Telomerase, a ribonucleoprotein enzyme, maintains telomeres-the protective DNA/protein complexes capping eukaryotic chromosomes- by replacing sequences lost during replication, thereby preventing catastrophic genome rearrangement. Appropriate telomere length is maintained, in part, by an elegant feedback mechanism in which proteins binding within the highly repetitive telomeric DNA negatively regulate telomerase access in cis. Remarkably, this feedback mechanism operates in Saccharomyces cerevisiae despite synthesis of heterogeneous TG-rich repeats by yeast telomerase. The P.I. and her colleagues have recently identified mutations in the catalytic subunit of yeast telomerase (est2LT alleles) that subtly alter telomere sequences and cause telomere overelongation. These novel mutants provide a unique system in which to study telomerase fidelity and the response of telomere homeostatic mechanisms to changes in enzyme function. The observation of subtle changes in the pattern of telomere repeat addition in est2LT strains raises the intriguing possibility that telomere lengthening results from decreased binding by proteins such as Rap1p that negatively regulate the activity of telomerase at telomeres. This hypothesis will be tested using genetic and biochemical approaches to examine the association and function of Rap1 protein at est2LT telomeres. Because little is understood about the properties of yeast telomerase that influence synthesis of particular patterns of telomeric repeats, mutations in the RNA template of telomerase will be utilized to identify the source of abnormal repeats generated by the mutant telomerase enzyme. Experiments are designed to examine telomerase processivity in vivo and to determine whether the frequency and/or extent of telomere addition are altered at native and broken DNA ends in est2LT strains. These experiments will provide significant insight into the patterns of template utilization by yeast telomerase and elucidate mechanisms through which subtle alterations in telomere sequence influence telomere length homeostasis.
Telomeres are DNA/protein complexes that protect the ends of eukaryotic chromosomes and facilitate complete chromosome replication. The repetitive DNA sequences of telomeres are maintained at a specific length by the regulated action of an enzyme called telomerase. This research will elucidate properties of the telomerase enzyme that affect the precise sequence and amount of telomeric DNA at chromosome ends. Such mechanisms are fundamental to the fidelity of chromosome maintenance. Part of the research project will be completed by a team of undergraduate students, including a summer participant from Barry University, a predominantly minority institution. A course designed to improve the mentorship skills of scientists at all levels of experience will augment research activities in the laboratory.
