Thoma Brian S, Wakasugi Mitsuo, Christensen Jesper, Reddy Madhava C, Vasquez Karen M
Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division Smithville, TX 78957, USA.
Nucleic Acids Res. 2005 May 24;33(9):2993-3001. doi: 10.1093/nar/gki610. Print 2005.
DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.
DNA链间交联（ICLs）是一种严重的损伤形式，它会阻碍DNA代谢过程，并可能导致细胞死亡或致癌。哺乳动物中DNA ICLs的修复机制尚未完全明确。我们之前报道过，核苷酸切除修复（NER）的关键蛋白复合物XPA-RPA能够识别DNA ICLs。我们现在报道利用三链体技术将位点特异性补骨脂素ICL定位到目标DNA底物上，以确定人类全基因组NER损伤识别复合物XPC-hHR23B是否能识别这种损伤。我们的结果表明，XPC-hHR23B能够识别补骨脂素ICLs，其结构与XPC-hHR23B已知结合的其他损伤有根本不同，且具有高亲和力和特异性。还观察到XPC-hHR23B和XPA-RPA蛋白复合物能同时结合补骨脂素ICLs，这不仅表明补骨脂素ICLs能被XPC-hHR23B单独识别，还表明XPA-RPA可能在受损DNA上与XPC-hHR23B协同相互作用，形成多聚体复合物。由于XPC-hHR23B和XPA-RPA参与DNA损伤的识别和验证，这些结果支持了以下假设：NER全基因组修复子途径各组分之间的相互作用对于识别哺乳动物基因组中的补骨脂素DNA ICLs至关重要。
