The versatile thymine DNA-glycosylase: a comparative characterization of the human, Drosophila and ®ssion yeast orthologs

Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G´T and G´U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spon-taneous deamination of 5-methylcytosine and cyto-sine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically signi®cant facts. (i) All eukaryotic TDG orthologs have broad and species-speci®c substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most ef®ciently processed substrates of all are uracil and 3,N4-ethenocytosine opposite guanine and 5-¯uorouracil in any double-stranded DNA context; (iii) 5-methyl-cytosine and thymine derivatives are processed with an appreciable ef®ciency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5¢-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the sub-strate and is not a stringent feature of this subfamily of DNA glycosylases. These ®ndings advance our current view on the role of TDG proteins and docu-ment that they have evolved with high structural ¯exibility to counter a broad range of DNA base damage in accordance with the speci®c needs of individual species