DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity
DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity
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DOI：
10.1158/0008-5472.can-06-1115
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发表时间：
2006-09-01
期刊：
影响因子：
11.2
通讯作者：
Wani, Altaf A.
中科院分区：
文献类型：
--
作者：
Li, Jinyou;Wang, Qi-En;Wani, Altaf A.
DNA损伤结合（DDB）活性的功能缺陷与核苷酸切除修复（NER）降低和癌症易感性增加有直接关系。DDB与cullin 4A（Cul 4A）形成复合物，现在已知cullin 4A使DDB 2、XPC和组蛋白H2 A泛素化。然而，DDB 1在NER中的确切作用尚不清楚。在这项研究中，我们发现DDB 1在人类细胞中的敲低损害了它们有效修复UV诱导的环丁烷嘧啶二聚体（CPD）的能力，但不损害6-4光产物（6-4PP）。广泛的核蛋白分级分离和染色质缔合分析表明，照射后，DDB 1蛋白易位从松散结合到紧密结合在体内染色质馏分和DDB 1易位需要的功能DDB 2蛋白的参与。DDB 1基因敲除还影响Cul 4A组分在体内UV损伤的染色质中向紧密结合形式的易位以及其在原位向局部损伤的核灶的募集。然而，DDB 1敲低对DDB 2的DNA损伤结合能力没有影响。这些数据表明，DDB 2可以结合到体内受损的DNA作为单体，而CuI 4A招聘损伤位点依赖于完全组装的复合物。我们的数据还表明，DDB 1是紫外线诱导的DDB 2泛素化和降解所必需的。总之，结果表明：（a）DDB 1对于CPD的有效NER至关重要;（B）DDB 1在桥接DDB 2和泛素连接酶Cul 4A中起作用;以及（c）DDB 1有助于将泛素连接酶活性募集到受损位点，以成功开始NER对损伤的处理。
Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas CuI4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER.