[37] However, this

was demonstrated only in vitro in a non-physiological concentration of MnCl2 using X-396 cost isolated RSS substrates and not in the physiological 12RSS and 23RSS pair.[37] The in vivo scenario is still unclear though it is commonly thought that Mg2+ is the physiological divalent metal ion involved in RAG-mediated cleavage. RAG1 and RAG2 are assisted by high mobility group proteins of the HMG-box family (HMGB1 and HMGB2) for bringing two signal ends together. The HMG proteins interact with the nonamer binding domain of RAG1 in the absence of DNA and enhance its intrinsic DNA bending activity.[38] Following resolution of the hairpin structure, the coding ends are joined to create the exon, which forms the antigen-binding region of the antigen receptors (Fig. 2c). The signal ends remain bound to RAGs, which in turn protect the IDO inhibitor ends from further nuclease digestion [36, 39] (Fig. 2c). The blunt-ended signal ends can be directly ligated without any modification, while the coding ends undergo further processing (Fig. 2c).[34,

35] The hairpin at the coding end is opened and joined together by non-homologous end joining (NHEJ), the DNA double-strand break repair pathway.[40, 41] Artemis, in conjunction with DNA-PKcs, acts as an endonuclease and resolves the hairpins formed during V(D)J recombination.[42] Ku heterodimer, consisting of Ku70 and Ku80, binds to the broken DNA ends and forms a complex with DNA-PKcs.[43] Artemis has an inherent 5′-3′ exonuclease activity, whereas in association with DNA-PKcs it acts as a 5′-3′ endonuclease.[42] The ends are filled in by the Pol X family of polymerases namely Pol μ and

PJ34 HCl Pol λ. Mice deficient in Pol μ are shown to have shorter immunoglobulin light chain V to J junctions,[44] while those lacking Pol λ have shorter immunoglobulin heavy-chain D to J and V to DJ junctions.[45] In addition, Pol μ plays a role in the processing of 3′ ends while Pol λ processes the 5′ ends.[45] This would suggest that Pol μ is involved in V(D)J recombination, but not Pol λ.[46] The ligase IV/XRCC4 complex ligates the processed ends[47, 48] with the help of XLF.[49, 50] Ku70, Ku80, XRCC4 and Ligase IV are considered to be the ‘core’ NHEJ factors as these proteins were conserved during evolution and are required for all known NHEJ reactions.[51, 52] These are also inevitable for the joining of both coding and signal ends. On the other hand, DNA-PKcs and Artemis are believed to have evolved more recently and are needed only for the joining of coding ends.[52] At the time of joining of V, D and J subexons, several modifications like insertions and deletions can occur at the junctions resulting in further increase in the antigen receptor diversity. Asymmetric hairpin opening at the coding ends due to nicking a few bases away from the terminus results in one DNA strand longer than the other.