Crystal structure and mutational analysis of human uracil-DNA glycosylase: Structural basis for specificity and catalysis

Abstract

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

“…AtUNG conserves critical residues involved in substrate recognition and catalysis in Family-1 enzymes (1). Amino acids Tyr-175 and Asn-231 correspond to residues that contribute to selectivity against other pyrimidines, including thymine, in human UNG (19). The conserved Leu-299 is involved in nucleotide flipping by penetrating in the minor groove and expelling uracil (19), and Asp-173 acts as a general base that activates water for attack of the N-glycosyl bond (20).…”

DNA glycosylases in the base excision repair of DNA

“…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.…”

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

“…S3). For the purpose of clarity, the names of the human UNG mutants were kept the same as described (27,35). To express AID and UNG from a single retroviral vector, hAID was first cloned into EcoRI and SalI1 sites of the retroviral expression vector pFB-IRES-GFP.…”