The purification of a mismatch-specific thymine-DNA glycosylase from HeLa cells

G/T mispairs that arise in the DNA of higher eukaryotes as a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine must be corrected to G/C pairs. We describe here the purification to apparent homogeneity of the enzyme that initiates this repair process by excising the mispaired thymine from the hetero-duplex to generate an apyrimidinic site. The enzymatic activity could be attributed to a 55-kDa polypeptide, which was purified from extracts of HeLa cells by a combination of conventional and DNA-affinity chromatography. The enzyme is a mismatch-specific thymine-DNA N-glycosylase, capable of hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of the DNA and a mispaired thymine. In addition to the G/T, the enzyme can remove thymine also from C/T and T/T mispairs in the order G/T >> C/T > T/T. It has no detectable endonucleolytic activity on apyrimidinic sites and does not catalyze the removal of thymine from A/T pairs or from single-stranded DNA.