DDB2, the xeroderma pigmentosum group E gene product, is directly ubiquitylated by Cullin 4A-based ubiquitin ligase complex

• Published in: DNA repair 2005-05, Vol.4 (5), p.537-545
• Main Authors: Matsuda, Noriyuki, Azuma, Keiko, Saijo, Masafumi, Iemura, Shun-ichiro, Hioki, Yusaku, Natsume, Tohru, Chiba, Tomoki, Tanaka, Kiyoji, Tanaka, Keiji
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Bacteriology; Biological and medical sciences; CHO Cells; Cricetinae; Cullin 4A; Cullin Proteins - genetics; Cullin Proteins - metabolism; DDB1; DDB2; Dermatology; DNA Repair; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Growth, nutrition, cell differenciation; HeLa Cells; Hereditary diseases of the skin. Congenital diseases of the skin. Haemangioma of the skin, of mucosae and of soft tissue; Humans; Immunoprecipitation; Medical sciences; Microbiology; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Mutation; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Nucleotide excision repair; Ubiquitin; Ubiquitin - metabolism; Ubiquitin-Protein Ligase Complexes - genetics; Ubiquitin-Protein Ligase Complexes - metabolism; Ultraviolet Rays; Xeroderma Pigmentosum - genetics; Xeroderma Pigmentosum - metabolism
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2004.12.012

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.