title: DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA.
abstract: The DDB protein complex, comprising the subunits DDB1 and DDB2, binds tightly to UV light-irradiated DNA. Mutations in DDB2 are responsible for xeroderma pigmentosum group E, a disorder with defects in nucleotide excision repair of DNA. Both subunits are also components of a complex involved in ubiquitin-mediated proteolysis. Cellular defects in DDB2 disable repair of the major UV radiation photoproduct in DNA, a cyclobutane pyrimidine dimer, but no significant direct binding of DDB to this photoproduct in DNA has ever been demonstrated. Thus, it has been uncertain how DDB could play a specific role in DNA repair of such damage. We investigated DDB function using highly purified proteins. Co-purified DDB1-DDB2 or DDB reconstituted with individual DDB1 and DDB2 subunits binds to damaged DNA as a ternary complex. We found that DDB can indeed recognize a cyclobutane pyrimidine dimer in DNA with an affinity (K(app)a) 6-fold higher than that of nondamaged DNA. The DDB1-DDB2 complex also bound with high specificity to a UV radiation-induced (6-4) photoproduct and to an apurinic site in DNA. Unexpectedly, DDB also bound avidly to DNA containing a 2- or 3-bp mismatch (and does not bind well to DNA containing larger mismatches). These data indicate that DDB does not detect lesions per se. It instead recognizes other structural features of damaged DNA, acting as a sensor that probes DNA for a subset of conformational changes. Lesions recognized may include those arising when translesion polymerases such as POLH incorporate bases across from DNA lesions caused by UV radiation.
authors.affiliation: University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Birgitte Ø
authors.initials: BØ
authors.lastname: Wittschieben
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.forename: Richard D
authors.initials: RD
authors.lastname: Wood
mesh_terms: D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011740:Pyrimidine Dimers / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: C091435:DDB1 protein, human; C117669:DDB2 protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins; D004247:DNA