Function of human FEN1 in telomere maintenance

描述（由申请人提供）：端粒保护染色体末端，对维持稳定的基因组至关重要。这种保护系统的失败会导致基因组不稳定，从而导致癌症和过早衰老等疾病。许多参与DNA修复、复制和重组的蛋白质与端粒相关，并介导端粒的保护功能。因此，重要的是要了解在体内的作用，DNA代谢蛋白质和这些蛋白质在端粒突变的后果。我们的长期目标是了解DNA复制因子如何有助于维持端粒的完整性。对酵母细胞的研究表明，DNA复制机制在端粒聚合酶介导的端粒维持中起着至关重要的作用。然而，在人类癌细胞中，复制机制的组成部分如何影响端粒酶活性和端粒结构却知之甚少。在这个提议中，我们将研究一个这样的因子，人类FEN 1蛋白，一种在DNA复制过程中冈崎片段正常成熟所需的核酸酶。我们的初步研究表明，hFEN 1参与在端粒末端产生单链G-突出端，并且hFEN 1的缺陷诱导癌细胞中的端粒缩短。假设：hFEN 1核酸酶在端粒DNA复制后降解富含C链的端粒DNA，产生单链G突出端。hFEN 1的缺乏可能损害G-突出端的产生并使端粒末端成为端粒酶的不良底物，导致癌细胞中端粒酶介导的端粒维持的破坏。具体目标：1.确定hFEN 1的核酸酶活性是否负责体内和体外G-突出端的产生。显性负性核酸酶缺陷型hFEN 1将在人细胞中过表达，并分析其对端粒结构和端粒长度的影响。将建立一个体外系统来检测hFEN 1蛋白加工端粒DNA末端的能力。2.确定hFEN 1对前导和滞后子端粒的影响。在一轮DNA复制后，将分离前导和滞后子端粒，并分析FEN 1缺乏对这两个子端粒的影响。3.确定hFEN 1对端粒酶向端粒募集的影响。免疫共沉淀法将用于检测在细胞周期的S期期间hFEN 1与端粒酶的物理相互作用。染色质免疫沉淀试验将用于检查FEN 1缺陷细胞中端粒酶向端粒募集的任何改变。这些研究结果将有助于我们理解癌细胞端粒的维持机制，并促进新的癌症治疗方法的发展。与公共卫生的相关性：维持端粒的完整性对癌细胞的无限生长至关重要。该提案旨在对癌细胞中的端粒维持、细胞生长控制和肿瘤发生产生新的见解。它可能会导致新的抗癌疗法的发展。

DESCRIPTION (provided by applicant): Telomeres protect chromosome ends and are critical for maintaining a stable genome. Failure of this protection system results in genome instability that leads to diseases such as cancer and premature aging. Many proteins involved in DNA repair, replication, and recombination are associated with telomeres and mediate the protective function of telomeres. It is therefore important to understand the in vivo roles of DNA metabolic proteins and the consequences of mutations in these proteins at telomeres. Our long-term goal is to understand how the DNA replication factors contribute to the maintenance of telomere integrity. A critical role of the DNA replication machinery in telomerase-mediated telomere maintenance has been suggested from studies of yeast cells. However, how the components of the replication machinery influence telomerase activity and telomere structure is poorly understood in human cancer cells. In this proposal, we will investigate one such factor, human FEN1 protein, a nuclease that is required for normal maturation of Okazaki fragments during DNA replication. Our preliminary studies demonstrate that hFEN1 is involved in generating the single- stranded G-overhangs at telomere ends, and that a deficiency in hFEN1 induces telomere shortening in cancer cells. Hypothesis: the hFEN1 nuclease degrades telomere DNA on the C-rich strand after telomere DNA replication, generating single-stranded G-overhangs. Deficiency in hFEN1 may compromise G-overhang generation and make telomere ends poor substrates for telomerase, leading to disruption of telomerase- mediated telomere maintenance in cancer cells. Specific aims: 1. To determine whether the nuclease activity of hFEN1 is responsible for G-overhang generation in vivo and in vitro. A dominant-negative nuclease- deficient hFEN1 will be overexpressed in human cells and its effect on telomere structure and telomere length will be analyzed. An in vitro system will be established to examine the ability of hFEN1 protein to process telomere DNA ends. 2. To determine the effect of hFEN1 on leading and lagging daughter telomeres. Leading and lagging daughter telomeres will be separated after one round of DNA replication and the effects of FEN1 deficiency on these two daughter telomeres will be analyzed. 3. To determine the effects of hFEN1 on the recruitment of telomerase to telomeres. A co-immunoprecipitation method will be used to detect physical interaction of hFEN1 with telomerase during the S phase of the cell cycle. The chromatin immunoprecipitation assay will be used to examine any alteration in the recruitment of telomerase to telomeres in FEN1 deficient cells. The findings from these studies will aid us in understanding telomere maintenance mechanism in cancer cells and facilitate the development of new cancer therapy. Relevance to public health: Maintenance of telomere integrity is essential for the unlimited growth of cancer cells. This proposal aims to yield new insights into telomere maintenance in cancer cells, cell growth control and oncogenesis. It may lead to the development of new anti-cancer therapy.