Investigating the role of nuclear organization in telomere length regulation

Telomeres are DNA-protein complexes that form the ends of linear chromosomes in eukaryotic organisms. The telomeric structure performs several functions that maintain genome integrity. First, telomeres protect chromosome ends from being recognized by the cell's DNA damage repair proteins, preventing fusion to other chromosome ends or to true double-stranded breaks. Second, the extension of telomeres by telomerase, a specialized reverse transcriptase that extends telomeres, buffers the genome from the loss of end DNA that occurs in every round of DNA replication due to the end-replication problem (Watson 1972). Telomerase-mediated elongation has remained an area of active investigation, and many of its details have been elucidated in the model organism Saccharomyces cerevisiae.;Telomeres in Saccharomyces cerevisiae cluster at a small number of foci at the nuclear periphery (Gotta et al 1996). Individual telomeres are more dynamic and leave the nuclear periphery throughout the cell cycle (Heun et al. 2001, Tham et al. 2001). In this thesis, I sought to investigate how telomerase is able to access telomeres from the bulk of peripheral telomeres. To determine whether elongating telomeres become spatially isolated from bulk telomeres at any point during the cell cycle, I developed a system where I could examine the nuclear position of shortened VII-L telomeres that become preferred targets for telomerase (Marcand et al. 1999). Using a single cell analysis approach, I correlated the distance of telomeres from the nuclear periphery with the cycle position for each cell. I detected no difference in the localization of short or wild-type length of telomeres at any point in the cell cycle. The data show that elongating telomeres do not move away from the nuclear periphery to be elongated by telomerase, and suggest that short telomeres are not required to leave the nuclear periphery to become recognized as preferred substrates for telomerase.;Telomere anchoring to the nuclear periphery in S phase is mediated by the nuclear membrane protein Mps3p. An N-terminal domain (amino acids 75-150) is necessary and sufficient for this anchoring function (Bupp et al 2007). I show that telomere length is unaffected in an mps3Delta75-150 mutant. Using the chromatin immunoprecipitation (ChIP) assay, I show that the binding of the telomerase subunits Est1p and Est2p to telomeres is unaffected in the mps3Delta75-150 mutant. The data suggest that Mps3p-mediated anchoring of telomeres to the nuclear periphery is not required for telomere length regulation.;Telomeres that are artificially tethered to the nuclear envelope are maintained at a length 75 bp shorter than wild-type telomeres (Mondoux et al 2007). It was hypothesized that tethering telomeres either restricts telomeres from leaving the periphery to be accessed by telomerase, or restricts the mobility of telomeres that is required for engaging the telomerase machinery. The results in this thesis support a model where telomerase is able to access its preferred targets, short telomeres, throughout the nuclear space, and do not require a specific interaction with the Mps3p to elongate telomeres. These data do not exclude the possibility that telomere mobility is critical to telomerase recruitment to short telomeres.