Synthesis, Structure and Repair of DNA Interstrand Crosslinks

项目概要 许多临床上重要的抗肿瘤药物，如顺铂、环磷酰胺（一种氮 芥末）或卡莫司汀（BCNU，一种氯乙基亚硝基脲）形成 DNA 链间交联 (ICL) 关键的细胞毒性病变。 ICL 共价连接 DNA 双链体的两条链，因此提供了有效的 阻断 DNA 复制和转录。尽管 ICL 形成剂在 治疗多种肿瘤时，ICL修复引起的耐药性的发生（以及 其他机制）和继发性肿瘤的发生仍然是重大问题。研究 旨在了解抗肿瘤药物形成的 ICL 引发的生物反应 用于生化和细胞生物学的位点特异性 ICL 的可用性有限，这阻碍了 研究。 我们开发了合成由氮形成的位点特异性 ICL 的新方法 芥末和氯乙基亚硝基脲克服了这一限制。这将使我们能够合成 结构多样的 ICL 并将其整合到更长的寡核苷酸和质粒中以供研究 ICL 修复术。与约翰内斯·沃尔特（哈佛大学医学院）实验室合作，这些 底物被用来建立第一个定义的生化系统，用于研究复制- 依赖 ICL 修复，显示 ICL 周围的切口和经过脱钩的跨损伤合成 ICL 作为关键步骤。随着初步研究探索跨损伤合成反应 聚合酶与 ICL 模板，这些研究为拟议的研究提供了基础 ICL 修复中的结构与功能关系。 这些研究的指导性假设是ICL结构的差异会影响跨损伤 特别是 ICL 修复中的合成和核苷酸切除修复步骤，并且这些差异 对于抗肿瘤化疗的治疗结果具有重要意义。在目标 1 中，我们建议 进一步努力合成通过主沟或碱基配对连接 DNA 的 ICL 表面，生成 ICL，导致 DNA 双链发生严重、中等、轻微或无扭曲 螺旋。我们还将进一步合成代表 ICL 修复中间体的 ICL 结构 研究它们是如何被 DNA 聚合酶加工的。在目标 2 中，我们将描述以下结构的特征 通过 NMR 光谱和分子动力学模拟对这些 ICL 进行分析，以获得详细的见解 各种 ICL 如何影响 DNA 结构。在目标 3 中，我们将研究这些结构上的多样性如何 ICL 在复制依赖性 ICL 修复中进行处理以及 ICL 的结构如何影响 它们如何被跨损伤合成聚合酶加工。我们期望这些研究将 揭示了结构不同的 ICL 在处理过程中的共性和重要差异 人体细胞。我们的研究应该为背后的机制提供重要的见解 肿瘤对癌症化疗中使用的交联剂的耐药性以及形成 继发性肿瘤。由于我们的研究涉及由抗肿瘤药物以及具有抗肿瘤作用的药物形成的 ICL 新颖的结构，它们可能导致开发具有改进性能的抗肿瘤药物。

PROJECT SUMMARY A number of clinically important antitumor agents such as cisplatin, cyclophosphamide (a nitrogen mustard) or carmustine (BCNU, a chloro ethyl nitroso urea) form DNA interstrand crosslinks (ICLs) as key cytotoxic lesions. ICLs covalently link two strands of a DNA duplex and therefore provide a potent block to DNA replication and transcription. Despite the enormous success of ICL-forming agents in treating a large variety of tumors, the occurrence of resistance caused by the repair of ICLs (and other mechanisms) and the occurrence of secondary tumors remain significant problems. Studies aimed at understanding the biological responses triggered by ICLs formed by antitumor agents have been hampered by the limited availability of site-specific ICLs for biochemical and cell biological studies. We have developed new methodology for the synthesis of site-specific ICLs formed by nitrogen mustards and chloro ethyl nitroso ureas to overcome this limitation. This will enable us to synthesize structurally diverse ICLs and incorporate them into longer oligonucleotides and plasmids for the study of ICL repair. In collaboration with the laboratory of Johannes Walter (Harvard Medical School) these substrates were used to establish the first defined biochemical system for the study of replication- dependent ICL repair, revealing incisions around the ICL and translesion synthesis past an unhooked ICL as key steps. Along with preliminary studies exploring the reactions of translesion synthesis polymerases with ICL templates, these studies provide the foundation for the proposed studies of structure-function relationships in ICL repair. The guiding hypothesis of these studies is that differences in ICL structure will affect the translesion synthesis and nucleotide excision repair steps in ICL repair in particular, and that these differences have important implication for therapeutic outcomes in antitumor chemotherapy. In Aim 1 we propose to further our efforts to synthesize ICLs that link the DNA through the major groove or base-pairing surfaces, generating ICLs that induce severe, intermediate, mild or no distortion in the DNA double helix. We will furthermore synthesize ICLs in structures that represent intermediates in ICL repair to study how they are processed by DNA polymerases. In Aim 2, we will characterize the structures of these ICLs by NMR spectroscopy and molecular dynamics simulations to gain detailed insights into how the various ICLs affect DNA structure. In Aim 3, we will investigate how these structurally diverse ICLs are processed in replication-dependent ICL repair and how the structures of the ICLs influence how they are processed by translesion synthesis polymerases. We expect that these studies will reveal commonalities and also important differences of how structurally diverse ICLs are processed in human cells. Our studies should provide important insights into the mechanisms that underlie resistance of tumors to crosslinking agents used in cancer chemotherapy as well as the formation of secondary tumors. Since our studies involve ICLs formed by antitumor agents as well as ones with novel structures, they could lead to the development of antitumor agents with improved properties.