Synthesis, Structure and Repair of DNA Interstrand Crosslinks

DNA 链间交联的合成、结构和修复

• 批准号: 8402673
• 负责人: Orlando D. Scharer
• 金额: $ 32.58万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8402673
• 关键词: Address; Affect; Base Pairing; Biochemical; Biological; Bypass; Carmustine; Cell physiology; Cells; Chemotherapy-Oncologic Procedure; Cisplatin; Clinical; Collaborations; Complex; Cyclophosphamide; DNA; DNA Adducts; DNA Interstrand Crosslinking; DNA Sequence; DNA Structure; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Development; Drug usage; Foundations; Genetic Transcription; Health; Human; Laboratories; Lead; Length; Lesion; Link; Major Groove; Mechlorethamine; Methodology; Molecular Analysis; NMR Spectroscopy; Neoplasm Metastasis; Nucleotide Excision Repair; Oligonucleotides; Organic Chemistry; Outcome; Pathway interactions; Pharmaceutical Preparations; Plasmids; Polymerase; Process; Property; Reaction; Resistance; Site; Structure; Structure-Activity Relationship; Surface; Surgical incisions; System; Technology; Testing; Therapeutic; Urea; Xenopus laevis; antitumor agent; base; chemotherapy; clinically relevant; crosslink; cytotoxic; flexibility; homologous recombination; improved; insight; medical schools; molecular dynamics; neoplastic cell; novel; repaired; resistance mechanism; response; success; tool; tumor

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Cisplatin, nitrogen mustards and chloro ethyl nitroso ureas are among the most successful drugs used in antitumor therapy. They exert their effect by forming DNA adducts called interstrand crosslinks as the most important therapeutic lesions. Despite the clinical success of these drugs, the occurrence of resistance in tumor cells and formation of secondary tumors are significant problems. Our studies aimed at understanding how structurally diverse DNA adducts formed by antitumor agents are processed in human cells will have significant impact on the understanding and overcoming of resistance of tumor cells to known drugs and might lead to the development of new antitumor agents with improved properties.

描述（由申请方提供）：许多临床上重要的抗肿瘤药物，如顺铂、环磷酰胺（一种氮芥）或卡莫司汀（BCNU，一种氯乙基亚硝基脲）形成DNA链间交联（ICL），作为关键细胞毒性病变。ICL共价连接DNA双链体的两条链，因此提供了对DNA复制和转录的有效阻断。尽管ICL形成剂在治疗多种肿瘤方面取得了巨大成功，但由ICL修复（和其他机制）引起的耐药性的发生和继发性肿瘤的发生仍然是重大问题。旨在了解由抗肿瘤药物形成的ICL引发的生物学反应的研究受到生物化学和细胞生物学位点特异性ICL的有限可用性的阻碍。 问题研究我们已经开发了新的方法来合成由氮芥和氯乙基亚硝基脲形成的位点特异性ICLs，以克服这一限制。这将使我们能够合成结构多样的ICL，并将它们整合到更长的寡核苷酸和质粒中，用于ICL修复的研究。与Johannes Walter（哈佛医学院）的实验室合作，这些底物被用于建立用于研究复制依赖性ICL修复的第一个确定的生化系统，揭示了ICL周围的切口和穿过脱钩的ICL的跨损伤合成作为关键步骤。沿着探索跨损伤合成聚合酶与ICL模板反应的初步研究，这些研究为ICL修复中结构-功能关系的拟议研究提供了基础。这些研究的指导假设是，ICL结构的差异将影响ICL修复中的跨损伤合成和核苷酸切除修复步骤，特别是这些差异对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大沟或碱基配对表面连接DNA的ICL，产生诱导DNA双螺旋中严重、中等、轻度或无扭曲的ICL。我们将进一步合成ICL的结构，代表ICL修复的中间体，以研究它们如何被DNA聚合酶加工。在目标2中，我们将通过NMR光谱和分子动力学模拟来表征这些ICL的结构，以详细了解各种ICL如何影响DNA结构。在目标3中，我们将研究这些结构不同的ICL如何在复制依赖性ICL修复中进行处理，以及ICL的结构如何影响它们如何通过translesion合成聚合酶进行处理。我们希望这些研究将揭示结构多样的ICL在人类细胞中加工的共性和重要差异。我们的研究应该提供重要的见解的机制，肿瘤耐药的交联剂用于癌症化疗以及继发性肿瘤的形成。由于我们的研究涉及由抗肿瘤剂形成的ICL以及具有新结构的ICL，因此它们可能导致开发具有改进性质的抗肿瘤剂。 公共卫生相关性：顺铂、氮芥和氯乙基亚硝基脲是抗肿瘤治疗中最成功的药物。它们通过形成称为链间交联的DNA加合物发挥作用，作为最重要的治疗损伤。尽管这些药物在临床上取得了成功，但肿瘤细胞中耐药性的发生和继发性肿瘤的形成是重大问题。我们的研究旨在了解抗肿瘤药物形成的结构多样的DNA加合物如何在人类细胞中加工，这将对理解和克服肿瘤细胞对已知药物的耐药性产生重大影响，并可能导致开发具有改进性质的新抗肿瘤药物。