The Ape1-NPM1 Axis and Telomere Maintenance

 描述（由申请人提供）：端粒需要特殊的机制来维持，这些机制或端粒结构本身的破坏几乎发生在所有癌症中。端粒DNA的损伤破坏端粒的维持，并可导致染色体重排。我们最近发现，Ape 1蛋白，中央球员的碱基切除DNA修复，是需要适当的端粒维持在正常哺乳动物细胞和肿瘤细胞系。Ape 1是保护性蛋白TRF 2正常结合所必需的，Ape 1缺乏导致POT 1的端粒结合增加。Ape 1的AP核酸内切酶活性（DNA修复功能）是维持端粒所必需的。虽然Ape 1在整个细胞核中发现，但它通过与核磷蛋白（NPM 1）相互作用优先在核仁中积累，通过Ape 1 N-末端附近赖氨酸的乙酰化在DNA损伤的细胞中释放。虽然DNA损伤和端粒破坏与Ape 1在机制上相关，但NPM 1对Ape 1的调节如何在这一过程中发挥作用尚不清楚。此外，发生在约1/3的急性髓细胞性白血病（AML）的NPM 1c+突变导致NPM 1重新定位到细胞质中，携带Ape 1。在此修订后的应用程序中，我们将研究NPM 1-Ape 1相互作用对端粒维持的影响，以及其在AML和其他癌症的病因学中可能发挥的作用。虽然Ape 1不能被消除，但我们已经建立了有效下调Ape 1的RNAi方案。与NPM 1的相互作用可以通过用丙氨酸（以模拟不带电荷的乙酰化状态）或丙氨酸（以防止乙酰化并保留电荷）取代Ape 1的关键N-末端赖氨酸（27，31，32，35）来控制。这些工具和表达NPM 1c+蛋白的细胞将用于确定Ape 1-NPM 1相互作用是否对正常条件下的端粒维持很重要，以及面对氧化或烷化剂引起的DNA损伤，这些损伤通过碱基切除修复处理。端粒长度和TRF 2蛋白的占据将通过我们已经建立的方法进行监测。

 DESCRIPTION (provided by applicant): Telomeres require special mechanisms for their maintenance, and disruption of these mechanisms, or of the telomeric structures themselves, occur in nearly all cancers. Damage to telomeric DNA disrupts telomere maintenance and can result in chromosome rearrangements. We found recently that Ape1 protein, the central player of base excision DNA repair, is required for proper telomere maintenance in both normal mammalian cells and in tumor lines. Ape1 is required for normal binding of the protective protein TRF2, and Ape1 deficiency leads to increased telomeric binding of POT1. The AP endonuclease activity (DNA repair function) of Ape1 is required for telomere maintenance. Although Ape1 is found throughout the nucleus, it accumulates preferentially in the nucleolus via interaction with nucleophosmin (NPM1), from which it is released in DNA-damaged cells through acetylation of lysines near the Ape1 N- terminus. While DNA damage and telomere disruption are mechanistically related by Ape1, it is unknown how the modulation of Ape1 by NPM1 plays a role in this process. Moreover, the NPM1c+ mutations that occur in about 1/3 of acute myelogenous leukemias (AML) lead to relocation of NPM1 to the cytoplasm, carrying Ape1 with it. In this revised application, we will examine the effects of the NPM1-Ape1 interaction on telomere maintenance, and its possible role in the etiology of AML and other cancers. Although Ape1 cannot be eliminated, we have established RNAi protocols for its efficient down-regulation. The interaction with NPM1 can be controlled by replacement of the key N-terminal lysines (27, 31, 32, 35) of Ape1 with alanines (to mimic the uncharged acetylated state) or arginines (to prevent actylation and retain charge). These tools, and cells expressing the NPM1c+ protein, will be used to determine whether the Ape1-NPM1 interaction is important for telomere maintenance under both normal conditions, and in the face of DNA damage by oxidative or alkylating agents, which generate lesions processed by base excision repair. Telomere length, and the occupation by TRF2 protein, will be monitored by methods we have already established.