Abstract
Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.
Highlights
Base excision repair is an important pathway for cytosine demethylation at the CpG dinucleotide for epigenetic control
We measured the activities of purified thymine DNA glycosylase (TDG) and oxoG DNA glycosylase 1 (OGG1) on variations of the altered CpG dinucleotide
The TDG-mediated base excision repair of the modified 5-mC (i.e. T, 5-hydroxymethyluracil, 5-formylcytosine, or 5-carboxylcytosine) at CpG dinucleotides is important for active DNA demethylation during cell development [17, 21, 57]
Summary
Base excision repair is an important pathway for cytosine demethylation at the CpG dinucleotide for epigenetic control. Results: A deaminated 5-methylcytosine (thymine) and an adjacent oxidized guanine (7,8-dihydro-8-oxoguanine) retard base excision repair of each lesion. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). The OGG1-initiated BER pathway is blocked due to the 5-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase  activities. In TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide
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