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The Cullin 4B–Based UV-Damaged DNA-Binding Protein Ligase Binds to UV-Damaged Chromatin and Ubiquitinates Histone H2A

Jennifer Guerrero-Santoro, Maria G. Kapetanaki, Ching L. Hsieh, Ilya Gorbachinsky, Arthur S. Levine and Vesna Rapi-Otrin

Department of Microbiology and Molecular Genetics, School of Medicine, and Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania

Requests for reprints: Vesna Rapi-Otrin, Hillman Cancer Center, Research Pavilion, Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-5962; Fax: 412-623-7761; E-mail: vesnaro{at}pitt.edu.

Key Words: CUL4B • DDB1 • DDB2 • ubiquitinated histone H2A • nucleotide excision repair • xeroderma pigmentosum group E

By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)–based ubiquitin ligase, DDB1-CUL4A^DDB2. We previously showed that DDB1-CUL4A^DDB2 ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A^DDB2 and DDB1-CUL4B^DDB2. To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B^DDB2 E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B^DDB2 is more efficient than DDB1-CUL4A^DDB2 in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B^DDB2 E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER. [Cancer Res 2008;68(13):5014–22]