The main role of human thymine-DNA glycosylase is removal of thymine produced by deamination of 5-methylcytosine and not removal of ethenocytosine

Metabolites of vinyl chloride react with cytosine in DNA to form 3,N-4-ethenocytosine. Recent studies suggest that ethenocytosine is repaired by the base excision repair pathway with the ethenobase being removed by thymine-DNA glycosylase. Here single turnover kinetics have been used to compare the excision of ethenocytosine by thymine-DNA glycosylase with the excision of thymine. The effect of flanking DNA sequence on the excision of ethenocytosine was also investigated. The 34-bp duplexes studied here fall into three categories. Ethenocytosine base-paired with guanine within a CpG site (i.e. CpG .C-is an element of-DNA) was by far the best substrate having a specificity constant (k(2)/K-d) of 25.1 x 10(6) M-1 S-1. The next best substrates were DNA duplexes containing TpG.C-is an element of, GpG.C-is an element of, and CpG.T. These had specificity constants 45-130 times smaller than CpG -'C-DNA. The worst substrates were DNA duplexes containing ApG-'C and TpG.T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG.C-is an element of-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G.T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G.C-is an element of base pair more easily than it can flip out thymine from a G.T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.