A NUCLEOTIDE-FLIPPING MECHANISM FROM THE STRUCTURE OF HUMAN URACIL-DNA GLYCOSYLASE BOUND TO DNA

ANY uracil bases in DNA, a result of either misincorporation or deamin ation of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzym es(1). Crystal structures of human(2,3) and viral(4) UDGs complexed wi th free uracil have indicated that the enzyme binds an extrahelical ur acil. Such binding of undamaged extra-helical bases has been seen in t he structures of two bacterial methyltransferases(5,6) and bacteriopha ge T4 endonuclease V (ref. 7). Here we characterize the DNA binding an d kinetics of several engineered human UDG mutants and present the cry stal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with it s damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through th e minor groove, and compression of the DNA backbone flanking the uraci l all result in the flipping-out of the damaged base from the DNA majo r groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productiv e complex specific for cleavage of uracil from DNA and also reveals th e basis for the enzyme-assisted nucleotide flipping by this critical D NA-repair enzyme.