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I'm studying V(D)J recombination. I think I have two incompatible books about explantaion of the process. Which is right?

  • In Molecular Biology of the Cell 5th Ed., firstly RAG (-1?) combines to RSSs of V and J genes independently. After that, a hairpin loop is formated by paring random(?) RAGs.
  • In Janeway's Immunobiology 8th Ed., RAG complex (RAG-1 and RAG-2) has two sites for RSSs from the beginning. And in the figure, V gene is firstly recruited for a site. J gene combines to the RAG complex after V gene.
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    $\begingroup$ It would be helpful if you could provide an actual excerpt from the text instead of a garbled summation. It's honestly hard to tell what your interpretation even is with some of the grammatical errors and use of incorrect terms to describe interactions. $\endgroup$ – InactionPotential Jun 26 '15 at 20:18
  • $\begingroup$ The question is whether components of the RAG complex bind the RSS of V and J segments independently and then come together to form the complete complex or whether the RAG complex assembles completely on one one RSS and then binds the second. $\endgroup$ – canadianer Jun 26 '15 at 21:00
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Excerpt from Cellular and Molecular Immunology, 8th ED, p.181-182. Don't up-vote me, i just thought this text offers a good, up-to-date explanation.

The Mechanism of V(D)J Recombination

Rearrangement of Ig and TCR genes represents a special kind of non-homologous DNA recombination event, mediated by the coordinated activities of several enzymes, some of which are found only in developing lymphocytes, whereas others are ubiquitous DNA double-stranded break repair (DSBR) enzymes.Although the mechanism of V(D)J recombination is fairly well understood and will be described here, how exactly specific loci are made accessible to the machinery involved in recombination remains to be determined. It is likely that the accessibility of the Ig and TCR loci to the enzymes that mediate recombination is regulated in developing B and T cells by several mechanisms, including epigenetic alterations in chromatin structure and DNA as discussed earlier, and basal transcriptional activity in the gene loci. The process of V(D)J recombination can be divided into four distinct events that flow sequentially from one to the next (Fig. 8-10):

1. Synapsis: Portions of the chromosome on which the antigen receptor gene is located are made accessible to the recombination machinery. Two selected coding segments and their adjacent RSSs are brought together by a chromosomal looping event and held in position for subsequent cleavage, processing, and joining.

2. Cleavage: Double-stranded breaks are enzymatically generated at RSS-coding sequence junctions by machinery that is lymphoid specific. Two proteins encoded by lymphoid-specific genes, called recombination-activating gene 1and recombination-activating gene 2(RAG1and RAG2), form a complex, containing two molecules of each protein, that plays an essential role in V(D)J recombination. The Rag-1/Rag-2complex is also known as the V(D)J recombinase.The Rag-1 protein, in a manner similar to a restriction endonuclease, recognizes the DNA sequence at the junction between a heptamer and a coding segment and cleaves it, but it is enzymatically active only when complexed with the Rag-2 protein. The Rag-2 protein may help link the Rag-1/Rag-2 tetramer to other proteins, including accessibility factors that bring these proteins to specific open receptor gene loci at specific times and at defined stages of lymphocyte development. Rag-1 and Rag-2 contribute to holding together gene segments during the process of chromosomal folding or synapsis. Rag-1 then makes a nick (on one DNA strand) between the coding end and the heptamer. The released 3′OH of the coding end then attacks a phosphodiester bond on the other DNA strand, forming a covalent hairpin. The signal end (including the heptamer and the rest of the RSS) does not form a hairpin and is generated as a blunt double-stranded DNA terminus that undergoes no further processing. This double-stranded break results in a closed hairpin of one coding segment being held in apposition to the closed hairpin of the other coding end and two blunt recombination signal ends being placed next to each other. Rag-1 and Rag-2, apart from generating the double-stranded breaks, also hold the hairpin ends and the blunt ends together before the modification of the coding ends and the process of ligation.

RAGgenes are lymphoid specific and are expressed only in developing B and T cells. Rag proteins are expressed mainly in the G0and G1 stages of the cell cycle and are inactivated in proliferating cells. It is thought that limiting DNA cleavage and recombination to the G 0 and G1 stages minimizes the risk of generating inappropriate DNA breaks during DNA replication or during mitosis. Mice without functional Rag1 or Rag2genes (Ragknockout mice) fail to develop B or T lymphocytes, and Rag-1 or Rag-2 deficiency is also a rare cause of SCID, in which patients also lack all lymphocytes.

3. Hairpin opening and end-processing: The broken coding ends are modified by the addition or removal of bases, and thus greater diversity is generated. After the formation of double-stranded breaks, hairpins must be resolved (opened up) at the coding junctions, and bases may be added to or removed from the coding ends to ensure even greater diversification. Artemisis an endonuclease that opens up the hairpins at the coding ends. In the absence of Artemis, hairpins cannot be opened, and mature T and B cells cannot be generated. Mutations in ARTEMIS are a rare cause of SCID, similar to patients with RAG1or RAG2mutations. (see Chapter 21). A lymphoid-specific enzyme, called terminal deoxynucleotidyl transferase (TdT), adds bases to broken DNA ends and will be discussed later in the chapter in the context of junctional diversity.

4. Joining: The broken coding ends as well as the signal ends are brought together and ligated by a double-stranded break repair process found in all cells that is called nonhomologous end joining. A number of ubiquitous factors participate in nonhomologous end joining. Ku70 and Ku80 are DNA end-binding proteins that bind to the breaks and recruit the catalytic subunit of DNA-dependent protein kinase (DNA-PK), a double-stranded DNA repair enzyme. This enzyme is defective in mice carrying the severe combined immunodeficiency (scid)mutation, and mutations in the gene encoding this enzyme have also been discovered in human SCID patients (see Chapter 21). Like Rag-deficient mice, scidmice fail to produce mature lymphocytes. DNA-PK also phosphorylates and activates Artemis, which, as mentioned before, is involved in end processing. Ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4, the latter being a non-catalytic but essential subunit of the ligase.

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Figure 8-10, Cellular and Molecular Immunology, 8e, p182.

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  • $\begingroup$ If you downvote you should include an explanation. $\endgroup$ – CKM Jun 26 '15 at 18:21
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    $\begingroup$ Upvoted to bring balance to the force. Also all RSSes are not identical in the TCR/BCR genes. The RSS structure is a conserved heptamer followed by either a 12 or 23bp unconserved spacer, followed by a conserved nonamer. An RSS (12) and RSS (23) recombine much more efficiently than two (12) or two (23) sites. The direction and location of the (12) vs (23) sites facilitates the desired juxtaposition of a V site with a D/J site. $\endgroup$ – InactionPotential Jun 26 '15 at 20:30

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