The structural basis of specific base-excision repair by uracil–DNA glycosylase

Abstract

The 1.75-A crystal structure of the uracil-DNA glycosylase from herpes simplex virus type-1 reveals a new fold, distantly related to dinucleotide-binding proteins. Complexes with a trideoxynucleotide, and with uracil, define the DNA-binding site and allow a detailed understanding of the exquisitely specific recognition of uracil in DNA. The overall structure suggests binding models for elongated single- and double-stranded DNA substrates. Conserved residues close to the uracil-binding site suggest a catalytic mechanism for hydrolytic base excision.

DNA glycosylases in the base excision repair of DNA

“…The complex of human UDG and uracilcontaining DNA has demonstrated the basis for the enzymeassisted nucleotide flipping [83]. The three-dimensional structure of human UDG is very similar to the structures of the Herpes simplex virus type-1 (HSV-1) enzyme [84] and UDG from E. coli (C. D. Mol, D. W. Mosbaugh and J. A. Tainer, personal communication), and consists of a single α\β domain containing eight α-helices and a central four-stranded parallel and twisted β-sheet.…”

“…Site-directed mutagenesis of human UDG has demonstrated that effective catalysis is critically dependent upon Gln"%%, Asp"%&, Tyr"%(, Asn#!% and His#'). In the buried active site, Asp"%& may rotate towards bound uracil and may activate a water molecule, with the resultant hydroxy nucleophile making a direct in-line attack on deoxyribose C-1h, as suggested in [84]. Gln"%% and Tyr"%( shield the active site from the bulk solvent, and the severely reduced k cat of a Tyr"%( Ala mutant suggests that bulk-water exclusion is important for catalysis.…”

“…The elucidation of the crystal structures and mutational studies of human [70,[81][82][83] and herpes-viral [84] UDGs have demonstrated the mechanism for the selective binding of uracil over the structurally closely related normal pyrimidine in DNA, and have revealed the catalytic mechanism. DNA binds along a positively charged groove in the enzyme, but the tight-fitting uracil-binding pocket located at the base of this groove is too deep and narrow to allow binding of DNA-uracil unless it is ' flipped out ' of the DNA helix.…”

“…Covalent intermediates are not observed with the monofunctional DNA glycosylases such as AlkA, for which the attacking nucleophile may be a water molecule which is activated by Asp#$) located at the same position as Asp"$) in EndoIII (Figures 7 and 8). Interestingly, nucleophilic attack by water activated by an invariant Asp residue is also likely to occur in the proposed catalytic mechanism of UDG [83,84], although this enzyme does not contain a HhH motif and is structurally unrelated to AlkA and EndoIII.…”

The acidity of uracil and uracil analogs in the gas phase: Four surprisingly acidic sites and biological implications

“…Hydrogen bonding modulates recognition of DNA and RNA bases, and the interaction energy between two bonded complementary nucleobases is dependent on the intrinsic basicity of the acceptor atoms as well as on the acidity of donor NH groups [1,2]. In addition, understanding the intrinsic reactivity of nucleic bases can shed light on key biosynthetic mechanisms in which nucleobases are substrates [3][4][5][6][7][8].…”