Human DNA Repair Enzymes for Redox and Alkylation Damage

描述：(申请人提供)细胞DNA被持续攻击 代谢源，如氧自由基和环境制剂 产生具有细胞毒性并威胁遗传稳定性的损伤。这些可怕的东西 DNA修复系统可以抵消这些影响。碱基切除修复(BER)行为 内源性DNA损伤和不同环境因素造成的损伤。 BER蛋白已被单独鉴定，但它们之间的配位 对BER蛋白及其表达的调控知之甚少。在……里面 哺乳动物细胞，BER的中心酶是无嘌呤(AP)内切酶 APEL，切割由DNA糖基酶或DNA损伤产生的AP位点 并去除阻断3‘末端的氧化脱氧核糖片段。这些 这些反应产生了用于DNA修复合成的活性引物。我们已经证明了 ABEL与其他BER酶相互作用，使它们与底物接触，并 刺激它们的活性：MyH糖基酶在上游的氧化错配 BER和DNA聚合酶B下游。我们将对这种蛋白质进行表征 互动网站，其他参与者的参与，如P53，以及 交互作用对误码率和特异度的影响。我们将进行工程设计 类人猿1相互作用缺陷衍生物及其与类人猿的关系 1-衍生肽在体内外干扰有效的误码率。阿佩尔 表达在细胞周期中受到调节，并对氧化作出反应 压力。我们将定义APE1启动子参与诱导的区域 通过氧化应激，并使用反义方法来测试 阿佩尔在诱导细胞对DNA损伤剂的抗性中。我们会研究 解决突变对酿酒酵母内源性AP位点的影响 特异性、病变“旁路”DNA聚合酶的处理及其影响 复制的极性。这些研究将提供关于 AP位点的误码率和突变的动态和交互过程。

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.