title: Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation.
abstract: BACKGROUND
  Base excision repair initiated by human thymine-DNA glycosylase (TDG) results in the generation of abasic sites (AP sites) in DNA. TDG remains bound to this unstable repair intermediate, indicating that its transmission to the downstream-acting AP endonuclease is a coordinated process. Previously, we established that posttranslational modification of TDG with Small Ubiquitin-like MOdifiers (SUMOs) facilitates the dissociation of the DNA glycosylase from the product AP site, but the underlying molecular mechanism remained unclear.
  
  
  RESULTS
  We now show that upon DNA interaction, TDG undergoes a dramatic conformational change, which involves its flexible N-terminal domain and accounts for the nonspecific DNA binding ability of the enzyme. This function is required for efficient processing of the G.T mismatch but then cooperates with the specific DNA contacts established in the active site pocket of TDG to prevent its dissociation from the product AP site after base release. SUMO1 conjugation to the C-terminal K330 of TDG modulates the DNA binding function of the N terminus to induce dissociation of the glycosylase from the AP site while it leaves the catalytic properties of base release in the active site pocket of the enzyme unaffected.
  
  
  CONCLUSIONS
  Our data provide insight into the molecular mechanism of SUMO modification mediated modulation of enzymatic properties of TDG. A conformational change, involving the N-terminal domain of TDG, provides unspecific DNA interactions that facilitate processing of a wider spectrum of substrates at the expense of enzymatic turnover. SUMOylation then reverses this structural change in the product bound TDG.
authors.affiliation: Centre for Biomedicine, Department of Biological Clinical Sciences, University of Basel, Basel, Switzerland.
authors.forename: Roland
authors.initials: R
authors.lastname: Steinacher
authors.forename: Primo
authors.initials: P
authors.lastname: Schär
mesh_terms: D015153:Blotting, Western; D017931:DNA Primers; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005822:Genetic Vectors; D006801:Humans; D008954:Models, Biological; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D044502:Thymine DNA Glycosylase / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D044502:Thymine DNA Glycosylase; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase