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（由适用提供）：导致DNA损伤的热剂的给药是消除患者癌症的主要方法。用DNA交联药（如顺铂）处理后，缺乏完全的肿瘤反应，这仍然是一个持续的临床问题。该项目的长期目标是更好地了解DNA损伤响应途径如何坐标以去除结合两个相反DNA链并促进对这些病变的抗性的交联。该提案的主要目的是确定转移DNA聚合酶Rev1和DNA聚合酶Zeta（POLζ）促进DNA Interstrandsstrand交叉链接（ICL）修复的分子机制。 REV1或POLζ缺陷细胞显示出相似的DNA修复缺陷，这些缺陷是衍生的FRM Fanconi贫血（FA）患者的特征，包括对ICL产生药物的高敏性和染色体畸变的积累。在这里，我们提供了证据表明，FA效应蛋白Fanci和Fancd2，特别是与Rev1或Rev3（Polζ的催化亚基）共免疫沉淀，并共同提高同源重组（HR）修复的效率。我们将测试中心假设，即蛋白质属于FA途径与Rev1andPolζ合作以促进DNA修复。长期以来，人们已经假设Rev1，Rev3和Rev7（Polζ辅助亚基）共同进行跨性DNA合成（TLS）和DNA修复，但是复合物中每个成分的功能意义却鲜为人知。在AIM 1中，我们将创建Rev1和Rev3的突变版本，该版本无法与Rev7相互作用或DNA聚合酶Delta的Pold2亚基，该DNA聚合酶三角洲与Rev3结合。我们将确定该复合物的哪些组成部分对于DNA修复和化学耐药性至关重要。在AIM 2中，我们将表征FANCI/FANCD2异二聚体与Rev1/Polζ络合物之间的相互作用。我们将研究FANCI/FANCD2和REV1/POLζ合作是否促进DNA修复并保持基因组稳定性。在AIM 3中，我们将检查其他FA蛋白是否与Rev1/Polζ复合物相关，并表征我们在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 NU MSH2不匹配修复蛋白和SLX4内核酸内核蛋白质蛋白质。我们将确定这些蛋白是否直接与Rev1/Polζ复合物结合并坐标以解决DNA中的ICL。该建议具有创新性，因为它将开发新的模型，以了解FA途径如何与跨性别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，以探索用于破坏该复合功能功能的小分子抑制剂开发。该提议很重要，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，并扩大了我们对细胞去除许多临床相关癌症药物产生的细胞毒性DNA水平的知识。提高我们对这些机制的理解可能会确定克服化学治疗剂抗肿瘤性的新策略。