24 | Chapter 2 for DNA cleavage55. The human and murine RAG2 is composed of only 527 residues and has no direct contribution to DNA cutting, but enhances the RAG1 DNA cleavage activity by hundreds of folds. The RAG2 is folded into a six-bladed β-propeller or Kelch-repeat structure56. The diverse domains in both RAGs can be categorized as either catalytically essential “core” domains or “non-core” domains, which are dispensable for the enzymatic activity but have a regulatory function57. For instance, non-core RAG2, consisting of approximately one-third of the full-length protein, mediates its nuclear localization, association with specific histone-modified chromatin, and facilitates the long-range recombination reactions58–60. The non-core RAG2 also contains a zinc-binding domain called plant homology domain (PHD), which coordinates with two zinc ions, and it specifically binds the histone H3 trimethylated on Lysine 4 (H3K4me3). Mutations in this region lead to severe defects in V(D)J recombination activity and failure of RAG2-PHD to bind H3K4me3 and has been linked to Omenn syndrome61. Non-core RAG1 increases the efficiency and fidelity of V(D)J recombination62,63. An important feature in the N-terminal of non-core RAG1 is the zinc-binding dimerization domain (ZDD). ZDD contains two sub-domains, the C2H2 zinc finger and the Really Interesting New Gene (RING) finger, which act as an E3 ubiquitin ligase mediating (auto)ubiquitination of Histone H3. Point mutations in this domain result in decreased V(D)J recombination activity64,65. Structural studies in mice and zebrafish revealed that RAG1 and RAG2 interact and form together a Y-shaped heterodimer of 230kDa size, the RAG complex is composed of two core subunits, forming a heterotetramer that holds the two paired DNA strands, where RAG1 catalyzes the DNA nicking step. RAG2 is in particular responsible for stabilizing the protein-protein interaction between the two halves of the RAG tetramer, and for conferring the specificity to the interaction of RAG1 with the DNA substrate66,67. Molecular mechanism of V(D)J recombination V(D)J recombination is initiated by the RAG1/2 endonuclease heterotetramer,56 introducing a single-stranded DNA nick in the RSSs flanking the two participating coding sequences (Figure 2A and 2B) The RSSs contain conserved palindromic heptamer (CACAGTG) and an AT-rich nonamer (ACAAAAACC), separated by 12 or 23 nucleotides of a less-conserved spacer sequence. The nucleotide sequence of the RSS may vary, however, the gene recombination can (normally) occur only between genes flanked by the RSS segments, and it is only efficient when it takes place between RSSs with different space lengths, this is also known as the “12/23 rule”. In the recombination of T-cell receptor beta and delta loci, additional spatial restrictions apply, so-called “beyond 12/23 restriction”68,69. The RSSs are limiting sequence structures required for V(D)J recombination; recombination on artificial substrates can be successfully achieved in vitro and in vivo when the substrates are flanked by canonical RSSs70,71. The orientation of RSS also determines if the joining of the coding segments proceeds by inversion or by deletion of the intervening sequence72.
RkJQdWJsaXNoZXIy MTk4NDMw