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

描述（由申请人提供）：使用导致DNA损伤的治疗药物是消除患者癌症的主要方法。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去除结合两个相反DNA链的交联，并促进对这些损伤的抵抗力。本文的主要目的是确定翻译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还是Pol？-缺陷细胞表现出类似的DNA修复缺陷，这是来自范可尼贫血（FA）患者的细胞的特征，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。本研究表明，FA效应蛋白FANCI和FANCD2特异性地与REV1或REV3 （Pol?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。我们将验证FA途径中的蛋白质与rev1和Pol？来促进DNA修复。长期以来，人们一直假设REV1， REV3和REV7 (Pol？辅助亚基)共同完成翻译DNA合成（TLS）和DNA修复，然而复合物中每个成分的功能意义尚不清楚。在Aim 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。我们将确定复合体的哪些成分对DNA修复和化学耐药性至关重要。在目标2中，我们将描述FANCI/FANCD2异源二聚体与REV1/Pol？复杂。我们将检查FANCI/FANCD2和REV1/Pol？共同促进DNA修复和维持基因组稳定。在Aim 3中，我们将研究是否有其他FA蛋白与REV1/Pol？我们在REV1或REV3免疫沉淀物中鉴定了另外三种蛋白质：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是否直接结合到REV1/Pol？复杂和合作来解决DNA中的icl。这一建议具有创新性，因为它将开发新的模型来理解FA途径如何与翻译DNA合成聚合酶相互作用，并有可能确定新的蛋白质-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。这一建议意义重大，因为所提出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病变机制的认识。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的新策略。

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.