Synthesis, Structure and Repair of DNA Interstrand Crosslinks

项目总结 一些临床上重要的抗肿瘤药物，如顺铂、环磷酰胺(一种氮化物 芥菜)或芥子碱(BCNU，一种氯乙基亚硝脲)形成DNA链间交联物(ICL)，如 关键的细胞毒性病变。ICL共价连接DNA双链的两条链，因此提供了一种有效的 阻断DNA复制和转录。尽管ICL成形剂在 治疗多种肿瘤，ICL修复引起的耐药性的发生(和 其他机制)和继发性肿瘤的发生仍然是重大问题。研究 为了了解由抗肿瘤药物形成的ICL所引发的生物反应 由于用于生化和细胞生物学的特定部位ICL的可获得性有限 学习。 我们已经开发了合成由氮形成的位点特异性ICL的新方法 芥子和氯乙基亚硝脲可以克服这一限制。这将使我们能够合成 结构多样化的ICL，并将它们整合到更长的寡核苷酸和质粒中进行研究 ICL修理部。与约翰尼斯·沃尔特(哈佛医学院)的实验室合作 底物被用来建立第一个明确的生化系统，用于研究复制- 依赖ICL修复，显示ICL周围的切口和跨病变合成通过未挂钩的 ICL作为关键步骤。伴随着探索跨病变合成反应的初步研究 具有ICL模板的聚合酶，这些研究为拟议的研究提供了基础 ICL修复中的结构-功能关系。 这些研究的指导性假设是ICL结构的差异将影响跨病变 尤其是ICL修复中的合成和核苷酸切除修复步骤，以及这些差异 对抗肿瘤化疗的疗效有重要意义。在目标1中，我们建议 为了进一步努力合成通过主槽或碱基配对连接DNA的ICL 表面，产生ICL，导致DNA双链严重、中度、轻微或无扭曲 螺旋。我们将进一步在代表ICL修复中间体的结构中合成ICL，以 研究它们是如何被DNA聚合酶处理的。在目标2中，我们将描述其结构 这些ICL通过核磁共振光谱和分子动力学模拟来获得详细的见解 不同的ICL如何影响DNA结构。在目标3中，我们将调查这些结构多样性是如何 ICL在复制依赖的ICL修复中被处理以及ICL的结构如何影响 它们是如何被跨损伤合成聚合酶处理的。我们预期这些研究将会 揭示结构多样化的ICL在处理方式上的共性和重要差异 人类细胞。我们的研究应该为了解其背后的机制提供重要的见解 肿瘤对化疗中使用的交联剂的抗药性及其形成 继发性肿瘤。由于我们的研究涉及由抗肿瘤药物形成的ICL以及 新的结构，它们可能导致具有更好性能的抗肿瘤药物的开发。

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.