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[This corrects the article DOI: 10.3389/fimmu.2022.871766.].

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Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is a B cell specific process required for the generation of specific and high affinity antibodies during the maturation of the immune response against foreign antigens. This process depends on the activity of both activation-induced cytidine deaminase (AID) and several DNA repair factors. AID-dependent SHM creates the full spectrum of mutations in Ig variable (V) regions equally distributed at G/C and A/T bases. In most mammalian cells, deamination of deoxycytidine into uracil during S phase induces targeted G/C mutagenesis using either direct replication of uracils or TLS mediated bypass, however only the machinery of activated B lymphocytes can generate A/T mutagenesis around AID-created uracils. The molecular mechanism behind the latter remains incompletely understood to date. However, the lack of a cellular model that reproduces both G/C and A/T mutation spectra constitutes the major hurdle to elucidating it. The few available B cell lines used thus far to study Ig SHM indeed undergo mainly G/C mutations, that make them inappropriate or of limited use. In this report, we show that in the Ramos cell line that undergoes constitutive G/C-biased SHM in culture, the low rate of A/T mutations is due to an imbalance in the ubiquitination/deubiquitination reaction of PCNA, with the deubiquitination reaction being predominant. The inhibition of the deubiquitinase complex USP1-UAF1 or the expression of constitutive fusion of ubiquitin to PCNA provides the missing clue required for DNA polymerase η recruitment and thereafter the introduction of A/T base pair (bp) mutations during the process of IgV gene diversification. This study reports the establishment of the first modified human B cell line that recapitulates the mechanism of SHM of Ig genes in vitro.

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The mechanism of (CAG)n repeat generation, and related expandable repeat diseases in non-dividing cells, is currently understood in terms of a DNA template-based DNA repair synthesis process involving hairpin stabilized slippage, local error-prone repair via MutSß (MSH2-MSH3) hairpin protective stabilization, then nascent strand extension by DNA polymerases-ß and -δ. We advance a very similar slipped hairpin-stabilized model involving MSH2-MSH3 with two key differences: the copying template may also be the nascent pre-mRNA with the repair pathway being mediated by the Y-family error-prone enzymes DNA polymerase-η and DNA polymerase-κ acting as reverse transcriptases. We argue that both DNA-based and RNA-based mechanisms could well be activated in affected non-dividing brain cells in vivo. Here, we compare the advantages of the RNA/RT-based model proposed by us as an adjunct to previously proposed models. In brief, our model depends upon dysregulated innate and adaptive immunity cascades involving AID/APOBEC and ADAR deaminases that are known to be involved in normal locus-specific immunoglobulin somatic hypermutation, cancer progression and somatic mutations at many off-target non-immunoglobulin sites across the genome: we explain how these processes could also play an active role in repeat expansion diseases at RNA polymerase II-transcribed genes.

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To identify the interchangeability of VH and VL framework region (FR) residues, we artificially introduced random mutations at all residue positions in a chicken monoclonal antibody, which has only one functional VH and Vλ gene. When we classified the amino acids into 5 groups by their physicochemical properties, all FR residues could be replaced by another group except L23 (C), H36 (W), H86 (D), H104 (G), and H106 (G). Eighty-two (50.9%), 48 (29.8%), 17 (10.6%), and 9 FR residues (5.6%) could be replaced by 4, 3, 2, and 1 group(s), individually, without significant loss of reactivity. We also confirmed a similar level of versatility with 2 different chicken antibodies. This high level of versatility on FR residues has not been predicted because it has not been observed in the 150 chicken antibodies that we previously generated or in the 1,269 naïve chicken VH sequences publically available. In conclusion, chicken antibody FR residues are highly interchangeable and this property can be applied for improving the physicochemical property of antibody including thermal stability, solubility and viscosity.

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To identify the interchangeability of V(H) and V(L) framework region (FR) residues, we artificially introduced random mutations at all residue positions in a chicken monoclonal antibody, which has only one functional V(H) and Vλ gene. When we classified the amino acids into 5 groups by their physicochemical properties, all FR residues could be replaced by another group except L23 (C), H36 (W), H86 (D), H104 (G), and H106 (G). Eighty-two (50.9%), 48 (29.8%), 17 (10.6%), and 9 FR residues (5.6%) could be replaced by 4, 3, 2, and 1 group(s), individually, without significant loss of reactivity. We also confirmed a similar level of versatility with 2 different chicken antibodies. This high level of versatility on FR residues has not been predicted because it has not been observed in the 150 chicken antibodies that we previously generated or in the 1,269 naïve chicken V(H) sequences publically available. In conclusion, chicken antibody FR residues are highly interchangeable and this property can be applied for improving the physicochemical property of antibody including thermal stability, solubility and viscosity.

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