Function of human FEN1 in telomere maintenance

描述(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的不稳定，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，了解DNA代谢蛋白在体内的作用以及端粒上这些蛋白突变的后果是很重要的。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复制机制在端粒酶介导的端粒维持中起着关键作用。然而，在人类癌细胞中，复制机制的组件如何影响端粒酶活性和端粒结构却知之甚少。在这个提案中，我们将研究一个这样的因素，人FEN1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，hFEN1参与了端粒末端单链G-突出的产生，并且hFEN1的缺陷导致癌细胞的端粒缩短。假设：hFEN1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-突出物。HFEN1的缺失可能会影响G-悬垂的产生，使端粒末端不利于端粒酶的底物，导致端粒酶介导的端粒维持在癌细胞中的中断。具体目的：1.确定hFEN1的核酸酶活性是否与体内和体外G-悬浮物的产生有关。在人类细胞中过表达显性负核酸酶缺陷的hFEN1，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hFEN1蛋白处理端粒DNA末端的能力。2.检测hFEN1对超前和滞后子体端粒的影响。在一轮DNA复制后，领先和落后的子体端粒将被分离，并将分析FEN1缺陷对这两个子体端粒的影响。3.检测hFEN1对端粒酶活性的影响。免疫共沉淀法将被用来检测在细胞周期的S阶段hFEN1与端粒酶的物理相互作用。染色质免疫沉淀试验将被用来检测FEN1缺陷细胞端粒酶重新募集到端粒的任何变化。这些研究结果将有助于我们了解肿瘤细胞的端粒维持机制，并有助于开发新的癌症治疗方法。与公共健康相关：维持端粒的完整性对于癌细胞的无限生长至关重要。这项提议旨在对癌细胞的端粒维持、细胞生长控制和肿瘤发生产生新的见解。这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.