CLONING AND EXPRESSION OF HUMAN G T MISMATCH-SPECIFIC THYMINE-DNA GLYCOSYLASE/

Hydrolytic deamination of 5-methylcytosine leads to the formation of G /T mismatches. We have shown previously that these G/T mispairs are co rrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, T DG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two dis tinct cDNA species that differ by 100 nucleotides at the 3'-untranslat ed region. These cDNAs contain a 410-amino acid open reading frame tha t encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously clo ned mouse cDNA sequence that could be shown to encode the murine TDG h omologue. No common amino acid sequence motifs between the G/T-specifi c enzyme and other DNA glycosylases could be found, suggesting that TD G belongs to a new class of base-excision repair enzymes.