IDENTITY AND FUNCTION OF VERTEBRATE TELOMERE PROTEINS

端粒和端粒酶是人类肿瘤发生中的关键因素，因为癌细胞中端粒酶的上调导致端粒的维持，并使细胞变得永生。虽然端粒酶是维持端粒所必需的，但端粒的实际结构也至关重要。这是因为端粒中的蛋白质决定了端粒酶是否能到达DNA末端。这些端粒蛋白还通过防止端到端的融合来维持染色体的完整性，并阻止DNA末端被识别为受损的DNA并触发细胞周期检查点。我们的目标是了解脊椎动物端粒的结构以及端粒蛋白在端粒功能中所起的作用。虽然酵母对端粒的结构和功能的了解越来越多，但对脊椎动物的端粒却知之甚少。这在很大程度上是因为脊椎动物细胞不受基因操作的影响。然而，由于现在可以在鸡细胞系(DT40)中实现有效的基因打靶，我们将使用这个鸡系统来产生端粒蛋白的基因敲除。这将使我们能够采用遗传和生化方法来确定单个端粒蛋白如何对端粒结构和功能做出贡献。我们还将使用敲除细胞作为工具来识别正常端粒功能所需的额外蛋白质。目的一是研究鸡和人端粒的染色质结构。我们将检查末端DNA结构，并确定它是否与hnRNPs结合。我们还将检查DNA包装，以及端粒蛋白沿端粒束长度的定位。目的2是通过反向遗传方法确定单个端粒蛋白的功能。我们将建立已知端粒蛋白的敲除细胞系，并分析其表型。然后，我们将检查突变蛋白的表达是否恢复了端粒功能。目的3是确定末端连接和重组通路是否参与端粒维持。我们将调查Ku70和Rad54是否是维持端粒所必需的，以及这些蛋白质是否是端粒的组成部分。目的4是确定正常端粒功能所需的其他蛋白质。我们将使用逆转录病毒表达文库来筛选端粒功能缺陷的高复制抑制基因。

Telomeres and telomerase are key players in human tumorigenesis because upregulation of telomerase in cancer cells leads to telomere maintenance and allows the cells to become immortal. While telomerase is needed for telomere maintenance, the actual structure of the telomere is also critically important. This is because the proteins that are part of the telomere determine whether telomerase has access to the DNA terminus. These telomere proteins also maintain chromosome integrity by preventing end-to-end fusions, and they stop the DNA termini from being recognized as damaged DNA and triggering cell-cycle checkpoints. Our goal is to understand the structure of vertebrate telomeres and the role that the telomere proteins play in telomere function. While telomere structure and function are becoming quite well understood in yeast, much less is known about vertebrate telomeres. This is largely because vertebrate cells have not been amenable to genetic manipulation. However, because efficient gene targeting can now be achieved in a chicken cell line (DT40), we will use this chicken system to generate gene knockouts of telomere proteins. This will enable us to take both genetic and biochemical approaches to determine how individual telomere proteins contribute to telomere structure and function. We will also use the knockout cells as a tool to identify additional proteins that are required for normal telomere function. Aim one is to characterize the chromatin structure at chicken and human telomeres. We will examine the terminal DNA structure and ascertain if it is bound by hnRNPs. We will also examine the DNA packaging, and positioning of telomere proteins along the length of the telomeric tract. Aim 2 is to determine the function of individual telomere proteins by reverse genetic approaches. We will make knockout cell lines for known telomere proteins and analyze their phenotype. We will then examine whether expression of mutant protein restores telomere function. Aim 3 is to determine whether end-joining and recombination pathways are involved in telomere maintenance. We will investigate whether Ku70 and Rad54 are required for telomere maintenance and whether these proteins are telomere components. Aim 4 is to identify additional proteins that are required for normal telomere function. We will use a retroviral cDNA expression library to screen for genes that act as high copy suppressors of defects in telomere function.