Once RAG cleaves, leaving hairpins of DNA, the hairpins have to be opened up by enzymes but it is done asymmetrically. Thus nucleotides are tainted and lost at the junctions. Approximately 2 out of 3 rearrangements are non-productive due to the frameshift. Then exonuclease cleaves the hairpin, leaving the cut asymmetric. Furthermore, uniquely to lymphocytes, terminal deoxynucleotidal transferase (TdT) would add up to 20 nucleotides randomly, without a template, to the free 3’termini of coding ends following their cleavage by RAG1/2 recombinases. This is described as the junctional diversity, which gives extra diversity in the CDR3 region of the antibody heavy chain. After ligating, there may be involvement of nucleolytic activity, removing nucleotides. It is important to note that the RAG-1 is inducing VDJ recombinase inefficiently. However when cotransfected with RAG2, it resulted in a huge increase in the frequency of recombination. This synergy of RAG-1 and RAG-2 can be seen when pHG200 was introduced into a standard recipient fibroblast, 3TGR, with either RAG cDNAs cloned in a mammalian expression vector, and no Cam clones that hybridise dot the oligonucleotides were observed. In contrast, hundreds of rearranged plasmids were recovered from fibroblasts transfected with both RAG-1 and RAG-2. There was over 1000fold increase in recombinase activity compared to the activity induced by either. Further evidence can be found when northern blot analysis was done on Rag-1 alone first, and they found the pattern of expression was quite similar to the expression of VDH recombinase activity. With using RAG-2 cDNA as a probe, an identical expression profile was found.Somatic hypermutation would also occur, involving error-prone DNA repair. Activation-Induced Cytidine deaminase (AID) deaminated a cytidine residue, creating a guanosine-uridine mismatch, which can be facilitated by 3 different pathways. The first is deoxyuridine is interpreted by the DNA replication machinery as if it were a deoxythymidine. His results in the creation of A-T pair in place of the original G-C pair in the daughter cells. The second is the mismatched uridine has been excised by one of the uridine DNA glycosylase enzymes, leaving an basic site. The uridine can then be replaced by any of the four bases, in a reaction known as short-patch base excision repair, which can be catalysed by one of the number of error-prone polymerases. The last one is mismatch repair enzymes can detect the mismatch and excise a longer stretch of the DNA surrounding the U-G couple. Error-prone polymerases are then recruited to the hypermutable site by proliferating cell nuclear antigen and these polymerases can introduce a number of mutations around the original mismatch. It is important to note that analysis of Ig variable region sequences revealed that some sequence motifs, such as DGYW/WRCH, are more likely to be targeted by mutational apparatus at the underlined G-C pair, also referred to as mutational hotspots. The DGYW motif is also found frequently in the CSR, which appears to be an important sequence to direct AID binding to certain parts of the DNA.Lastly isotope switching recombination can also contribute to the diversity. Last switching occurs by the induction of recombination between donor and acceptor switch regions located 2kb to 3kb upstream from each CH region (except for Cepisilon). CSR occurs by end-joining mechanism similar to SHM, where the process is initiated by AID. The AID enzyme initiates class switch regions (CSR) by delaminating cytidine residues within the switch regions upstream of Cv and Cepsilon on both strands. This leads to the formation fo double-stranded breaks within both S regions that are resolved by DNA repair mechanisms, with the loss of the intervening DNA sequence. Furthermore, the process of CSR can potentially occur more than once during the lifetime of the cell. For example, an initial CSR vent can switch the cell from making IgM to synthesise IgG1, and a second CSR can switch it to making IgE or IgA.However, the ability to generate random recognition receptors is a double edge sword. It could resulting in some recognising and target the host. 24% of naive B cell antibody specificities in the peripheral blood were found to be polyreactive/autoreactive, and 7% of circulating naive B cells are anergia, which would be an indication that they are possibly self-reactive. As such, the immune system must somehow avoid recognising and destroying host cells. A good example could be seen when a study was done using Tdt-/- mice showing autoantibodies in SLE can raise ein the periphery from non-autoantigen reactive B cells by somatic hypermutation.This principle which relies on self/nonself discrimination is called self-tolerance. Self-Tolerance is a state of unresponsiveness to self-antigens, which refers to the mechanism that control self/non-self discrimination which protect an individual from anti-self immune attacks by anti-self immune attacks by self-reactive lymphocytes. Autoimmune disease involve the failure of the elimination and inhibition mechanism that maintain self-tolerance by the immune system. For safe guard against the autoimmunity, negative selection of anti-self B cells can occur in the bone marrow via 3 methods. First is the clonal deletion fo immature-high affinity self-reactive B cells by apoptosis if self-antigen is multivalent and immobilised e.g. expressed on cell membranes. So weakly reactive clones survive and enter the periphery. The second is receptor editing. A different light chain pairs with the heavy chain in immature B cells to eliminate self-specificity when the self antigen is multivalent and cross-links BCR. The third is anergy, where self reactive immature B cells exposed to large amounts of soluble, weakly cross-linking, low valence self antigen in the bone marrow migrate to the periphery where they are anergic. These processes would would allow only 10% of B-cells to enter the bloodstream.