Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA

EMBO J. 1998 Sep 1;17(17):5214-26. doi: 10.1093/emboj/17.17.5214.

Abstract

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Crystallography, X-Ray
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Glycosylases*
  • DNA Repair*
  • Humans
  • Models, Genetic
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • N-Glycosyl Hydrolases / chemistry*
  • N-Glycosyl Hydrolases / genetics
  • N-Glycosyl Hydrolases / metabolism
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / chemistry*
  • Oligodeoxyribonucleotides / metabolism
  • Peptide Fragments / metabolism
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Uracil-DNA Glycosidase

Substances

  • Oligodeoxyribonucleotides
  • Peptide Fragments
  • Recombinant Proteins
  • DNA
  • DNA Glycosylases
  • N-Glycosyl Hydrolases
  • Uracil-DNA Glycosidase

Associated data

  • PDB/1AKZ