Synthesis, Structure and Repair of DNA Interstrand Crosslinks

项目摘要 许多临床上重要的抗肿瘤剂，如顺铂、环磷酰胺（氮 芥子气）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形成剂在治疗中取得了巨大的成功， 治疗多种肿瘤，由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要的问题。研究 旨在了解由抗肿瘤药物形成的ICL引发的生物学反应， 由于用于生物化学和细胞生物学的位点特异性ICL的可用性有限， 问题研究 我们开发了一种新的方法来合成由氮形成的位点特异性ICL mu和氯乙基亚硝基脲以克服该限制。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进行研究 ICL修复与Johannes Walter（哈佛医学院）的实验室合作， 底物被用来建立第一个确定的生物化学系统，用于复制的研究- 依赖性ICL修复，显示ICL周围的切口和通过脱钩的经病变合成 ICL作为关键步骤。沿着探索跨损伤合成反应的初步研究 ICL模板的聚合酶，这些研究提供了基础的拟议研究 ICL修复中的结构-功能关系。 这些研究的指导假设是ICL结构的差异将影响跨病变 特别是ICL修复中的核苷酸合成和切除修复步骤， 对抗肿瘤化疗的治疗结果具有重要意义。在目标1中，我们提出 为了进一步合成ICL，通过大沟或碱基配对连接DNA， 表面，产生ICL，诱导DNA双链中的严重、中度、轻度或无扭曲 螺旋。我们将进一步合成ICL，其结构代表ICL修复的中间体， 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： 这些ICL通过NMR光谱和分子动力学模拟，以获得详细的见解， 各种ICLs如何影响DNA结构在目标3中，我们将研究这些结构多样的 ICL在复制依赖的ICL修复中被加工，以及ICL的结构如何影响ICL的修复。 它们如何被跨损伤合成聚合酶加工。我们希望这些研究将 揭示了结构多样的ICL如何处理的共性和重要差异， 人体细胞我们的研究应该为我们提供重要的见解， 肿瘤对癌症化疗中使用的交联剂的抗性以及 继发性肿瘤由于我们的研究涉及由抗肿瘤药物形成的ICL以及与抗肿瘤药物形成的ICL 新的结构，它们可以导致开发具有改进性质的抗肿瘤剂。

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.