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.