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）：给予导致DNA损伤的治疗剂是消除患者癌症的主要方法。用DNA交联剂如顺铂治疗后缺乏完全的肿瘤反应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损伤反应途径如何合作，以消除结合两条相反DNA链的交联，并促进对这些病变的抵抗。本提案的主要目的是确定跨损伤DNA聚合酶REV 1和DNA聚合酶zeta（Pol zeta）促进DNA链间交联（ICL）修复的分子机制。REV 1或Pol β缺陷细胞显示出类似的DNA修复缺陷，这是范可尼贫血（FA）患者衍生的细胞的特征，包括对产生ICL的药物的严重超敏反应和染色体畸变的积累。在这里，我们提出的证据表明，FA效应蛋白，FANCI和FANCD 2，特异性免疫共沉淀与REV 1或REV 3（催化亚基的Pol γ），并一起增加同源重组（HR）修复的效率。我们将测试的核心假设，属于FA途径的蛋白质与REV 1和Pol β 1合作，以促进DNA修复。长期以来，人们一直假设REV 1，REV 3和REV 7（Poly的辅助亚基）一起进行跨损伤DNA合成（TLS）和DNA修复，但对复合物中每个组分的功能意义知之甚少。在目标1中，我们将创建REV 1和REV 3的突变体版本，它们不能与REV 7或DNA聚合酶δ的POLD 2亚基相互作用，而POLD 2亚基与REV 3结合。我们将确定复合物的哪些组分对DNA修复和耐药性至关重要。在目的2中，我们将表征FANCI/FANCD 2异二聚体与REV 1/Pol β复合物之间的相互作用。我们将研究FANCI/FANCD 2和REV 1/Pol β是否合作促进DNA修复和维持基因组稳定性。在目标3中，我们将研究是否有额外的FA蛋白与REV 1/Pol蛋白复合物相关，并表征我们在REV 1或REV 3免疫沉淀物中鉴定的三种额外蛋白：DNA聚合酶Nu、MSH 2错配修复蛋白和SLX 4核酸内切酶支架蛋白。我们将确定这些蛋白质是否直接结合到REV 1/Pol蛋白复合物，并合作解决DNA中的ICLs。该提案具有创新性，因为它将开发新的模型来了解FA途径如何与translesion 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，以探索旨在破坏该复合物功能的小分子抑制剂的开发。这一提议是重要的，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，并拓宽我们对细胞去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发现克服肿瘤对化疗药物耐药性的新策略。