Research

The goal of our laboratory is to understand what roles telomeres play in chromosome stability and segregation and how this information is communicated to the cell cycle machinery. We approach these problems by studying the regulation of yeast telomere length and the proteins that are part of yeast telomeric chromatin using yeast genetics and molecular biological and biochemical approaches. These studies on telomere proteins have led us into a separate project investigating how these proteins regulate yeast life span.

Telomeres are the DNA-protein complexes required for the complete replication and stability of chromosome ends. The structure of telomeres in most organisms is a tandem array of short repeated sequences where the strand that makes up the 3' end of the chromosome contains many G residues. In humans and other vertebrates this simple repeat is T 2 AG 3 , and in the budding yeast Saccharomyces cerevisiae it is TG 1-3 . These DNA sequences are the only ones required for telomere function in humans and S. cerevisiae . The number of these repeats, or telomere length, is regulated in both organisms, presumably by balancing lengthening and shortening activities. In yeast, the TG 1-3 tracts are 275 to 400 bp in length and are bound by the protein Rap1p. Mutations in the genes for TEL1 , an ortholog of the human ATM gene involved in DNA-damage regulation of the cell cycle, HDF1 , an ortholog of the mammalian Ku70 protein that binds with Ku80 to double-strand DNA breaks, and TEL2 , a gene of unknown function, can cause yeast to maintain their TG 1-3 tracts at 100 to 150 bp. The TEL1 and TEL2 genes appear to function in the same genetic pathway while HDF1 acts in a different pathway.

We cloned the TEL2 gene and showed that it is essential for life (Runge and Zakian, 1996). The TEL2 protein can bind to both double-stranded and single- stranded telomeric DNA in vitro through a 60 amino acid DNA binding domain (Kota and Runge, 1998; Kota and Runge, 1999; R. Kota unpublished data). Basal expression of genes near telomeres are repressed, a phenomenon called telomere position effect or telomere silencing, a property of telomeric chromatin structure. The tel2-1 mutation, which causes short telomeres, also reduces TPE, while telomere shortening caused by the tel1 mutation does not, suggesting a specific role for TEL2 in determining telomere chromatin structure. We are currently investigating TEL2 function using our recently isolated tel2 temperature-sensitive mutants.

We have investigated telomere length directly by constructing synthetic telomeres, using them to replace a normal yeast chromosomal telomere and then monitoring telomere length in these synthetic constructs. We and others have found that yeast measure telomere length by counting the number of Rap1p molecules at the chromosome end. We have proposed a model that these molecules are counted by forming a structure that blocks telomere lengthening and that this structure is dependent upon a critical number of Rap1p molecules (Ray and Runge, 1999a), which we are currently testing. We have also used this system to examine yeast mutants lacking the TEL1 and HDF1 genes. We have found that while hdf1 cells count Rap1p molecules while maintaining their 100 bp TG 1-3 repeats, tel1 mutants do not (Ray and Runge, 1999b). Thus, tel1 cells maintain a nearly constant 100 bp TG 1-3 repeat length by using an alternative or "backup" telomere length regulatory mechanism. The nature of this backup mechanism and the nature of the proposed structure that regulates telomere length is currently being investigated. During the course of this work, we also discovered a new function for the telomere binding protein Rap1p: the ability to stimulate the elongation of short telomeres (Ray and Runge, 1998).

Genes placed near telomeres are transcriptionally silenced by the action of proteins called Sir2p, Sir3p and Sir4p. The Sir proteins also transcriptionally silence the yeast silent mating type cassettes and the rDNA repeats. Sir proteins are present in limiting amounts in yeast cells, so that the increases in the silencing of one locus occurs at the expense of silencing at another locus. For example, yeast cells can only divide ~25 times before they die and as cells approach the end of their life span, Sir proteins leave telomeres and migrate to the nucleolus. Telomere and silent mating type silencing are thus lost in old cells. We have recently found that the level of Sir3p phosphorylation is related to its localization: highly phosphorylated Sir3p is localized at telomeres while lack of Sir3p phosphorylation decreases telomere silencing, increases rDNA and silent mating type cassette silencing and increases life span (Roy and Runge, 2000). We are currently analyzing a variety of newly isolated mutants that decrease silencing at telomeres and increase silencing at rDNA and silent mating type cassettes. One of these mutants, slt2 , allowed us to identify the Slt2p MAP kinase as a kinase that phosphorylates Sir3p in response to continuous cell growth, and this phosphorylation event shortens yeast life span (Ray et al., 2003).