Mechanisms of sensing and regulating telomere length in budding yeast

Telomeres are protein-DNA structures at the ends of linear chromosomes. Crucial for faithful genome maintenance and inheritance, telomeres affect cellular aging, apoptosis, and cancer development. Telomeres are composed of a repetitive sequence synthesized by the enzyme telomerase. Telomere length is maintained within a defined range that is essential to proper telomere function and is an important determinant of the proliferative capacity of a cell. The goal of the work described in this thesis was to further elucidate the molecular mechanism of telomere length regulation. Questions relevant to this problem are: (1) how does telomeric tract length determine the structure and composition of the telomere, (2) how is telomere length sensed, and (3) how do telomere length sensing and telomere structure determine whether or not telomerase acts to elongate the telomere? This thesis addresses these questions of telomere structure and mechanisms of telomere length regulation.; Saccharomyces cerevisiae telomeres composed of (TG) 1-3 sequence are maintained between 250-350 base pairs. The principal double-stranded telomeric DNA binding protein is Rap1. Telomere-bound Rap1 negatively regulates telomere length, and telomere length is sensed by the number of Rap1 molecules at the telomere through a Rap1 counting mechanism. Rif1 and Rif2 also negatively regulate telomere length, and they associate with the telomere through interactions with the C-terminus of Rap1. We show that Rif1 and Rif2 have Rap1-independent modes of telomere length regulation and that a Rif protein counting model regulates telomere length. We also demonstrate that heterologous protein-protein interactions at the telomere can substitute for the Rif proteins to regulate telomere length. We describe a series of experiments that support a model for Rif2 function as a protein that initiates the nucleation and spreading of higher-order telomere structure. To investigate the molecular basis of length regulation by these proteins, in vitro studies were undertaken. We show that purified Rap1 protein is a monomer with an elongated shape, and that Rap1 associates non-cooperatively with arrays of DNA binding sites. We also find that purified Rif2 associates with Rap1/DNA complexes but not with Rap1 alone. We present low-resolution microscopy images of Rap1/DNA complexes.