title: Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors.
abstract: 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 catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base per se. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2'-hydrogen atoms with fluorine in the substrate 2'-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase-DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an approximately 20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3' side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: O D
authors.initials: OD
authors.lastname: Schärer
authors.forename: T
authors.initials: T
authors.lastname: Kawate
authors.forename: P
authors.initials: P
authors.lastname: Gallinari
authors.forename: J
authors.initials: J
authors.lastname: Jiricny
authors.forename: G L
authors.initials: GL
authors.lastname: Verdine
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D003850:Deoxyribonuclease I; D003857:Deoxyuridine; D015195:Drug Design; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005472:Fluorouracil / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D043224:Deoxyribonuclease (Pyrimidine Dimer); D005472:Fluorouracil; D003857:Deoxyuridine