Abstract
Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.
Highlights
Activation induced deaminase (AID) is essential for both Ig somatic hypermutation (SHM) and class switch recombination (CSR) in mature B cells [1, 2]
AID initiates SHM and CSR by deaminating deoxycytidine to deoxyuracil that is processed by base excision repair (BER) and mismatch repair (MMR) [reviewed in [4,5,6]]
The BER pathway facilitates the excision of dU bases by a uracil DNA glycosylase (UNG) leaving an abasic site that is cleaved by apurinic/apyrimidinic endonuclease (APE) producing a single strand break
Summary
Activation induced deaminase (AID) is essential for both Ig somatic hypermutation (SHM) and class switch recombination (CSR) in mature B cells [1, 2]. There are four uracil DNA glycosylases, that are capable of recognizing and removing dU in U:G mismatches, UNG, SMUG1, TDG, and methyl binding domain protein 4 (MBD4) [8, 9]. MBD4 in Somatic Hypermutation studies show that UNG deficiency in mice [10] and humans [11] reduces CSR 90–95% and perturbs SHM mutation spectra but does not alter SHM frequency. A recent study suggests that during SHM TDG and SMUG1 provide uracil glycosylase activity in the absence of UNG [14]. The predicted three-dimensional structure of the chicken MBD4 uracil glycosylase domain is essentially identical with that of human making it highly likely that it has functional activity. Loss of the highly conserved amino acids in the uracil glycosylase domain led to increase of SHM frequency in the DT40 deletion variants as compared to control cells.
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