Uracil DNA Glycosylase UNG2 carrying the germline mutation Arg88Cys: Potential implications for adaptive immunity and cancer
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Uracil is normally found in RNA where it replaces the DNA base thymine. However, it can occur in DNA if misincorporated in place of thymine, or as a result of deamination of cytosine. Uracil is also a normal intermediate in B-cells during antibody maturation, when cytosine gets enzymatically deaminated by AID. UNG2 is the quantitatively dominating glycosylase in the process of removing uracil from nuclear DNA and initiate base excision repair (BER) during replication and antibody maturation. During normal BER, a glycosylase removes the damaged base, and a high fidelity polymerase incorporates the correct base in the apurinic/apyrimidinic site before the strand is religated. Conversely, during antibody maturation, processing of AID-initiated uracil become error prone. This introduces mutations in the variable regions of the immunoglobulin genes to increase antibody specificity and strand breaks in the switch regions to alter antibody effector functions. Various mutations in the UNG gene can result in hyper IgM syndrome (HIGM) in humans, which is recognized by increased amounts of serum IgM and low or absent levels of IgA, IgE and IgG, causing immunodeficiency. In a patient that suffers from this disease there has been discovered a heterozygous mutation leading to an arginine to cysteine substitution at position 88 in the N-terminal unstructured domain of UNG. This mutation lies within the RPA-binding motif and impairs UNG2 interaction with RPA. This thesis was aimed at demonstrating whether UNG protein from the mutated allele was expressed in B-cells from this HIGM patient. By utilizing proteomics tools such as 1D and 2D PAGE and mass spectrometry, we found convincing evidence proving protein expression from both WT and mutated allele.