Novel mechanisms by which REV1 and POLZ affect response to anticancer agents

REV1 和 POLZ 影响抗癌药物反应的新机制

基本信息

批准号：
8696313
负责人：
Christine Elizabeth Canman
金额：
$ 31.49万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-10 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8696313
关键词：
Affect Aftercare Antineoplastic Agents Binding Binding Sites Biological Assay Bread Catalytic Domain Cells Characteristics Chromosome abnormality Cisplatin Clinical Complex Congenital Abnormality DNA DNA Crosslinking Agent DNA Damage DNA Interstrand Crosslinking DNA Polymerase III DNA Repair DNA Repair Disorder DNA biosynthesis DNA lesion DNA polymerase zeta DNA-Directed DNA Polymerase Data Development Fanconi anemia protein Fanconi&apos s Anemia Funding Genes Genome Stability Goals Human Hypersensitivity Knowledge Lead Lesion Link MSH2 gene Malignant Neoplasms Measures Mismatch Repair Mitomycins Modeling Molecular Mutate Pancytopenia Pathway interactions Patients Pharmaceutical Preparations Platinum Play Poly(ADP-ribose) Polymerases Polymerase Predisposition Process Protein Binding Protein Family Proteins Radial Refractory Relapse Reporting Research Resistance Resolution Role Scaffolding Protein Site Site-Directed Mutagenesis Structure Syndrome Testing Therapeutic Agents Ubiquitination Work analog base chemotherapeutic agent clinically relevant crosslink cytotoxic design ds-DNA endonuclease homologous recombination human DNA human disease inhibitor/antagonist innovation mutant neoplastic cell novel novel strategies prevent protein expression protein protein interaction public health relevance recombinational repair repaired response small molecule therapeutic effectiveness tumor

项目摘要

DESCRIPTION (provided by applicant): The administration of therapeutic agents that cause DNA damage is a major approach to eliminate cancer in patients. The lack of complete tumor response following treatment with DNA crosslinking agents like cisplatin continues to be a persistent clinical problem. The long term goal of this project is to better understand how DNA damage response pathways cooperate to remove crosslinks that bind two opposite DNA strands and promote resistance to these lesions. The major objective of this proposal is to determine the molecular mechanisms by which the translesion DNA polymerases REV1 and DNA polymerase zeta (Pol?) facilitate DNA interstrand crosslink (ICL) repair. REV1 or Pol?-deficient cells display similar DNA repair deficiencies that are characteristic of cells derived frm Fanconi anemia (FA) patients, including profound hypersensitivity to ICL- generating drugs and the accumulation of chromosomal aberrations. Here we present evidence that the FA effector proteins, FANCI and FANCD2, specifically co-immunoprecipitate with REV1 or REV3 (the catalytic subunit of Pol?) and together increase the efficiency of homologous recombination (HR) repair. We will test the central hypothesis that proteins belonging to the FA pathway cooperate with REV1and Pol? to facilitate DNA repair. It has long been hypothesized that REV1, REV3 and REV7 (the Pol? accessory subunit) work together to perform translesion DNA synthesis (TLS) and DNA repair, however the functional significance of each component in the complex is poorly understood. In Aim 1, we will create mutant versions of REV1 and REV3 that cannot interact with REV7 or the POLD2 subunit of DNA polymerase delta, which binds to REV3. We will determine which components of the complex are essential for DNA repair and chemoresistance. In Aim 2, we will characterize the interactions between the FANCI/FANCD2 heterodimer and the REV1/Pol? complex. We will examine whether FANCI/FANCD2 and REV1/Pol? cooperate to facilitate DNA repair and maintain genomic stability. In Aim 3, we will examine whether additional FA proteins associate with the REV1/Pol? complex and characterize three additional proteins we identified in REV1 or REV3 immunoprecipitates: DNA polymerase Nu the MSH2 mismatch repair protein, and the SLX4 endonuclease scaffold protein. We will determine whether these proteins directly bind to the REV1/Pol? complex and cooperate to resolve ICLs in DNA. This proposal is innovative since it will develop new models for understanding how the FA pathway interacts with translesion DNA synthesis polymerases and potentially identify new protein-protein interactions to explore for small molecule inhibitor development designed to disrupt the function of this complex. This proposal is significant because the proposed studies will further characterize the complex protein-protein interactions within the Fanconi anemia pathway and broaden our knowledge of the mechanisms by which cells remove the cytotoxic DNA lesions created by many clinically relevant cancer drugs. Increasing our understanding of these mechanisms may identify new strategies for overcoming tumor resistance to chemotherapeutic agents.

描述（由申请人提供）：施用引起DNA损伤的治疗剂是消除患者癌症的主要方法。使用顺铂等 DNA 交联剂治疗后缺乏完全的肿瘤反应仍然是一个持续存在的临床问题。该项目的长期目标是更好地了解 DNA 损伤反应途径如何合作消除结合两条相反 DNA 链的交联并促进对这些损伤的抵抗力。该提案的主要目标是确定跨损伤 DNA 聚合酶 REV1 和 DNA 聚合酶 zeta (Pol?) 促进 DNA 链间交联 (ICL) 修复的分子机制。 REV1 或 Pol? 缺陷细胞表现出类似的 DNA 修复缺陷，这是范可尼贫血 (FA) 患者细胞的特征，包括对 ICL 生成药物的严重过敏和染色体畸变的积累。在这里，我们提供的证据表明 FA 效应蛋白 FANCI 和 FANCD2 与 REV1 或 REV3（Pol? 的催化亚基）特异性共免疫沉淀，并共同提高同源重组 (HR) 修复的效率。我们将检验中心假设，即属于 FA 途径的蛋白质与 REV1 和 Pol？以促进DNA修复。长期以来，人们一直假设 REV1、REV3 和 REV7（Pol? 辅助亚基）共同作用来执行跨损伤 DNA 合成 (TLS) 和 DNA 修复，但复合物中每个组件的功能意义却知之甚少。在目标 1 中，我们将创建 REV1 和 REV3 的突变版本，它们不能与 REV7 或 DNA 聚合酶 delta 的 POLD2 亚基相互作用，后者与 REV3 结合。我们将确定复合物的哪些成分对于 DNA 修复和化学耐药性至关重要。在目标 2 中，我们将表征 FANCI/FANCD2 异二聚体和 REV1/Pol? 之间的相互作用。复杂的。我们将检查 FANCI/FANCD2 和 REV1/Pol 是否？合作促进DNA修复并维持基因组稳定性。在目标 3 中，我们将检查其他 FA 蛋白是否与 REV1/Pol 相关？复合物并表征了我们在 REV1 或 REV3 免疫沉淀物中鉴定出的另外三种蛋白质：DNA 聚合酶 Nu、MSH2 错配修复蛋白和 SLX4 核酸内切酶支架蛋白。我们将确定这些蛋白质是否直接与 REV1/Pol 结合？复杂并合作解决 DNA 中的 ICL。该提案具有创新性，因为它将开发新模型来了解 FA 途径如何与跨损伤 DNA 合成聚合酶相互作用，并有可能识别新的蛋白质-蛋白质相互作用，以探索旨在破坏该复合物功能的小分子抑制剂的开发。这项提议意义重大，因为拟议的研究将进一步表征范可尼贫血途径中复杂的蛋白质-蛋白质相互作用，并拓宽我们对细胞消除许多临床相关癌症药物产生的细胞毒性 DNA 损伤的机制的了解。增加我们对这些机制的理解可能会找到克服肿瘤对化疗药物耐药性的新策略。