MECHANISMS OF MAMMALIAN DNA REPAIR

哺乳动物 DNA 修复机制

基本信息

批准号：
2733101
负责人：
JOHN J. TURCHI
金额：
$ 10.49万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2000-06-30
项目状态：
已结题

项目摘要

cis-Diamminedichloroplatinum(II) (cis-DDP) is a widely prescribed chemotherapeutic drug used to treat a variety of malignancies. A major limitation of cis-DDP treatment is recurrence and subsequent resistance. Resistance to cis-DDP treatment is, in part, a result of the ability to repair cis-DDP-DNA adducts. Understanding the basic mechanism of cis-DDP repair and the regulation of repair specific protein expression is essential to identify how cis-DDP resistance occurs and to develop effective therapies. Therefore, our goal is to determine the enzymatic pathway responsible for the repair of cis-DDP DNA adducts. Our approach is to purify proteins from mammalian cells, which are likely to be involved in repair of cis-DDP-DNA adducts based on their affinity for cis-DDP damaged DNA. These include human helicase E, damage recognition proteins (DRPs), a recently identified DNA dependent ATPase and damage incision proteins (DIPs). Extensive characterization of the individual proteins will be performed with a focus on how they interact with cis-DDP damaged DNA. DRPs and DIPs will also be characterized with respect to specificity for different types of DNA damage. Specifically, their binding and incision activities will be tested on thymine dimers, psoralen, 2-aminofluorine and cc1065 DNA adducts. Characterization of helicase E includes activity on cis-DDP and UV damaged DNA substrates and the ability to form a functional complex with DNA polymerase epsilon to perform repair synthesis. The effect of DRPs bound to damaged DNA on helicase activity will also be assessed. Initial characterization of ATPase-I will include determining if other DNA metabolizing activities, specifically helicase, ligase or nuclease, are associated with ATPase-I activity. In addition, the activity of ATPase-I will be assessed using a variety of synthetic DNA substrates designed to mimic structures found in DNA replication and repair. ATPase-I activity will also be measured using UV and cis-DDP damage DNA substrates. The effect of DRPs bound to damaged DNA on ATPase-I activity will also be evaluated to assess how the proteins interact. Antibodies will be raised against the two DRPs and ATPase-I and used to assess the role of each protein in an in vitro DNA repair assay and a Xenopus ooyte excision repair system. The level of expression of these proteins will be assessed in cis-DDP resistant and sensitive cell lines and XP cells. Immunoaffinity purification protocols will be established to aid in the purification of these proteins from cis-DDP resistant cell lines and XP cells, which will then allow direct comparison of the activities from the different cell lines.

顺式二氯二氨铂 (II) (顺式 DDP) 是一种广泛使用的药物用于治疗多种恶性肿瘤的化疗药物。一个专业 cis-DDP 治疗的局限性是复发和随后的耐药性。对顺式 DDP 治疗的耐药性部分是由于以下能力的结果：修复顺式-DDP-DNA 加合物。了解cis-DDP的基本机制修复和修复特异性蛋白表达的调节是对于确定顺式 DDP 耐药性如何发生和发展至关重要有效的疗法。因此，我们的目标是确定酶促负责修复顺式 DDP DNA 加合物的途径。我们的方法是从哺乳动物细胞中纯化可能涉及的蛋白质基于其对 cis-DDP 的亲和力修复 cis-DDP-DNA 加合物受损的DNA。其中包括人类解旋酶 E、损伤识别蛋白 (DRP)，最近发现的一种 DNA 依赖性 ATP 酶和损伤切口蛋白质（DIP）。将对单个蛋白质进行广泛的表征重点是它们如何与顺式 DDP 损伤的 DNA 相互作用。 DRP 和 DIP 还将针对不同类型的特异性进行表征 DNA 损伤。具体来说，它们的结合和切割活动将是对胸腺嘧啶二聚体、补骨脂素、2-氨基氟和 cc1065 DNA 进行了测试加合物。解旋酶 E 的表征包括对 cis-DDP 的活性和紫外线损伤的 DNA 底物和形成功能复合物的能力用DNA聚合酶epsilon进行修复合成。的效果还将评估与受损 DNA 结合的 DRP 的解旋酶活性。 ATPase-I 的初步表征将包括确定是否有其他 DNA 代谢活性，特别是解旋酶、连接酶或核酸酶，与 ATPase-I 活性相关。此外，ATPase-I 的活性将使用各种合成 DNA 底物进行评估，旨在模仿 DNA 复制和修复中发现的结构。 ATP酶-I活性还将使用 UV 和 cis-DDP 损伤 DNA 底物进行测量。这与受损 DNA 结合的 DRP 对 ATPase-I 活性的影响也将是评估蛋白质如何相互作用。将产生针对两种 DRP 和 ATPase-I 的抗体，并用于评估每种蛋白质在体外 DNA 修复测定中的作用，并非洲爪蟾卵母切除修复系统。这些的表达水平将在顺式 DDP 抗性和敏感细胞系中评估蛋白质和 XP 细胞。将建立免疫亲和纯化方案有助于从顺式 DDP 抗性细胞中纯化这些蛋白质线和 XP 单元格，然后可以直接比较来自不同细胞系的活性。