, a restriction known as the 12/23 rule.
V(D)J recombination can be divided into two phases, DNA cleavage and DNA joining. DNA cleavage requires two lymphocyte-specific factors, the products of the recombination activating genes, RAG1 and RAG2, which together recognise the RSSs and create double strand breaks at the RSS-coding segment junctions [PUBMED:11961538]. RAG-mediated DNA cleavage occurs in a synaptic complex termed the paired complex, which is constituted from two distinct RSS-RAG complexes, a 12-SC and a 23-SC (where SC stands for signal complex). The DNA cleavage reaction involves two distinct enzymatic steps, initial nicking that creates a 3'-OH between a coding segment and its RSS, followed by hairpin formation in which the newly created 3'-OH attacks a phosphodiester bond on the opposite DNA strand. This generates a blunt, 5' phosphorylated signal end containing all of the RSS elements, and a covalently sealed hairpin coding end.
The second phase of V(D)J recombination, in which broken DNA fragments are processed and joined, is less well characterised. Signal ends are typically joined precisely to form a signal joint, whereas joining of the coding ends requires the hairpin structure to be opened and typically involves nucleotide addition and deletion before formation of the coding joint. The factors involved in these processes include ubiquitously expressed proteins involved in the repair of DNA double strand breaks by nonhomologous end joining, terminal deoxynucleotidyl transferase, and Artemis protein.
In addition to their critical roles in RSS recognition and DNA cleavage, the RAG proteins may perform two distinct types of functions in the postcleavage phase of V(D)J. A structural function has been inferred from the finding that, after DNA cleavage in vitro, the DNA ends remain associated with the RAG proteins in a "four end" complex known as the cleaved signal complex. After release of the coding ends in vitro, and after coding joint formation in vivo, the RAG proteins remain in a stable signal end complex (SEC) containing the two signal ends. These postcleavage complexes may serve as essential scaffolds for the second phase of the reaction, with the RAG proteins acting to organise the DNA processing and joining events.
The second type of RAG protein-mediated postcleavage activity is the catalysis of phosphodiester bond hydrolysis and strand transfer reactions. The RAG proteins are capable of opening hairpin coding ends in vitro. The RAG proteins also show 3' flap endonuclease activity that may contribute to coding end processing/joining and can utilise the 3' OH group on the signal ends to attack hairpin coding ends (forming hybrid or open/shut joints) or virtually any DNA duplex (forming a transposition product).
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|Cellular component||nucleus (GO:0005634)|
|Molecular function||DNA binding (GO:0003677)|
|Biological process||DNA recombination (GO:0006310)|
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|Seed source:||Pfam-B_4702 (release 6.5)|
|Number in seed:||18|
|Number in full:||5792|
|Average length of the domain:||268.30 aa|
|Average identity of full alignment:||58 %|
|Average coverage of the sequence by the domain:||91.61 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 23193494 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||9|
|Download:||download the raw HMM for this family|
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