Characterizing effects of sumoylation on Thymine-DNA Glycosylase base excision repair activity in vivo
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Date
2014-06-30
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Johns Hopkins University
Abstract
Thymine-DNA glycosylase (TDG) is an enzyme that recognizes and repairs G/T and G/U mismatches in the base excision repair (BER) pathway. Its BER function is important in maintaining genome integrity and in regulating DNA gene expression through DNA demethylation of 5-methylcystosine (5mC). It has been proposed that dynamic sumoylation-desumoylation is required for efficient enzymatic turnover of TDG in vitro. TDG sumoylation alleviates product inhibition at the abasic site after enzyme processing and allows dissociation of TDG from DNA. Based on this model, subsequent desumoylation of TDG is required for complete enzymatic turnover. The effect of sumoylation on TDG activity in vivo, however, has not been studied. Here we have devised an in vivo assay to study the role of sumoylation in regulating TDG activity. Ten- eleven translocation (TET) enzyme iteratively oxidizes 5-mC of genomic DNA to a final product of 5-carboxylcytosine (5caC). Because TDG is the only enzyme capable of recognizing and repairing 5caC, we co-expressed TDG sumoylation mutant constructs with TET in HEK293T cells and measured 5caC levels of genomic DNA to monitor TDG activity. Our results showed that sumoylation does not affect TDG BER activity in vivo. We also characterized the specific-SUMO isopeptidases involved in TDG desumoylation. Co-expressing SENP1 and 2 constructs with wild-type TDG demonstrated that SENP1 preferentially desumoylates TDG. Additionally, only the catalytic domain of SENP1 is required for efficient TDG desumoylation. Our findings provide important insight into the molecular mechanisms that regulate key biological processes involving TDG activity such as gene regulation, development, and maintenance in genomic integrity.
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Keywords
Thymine-DNA Glycosylase, TDG, sumoylation, SUMO, DNA repair, Base excision repair, BER