Human DNA Repair Enzymes for Redox and Alkylation Damage

描述：（由申请人提供）细胞DNA持续受到以下物质的攻击： 代谢源如氧自由基和环境因子， 产生细胞毒性的损害并威胁遗传稳定性。这些可怕 DNA修复系统会抵消这些影响。碱基切除修复（BER） 内源性DNA损伤和各种环境因素引起的损伤。 BER蛋白质已经被单独表征，但是它们之间的协调 BER蛋白及其表达的调节知之甚少。在 在哺乳动物细胞中，BER的中心酶是脱嘌呤（AP）内切酶 Apel，切割由DNA糖基化酶或DNA损伤产生的AP位点 试剂并去除封闭3'末端的氧化脱氧核糖片段。这些 反应产生用于DNA修复合成的活性引物。我们已经证明 Apel与其他BER酶相互作用，使它们与其底物结合， 刺激其活性：Myh糖基化酶在 BER和下游的DNA聚合酶B。我们将描述这种蛋白质 互动网站，其他玩家的参与，如p53，以及 相互作用对BER速率和特异性的影响。我们将工程师 Ape 1的相互作用缺陷衍生物，并测试它们或Ape 1-衍生肽在体外和体内干扰有效BER。阿佩尔 表达在细胞周期中受到调节，并响应于氧化 应力我们将定义参与诱导的APE 1启动子区域， 通过氧化应激，并使用反义方法来测试的重要性， Apel可诱导细胞对DNA损伤剂的抗性。我们会研究 内源性AP位点在S.酿酒酵母通过寻址突变 特异性，病变“旁路”DNA聚合酶的加工，以及 复制极性。这些研究将提供一个全面的图片， BER和AP位点突变的动态和相互作用过程。

DESCRIPTION: (PROVIDED BY APPLICANT) Cellular DNA is continuously assaulted by metabolic sources such as oxygen radicals and by environmental agents to generate damage that is cytotoxic and threatens genetic stability. These dire effects are counteracted by DNA repair systems. Base excision repair (BER) acts on both endogenous DNA damage and lesions from diverse environmental agents. BER proteins have been individually characterized, but the coordination among BER proteins and the modulation of their expression are poorly understood. In mammalian cells, the central enzyme of BER is the apurinic (AP) endonuclease Apel, which incises AP sites produced by DNA glycosylases or DNA-damaging agents and removes oxidative deoxyribose fragments that block 3' termini. These reactions produce active primers for DNA repair synthesis. We have shown that Apel interacts with other BER enzymes to engage them with their substrates and stimulate their activities: Myh glycosylase for oxidative mispairs upstream in BER, and DNA polymerase B downstream. We will characterize the protein interaction sites, the participation of other players such as p53, and the effects of interactions on the rate and specificity of BER. We will engineer interaction-defective derivatives of Ape 1 and test whether they or Ape 1-derived peptides interfere with efficient BER in vitro and in vivo. Apel expression is regulated during the cell cycle and in response to oxidative stress. We will define the regions of the APE1 promoter involved in induction by oxidative stress, and use an antisense approach to test the importance of Apel in inducible cellular resistance to DNA-damaging agents. We will study the consequences of endogenous AP sites in S. cerevisiae by addressing mutational specificity, processing by lesion "bypass" DNA polymerases, and the influence of replication polarity. These studies will provide a holistic picture of the dynamic and interactive processes of BER and mutagenesis by AP sites.