Exploring the Functions of the Tpz1 TEL-Patch and the Rif1 Protein in Fission Yeast Telomere Maintenance

In most eukaryotic species, the ends of chromosomes are protected by a repetitive stretch of GT rich non-coding DNA-protein protein structure called the telomere. This region buffers the end of the chromosome against DNA loss during replication. This loss of sequence during replication is known as the End Replication Problem. This imposes a replicative lifespan of a cell. After too many rounds of replication and cell division, a cell loses all of the protective telomere repeats and it triggers a DNA damage response leading to cell senescence before its chromosomal DNA is damaged. Dysregulation of this process or inhibition of the DNA damage response can lead to genomic instability and promote cancer cell formation. Disfunction of telomeres has also been reported to cause advanced aging phenotypes in humans. The telomerase protein solves this End Replication Problem by adding repeats to the telomere. Telomerase is activated in human germline cells and stem cells and is silent in somatic cells. However, telomerase is reactivated in immortalized cancer cells to allow them to divide indefinitely without loss of the telomere. The telomere associated proteins, known collectively as the Shelterin Complex, protect the telomere from being recognized as DNA damage and modulate telomerase recruitment to the telomere in cells where it is active. These proteins, along with other protein factors that they recruit, control telomerase recruitment to the telomere, regulating telomere length. They also protect the telomere and attenuate the DNA damage response. Our goal is to understand how the Shelterin proteins regulate telomerase recruitment and telomere length in the Schizosaccharomyces pombe model system. This single celled eukaryote provides a good model system for telomere research as it has a high degree of conserved proteins and functions with the human Shelterin complex. As a single celled organism, S. pombe needs to maintain a tight regulation of telomerase recruitment and activity to ensure proper DNA replication and cell division. However, it is also able to circularize its chromosomes once telomere repeats are lost, thereby enabling the cell to continue growing with genetic perturbations that would be lethal to other eukaryotic cells, and allowing me to assay the effects of more severe telomere dysfunctions. An important protein in the Shelterin complex is Tpz1. This protein sits at the interface between the ssDNA binding Pot1 protein and the Poz1 bridge to the dsDNA binding Rap1 and Taz1 proteins and has been implicated in telomerase recruitment and activation at the telomere. Another important regulator of telomere length is the Rif1 protein. This protein plays a conserved global role in regulation of replication timing in the cell and has an effect on telomere length in yeast. The thesis presented here contains two major projects to better understand telomere regulation: 1) Exploration of the role of the S. pombe Tpz1 TEL-patch in telomerase activation and engagement at the telomere, and 2) characterization of the functional domains of the Rif1 protein and their roles in Rif1 interaction with other protein partners, Rif1-DNA binding, and Rif1’s ability to control replication timing. I show that the Tpz1 TEL-patch is responsible for telomerase engagement at the telomere. It facilitates tight binding of telomerase which allows its catalytic activity. I also dissect the functional domains of the Rif1 protein and the role of the conserved HOOK domain in Rif1 recruitment throughout the genome. This HOOK domain is necessary for Rif1 recruitment to the telomere through the Taz1 protein as well as Rif1 binding to DNA at Taz1-independent genomic locations. Overall, this work sheds light on the important roles of the Tpz1 and Rif1 proteins in telomere length regulation.