Kapetanaki Maria G, Guerrero-Santoro Jennifer, Bisi Dawn C, Hsieh Ching L, Rapić-Otrin Vesna, Levine Arthur S
Department of Molecular Genetics and Biochemistry, School of Medicine, and Cancer Institute, University of Pittsburgh, Hillman Cancer Center, Research Pavilion, Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213, USA.
Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2588-93. doi: 10.1073/pnas.0511160103. Epub 2006 Feb 10.
Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4A(DDB2). The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4A(DDB2) complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4A(DDB2) ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.
着色性干皮病（XP）是一种遗传性人类疾病，其特征在于核苷酸切除修复（NER）缺陷和皮肤癌的发生。来自E组XP（XP-E）患者的细胞在紫外线损伤的DNA结合蛋白复合物（UV-DDB）中存在缺陷，该复合物参与NER的损伤识别步骤。UV-DDB由两个亚基组成，分别是DDB1和DDB2基因的产物。DDB2基因突变是XP-E潜在缺陷的原因。UV-DDB复合物是新鉴定的基于cullin 4A的泛素E3连接酶DDB1-CUL4A（DDB2）的一个组成部分。E3泛素连接酶识别特定底物并介导其泛素化，以调节蛋白质活性或通过蛋白酶体途径靶向蛋白质进行降解。在本研究中，我们探讨了基于UV-DDB的E3在NER中的作用并寻找一种生理底物。我们证明，天然染色质中的单泛素化组蛋白H2A在紫外线照射后与内源性DDB1-CUL4A（DDB2）复合物共免疫沉淀。此外，与修复功能正常的细胞相比，DDB2中的突变会改变DDB1-CUL4A（DDB2）连接酶的形成和结合活性，同时在紫外线处理XP-E细胞后H2A的单泛素化受损。这一发现表明，作为基于DDB1-CUL4A的连接酶的底物受体，DDB2以光损伤结合依赖的方式特异性地将组蛋白H2A靶向单泛素化。鉴于在紫外线损伤的DNA位点处单泛素化组蛋白H2A的缺失与XP-E细胞中全基因组修复的减少有关，本研究表明，由XPE因子介导的组蛋白修饰促进了NER的起始。
