HUMAN DNA REPAIR ENZYMES FOR REDOX AND ALKYLATION DAMAGE

描述：DNA天生不稳定，但也会受到代谢物的攻击 和环境诱变剂，如氧自由基和烷化剂， 它们形成了各种类型的基本(AP)站点。AP站点可能具有致突变性 这样就损害了遗传稳定性。启动AP站点的修复 普遍存在的“II类”AP内切酶。它们的生物学功能有 在微生物中进行了测试，但它们很可能是一线防御 人类体内的酶也是如此。他将定义主要的生物角色 哺乳动物的AP内切酶，由类人猿基因编码，在 几种相辅相成的方式。他将建立APE缺陷细胞系 猩猩间交配产生的小鼠胚胎组织 (“基因敲除”)杂合子；纯合子基因敲除胚胎不发育 超过第5天，但从它们衍生的细胞可以在体外培养。这个 将测试细胞系对与AP相关的诱变剂的敏感性 现场生产(如烷基化剂)以及其他试剂。Dr。 Demple将测量这些品系的自发突变频率以获得 洞察类人猿在纠正内源性DNA损伤中的作用。他会的 检测重组基因的引入是否逆转了表型 野生型类人猿，或编码修复缺陷蛋白的突变衍生品 或在体外影响转录因子结合的“refl”活性。他 还将探索类人猿与其他修复和非修复的相互作用 蛋白质采用酵母双杂交和噬菌体展示方法。他已经这么做了 证明了APE与DNA聚合酶B相互作用，DNA聚合酶B催化 接下来的两步是碱基切除修复。他将探索这一点的意义 这种相互作用是通过识别这两种蛋白质的区域来实现的 互动。Demple博士将隔离无法相互作用的类人猿衍生品 并评估它们支持正常修复的能力 活着。因此，这是一个研究哺乳动物AP的作用的建议。 DNA修复和修复中的核酸内切酶(APE基因产物) 氧化c-jun或c-fos的DNA结合活性(ref1活性 猩猩)。申请人提议建立APE缺陷细胞系 Ref1或DNA修复活性的差异表达及其特征 这些品系的DNA修复能力和自发突变能力 对AP-1或NFkB活性也有影响。APE蛋白与Ape蛋白的相互作用 DNA聚合酶β和其他蛋白质(通过酵母双杂交检测 系统或噬菌体展示)将被表征。

DESCRIPTION: DNA is inherently unstable, but also attacked by metabolites and environmental mutagens such as oxygen radicals and alkylating agents, which form various types of abasic (AP) sites. AP sites can be mutagenic and so compromise genetic stability. Repair of AP sites is initiated ubiquitous "class II" AP endonucleases. Their biological functions have been tested in microorganisms, but they are likely front-line defense enzymes in humans, too. He will define the biological role of the main mammalian AP endonuclease, encoded by the APE gene, approaching this goal in several complementary ways. He will establish Ape-deficient cell lines from mouse embryonic tissues arising from mating between APE disruption ("knockout") heterozygotes; homozygous knockout embryos do not develop beyond day 5, but cells derived from them can be cultured in vitro. The cell lines will be tested for sensitivity to mutagens associated with AP site production (e.g., alkylating agents), as well as other agents. Dr. Demple will measure spontaneous mutation frequencies for these lines to gain insight into the role of Ape in correcting endogenous DNA damage. He will test whether the phenotypes are reversed by introduction of recombinant wild-type APE, or mutant derivatives encoding proteins defective in repair or "Refl" activity that affects transcription factor binding in vitro. He will also explore interactions of Ape with other repair and non-repair proteins using yeast two-hybrid and phage display methods. He have already demonstrated that Ape interacts with DNA polymerase B, which catalyzes the next two steps in base excision repair. He will explore the significance of this interaction by identifying regions of the two proteins needed for this interaction. Dr. Demple will isolate Ape derivatives unable to interact with the polymerase and assess their ability to support normal repair in vivo. Thus, this is a proposal to study the role of the mammalian AP endonuclease (the product of the Ape gene) in DNA repair and in restoration of the DNA binding activity oxidized c-jun or c-fos (the ref1 activity of Ape). The applicant proposes to generate Ape-deficient cell lines differentially expressing ref1 or DNA repair activities and characterize these lines with respect to DNA repair capacity and spontaneous mutagenesis and also for AP-1 or NFkB activity. The interaction of the Ape protein with DNA polymerase beta and other proteins (detected by the yeast two-hybrid system or phage display) will be characterized.