Telomeres, the natural termini of linear chromosomes, are crucial for genome stability and cell proliferation. Telomere length reservoirs which are inadequate to sustain organ function in humans can be a substantial contributing factor to degenerative organ failure, referred to as "syndromes of telomere shortening". Recent evidence indicates that defects in three telomere-related complexes - telomerase, shelterin and a heterotrimeric RPA-like complex - can each give rise to the same detrimental consequences in humans. This third activity was first identified in budding yeast as a telomere-dedicated version of the canonical RPA complex (referred to as the t-RPA complex);it has long been assumed to function as an end protection factor, by protecting chromosome termini from unregulated resection while bound to the terminal G-strand overhang of fully replicated telomeres. This application proposes instead that the t-RPA complex performs its functions at telomeres during DNA replication by promoting passage of the replisome through telomeric duplex DNA. In this model, the essential function of the t-RPA complex is to prevent stalling and subsequent collapse of the replication fork, by modifying the DNA replication machinery to form a telomere- specific replisome. When fork collapse does occur, this model further proposes that telomerase is recruited to the site of fork collapse, to prevent the accumulation of prematurely truncated termini. This will be tested by the following three approaches. First, the mechanism by which the t-RPA complex promotes duplex telomeric DNA replication will be elucidated by constructing a comprehensive genetic map of the functional surface of the t-RPA complex, identification of protein interactions that mediate t-RPA-specific activities and construction of at-RPA-specific epistasis network. Second, the functional interplay between the t-RPA and RPA complexes at telomeres will be examined by asking whether the t-RPA complex replaces RPA as the replisome encounters repetitive telomeric DNA, or whether t-RPA becomes an additional component of the replisome. Third, a novel assay designed to capture replication errors that occur during a single cell cycle will be used to determine whether collapsed replication forks are elongated by telomerase;in addition, proteins previously thought to regulate recruitment of telomerase to fully replicated ends will be examined for whether they instead impact telomere length through effects on replication fork stalling/collapse. All three of these approaches will be aided by an assay that monitors sequence changes that arise in response to replication errors in duplex telomeric DNA at single-nucleotide resolution, which provides a level of analysis not previously possible with conventional protocols. This integrated approach will elucidate how the t-RPA complex interfaces with the DNA replication machinery to ensure faithful progression of the replisome through duplex telomeric DNA and thereby ensure telomere homeostasis.
Telomeres, specialized termini that cap the ends of chromosomes, need to be maintained in order to promote cell proliferation. Inadequate telomere function accounts for a wide range of pathophysiologies among a broad segment of the human population, referred to as syndromes of telomere shortening. The goal of this application is to provide a mechanistic basis for understanding how a crucial telomere complex helps promote fully functional chromosome ends.