title: DDB2, the xeroderma pigmentosum group E gene product, is directly ubiquitylated by Cullin 4A-based ubiquitin ligase complex.
abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to UV irradiation and high incidence of skin cancer caused by inherited defects in DNA repair. Mutational malfunction of damaged-DNA binding protein 2 (DDB2) causes the XP complementation group E (XP-E). DDB2 together with DDB1 comprises a heterodimer called DDB complex, which is involved in damaged-DNA binding and nucleotide excision repair. Interestingly, by screening for a cellular protein(s) that interacts with Cullin 4A (Cul4A), a key component of the ubiquitin ligase complex, we identified DDB1. Immunoprecipitation confirmed that Cul4A interacts with DDB1 and also associates with DDB2. To date, it has been reported that DDB2 is rapidly degraded after UV irradiation and that overproduction of Cul4A stimulates the ubiquitylation of DDB2 in the cells. However, as biochemical analysis using pure Cul4A-containing E3 is missing, it is still unknown whether the Cul4A complex directly ubiquitylates DDB2 or not. We thus purified the Cul4A-containing E3 complex to near homogeneity and attempted to ubiquitylate DDB2 in vitro. The ubiquitylation of DDB2 was reconstituted using this pure E3 complex, indicating that DDB-Cul4A E3 complex in itself can ubiquitylate DDB2 directly. We also showed that an amino acid substitution, K244E, in DDB2 derived from a XP-E patient did not affect its ubiquitylation.
authors.affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Matsuda
authors.forename: Keiko
authors.initials: K
authors.lastname: Azuma
authors.forename: Masafumi
authors.initials: M
authors.lastname: Saijo
authors.forename: Shun-Ichiro
authors.initials: S
authors.lastname: Iemura
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Hioki
authors.forename: Tohru
authors.initials: T
authors.lastname: Natsume
authors.forename: Tomoki
authors.initials: T
authors.lastname: Chiba
authors.forename: Kiyoji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics* / Q000378:metabolism
chemical_list: C114234:CUL4A protein, human; D044842:Cullin Proteins; C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes