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

• 批准号: 8827263
• 负责人: Christine Elizabeth Canman
• 金额: $ 31.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/8827263
• 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' s Anemia; Funding; Genes; Genome Stability; Goals; Health; 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; recombinational repair; repaired; response; small molecule; therapeutic effectiveness; tumor

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 (Polζ) facilitate DNA interstrand crosslink (ICL) repair. REV1 or Polζ-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 Polζ) 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 Polζ to facilitate DNA repair. It has long been hypothesized that REV1, REV3 and REV7 (the Polζ 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/Polζ complex. We will examine whether FANCI/FANCD2 and REV1/Polζ cooperate to facilitate DNA repair and maintain genomic stability. In Aim 3, we will examine whether additional FA proteins associate with the REV1/Polζ 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/Polζ 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)：治疗药物的管理会导致DNA损伤，是消除患者癌症的主要方法。顺铂等DNA交联剂治疗后缺乏完整的肿瘤反应仍然是一个长期存在的临床问题。该项目的长期目标是更好地了解DNA损伤反应通路如何合作，以消除结合两条相反DNA链的交联链，并促进对这些损伤的抗性。该建议的主要目的是确定跨损伤DNA聚合酶Rev1和DNA聚合酶Zeta(POLζ)促进DNA链间交联修复的分子机制。REV1或POLζ缺陷细胞表现出与范可尼贫血(FA)患者细胞相似的DNA修复缺陷，包括对产生ICL的药物高度过敏和染色体异常堆积。在这里，我们提出了证据，FA效应蛋白FANCI和FANCD2，特异性地与REV1或REV3(POLζ的催化亚单位)共沉淀，共同提高同源重组(HR)修复的效率。我们将检验这一中心假设，即属于FA途径的蛋白质与REV1和POLζ合作促进DNA修复。长期以来，人们一直认为Rev1、Rev3和Rev7(POLDNA辅助亚单位)共同作用进行跨损伤ζ合成和DNA修复，然而，人们对该复合体中每个组成部分的功能意义知之甚少。在目标1中，我们将创建Rev1和Rev3的突变版本，它们不能与Rev7或与Rev3结合的DNA聚合酶Delta的POLD2亚单位相互作用。我们将确定该复合体的哪些成分是DNA修复和化疗耐药所必需的。在目标2中，我们将表征FANCI/FANCD2异二聚体与Rev1/POLζ复合体之间的相互作用。我们将研究FANCI/FANCD2和REV1/POLζ是否合作促进DNA修复和维持基因组稳定。在目标3中，我们将研究额外的FA蛋白是否与Rev1/Polζ复合体有关，并鉴定我们在Rev1或Rev3免疫沉淀中鉴定的另外三种蛋白质：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/PolDNA复合体结合，并合作解析ζ中的ICL。这一建议是创新的，因为它将开发新的模型来了解FA途径如何与跨损伤DNA合成聚合酶相互作用，并可能识别新的蛋白质-蛋白质相互作用，以探索旨在破坏这种复合体功能的小分子抑制剂的开发。这一建议意义重大，因为拟议的研究将进一步表征Fanconi贫血途径中复杂的蛋白质-蛋白质相互作用，并拓宽我们对细胞清除许多临床相关癌症药物造成的细胞毒性DNA损伤的机制的了解。增加我们对这些机制的理解，可能会发现克服肿瘤对化疗药物耐药性的新策略。