INVESTIGATION OF THE MECHANISMS OF DNA-BINDING OF THE HUMAN G T GLYCOSYLASE USING DESIGNED INHIBITORS/

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch- specific thymine DNA glycosylase (TDG or G/T glycosylase), which catal yzes specific excision of the thymine base through N-glycosidic bond h ydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant p airing of two normal bases rather than a damaged base per se. An impor tant structural issue is thus to understand how the enzyme specificall y targets the T (or U) residue of the mismatched base pair, Our approa ch toward the study of substrate recognition and processing by catalyt ic DNA binding proteins has been to modify the substrate so as to pres erve recognition of the base but to prevent its excision, Here we repo rt that replacement of 2'-hydrogen atoms with fluorine in the substrat e 2'-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, th ereby leading to the formation of a tight, specific glycosylase-DNA co mplex, Biochemical characterization of these complexes reveals that th e enzyme protects an approximate to 20-bp stretch of the substrate fro m DNase I cleavage, and directly contacts a G residue on the 3' side o f the mismatched U derivative. These studies provide a mechanistic rat ionale for the preferential repair of deaminated CpG sites and pave th e way for future high-resolution studies of TDG bound to DNA.