2 Role of RAG1 and RAG2 in B-cell development | 25 Figure 2. (A) Schematic representation of DNA cleavage process during V(D)J recombination. RAG1 and RAG2, assisted by high-mobility group proteins (HMGB), binds the V(D)J segments (here represented by the yellow/purple box) at recombination signal sequences (RSS) (represented by the yellow/purple triangles). Next, a synapse is formed and nicked by the RAG1/2 at the RSS. The cleaved DNA is then repaired in the process of non-homologous end joining (NHEJ), leading to the formation of a coding joint (the recombines loci) and a formation of a signal joint, also called the excised signal circle “ESC” (the intervening loci) (B) A more detailed schematic representation of the molecular mechanism of V(D)J recombination. RAG1/2 recombinase introduces a nick on one DNA strand. Subsequently, the free -OH group, created by the nick, attacks the other DNA strand by transesterification, leading to the formation of a break at the other DNA strand, which ultimately creates a double-stranded DNA break (DSB). A hairpin is formed at the broken end, and the Ku70 and Ku80 bind and stabilize the loose DNA end, they also attract other proteins involved in the DNA repair process. To resolve the DSB, first, the hairpin is opened by Artemis, which in the presence of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) acts as an endonuclease. Upon hairpin opening, the shorter DNA strand is extended by the addition of palindromic nucleotides (P-nucleotides) at the coding end, and the terminal deoxynucleotidyl transferase (TdT) further diversifies the junction by catalyzing the addition of non-templated nucleotides (N-nucleotides). The ends are filled by the Polymerase X family of polymerases (Pol μ and Pol λ) and finally, the broken DNA is sealed by Ligase IV/XRCC4. Created with BioRender.com
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