TELOMERASE RNP STRUCTURE/FUNCTION

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Eukaryotic chromosome ends are capped by teleomeres, DNA-protein structures essential for genome stability. The primary mechanism for synthesizing teleomeres is telomerase, an unusual ribonucleoprotein (RNP) with reverse transcriptase activity. In higher eukaryotes, telomerase expression is highly regulated and strongly correlated with cells having unlimited proliferation capacity. For example, more than 90 percent of human tumors are telomerase positive. Although these observations imply that telomerase plays a critical role in eukaryotes, surprisingly little is known about the subunit composition of this enzyme or its interactions in vivo. We are studying telomerase in a ciliated protozoan, Euplotes crassus. Because of the unique organization of the genome, ciliates such as Euplotes are an extremely rich source of telomeres and one of the few systems where both telomere maintenance and de novo formation are readily observed. We hypothesize that the E. crassus telomerase exists as a holoenzyme, comprised of a core RNP and factors that modify enzyme behavior. Our hypothesis is based on preliminary data showing 1) that telomerase performs de novo synthesis in only one stage of the life-cycle and this reaction is mediated by a trans-acting factor; and 2) that telomerase exists in higher order complexes exhibiting biochemical properties distinct from the core RNP particle. The long-term goal of this project is to elucidate the function and interactions of telomerase RNP constituents. Using conventional chromatography, affinity purification and molecular biological approaches, we propose to identify the core protein subunits of the E. crassus telomerase RNP, clone the corresponding genes and generate recombinant proteins (Aim 1). The function of telomerase- associated proteins will be examined using a variety of biochemical strategies, including development of an vitro reconstitution system (Aim 2). We will analyze higher order telomerase complexes that may correspond to a telomerase holoenzyme, by purifying these complexes, analyzing their constituents and examining their biochemical properties (Aim 3). Finally, we will investigate the molecular basis for the developmentally programmed switch in telomerase-DNA interactions. These studies include assaying for structural variations in the core RNP from two stages of the life cycle and characterizing the trans-acting factor implicated in de novo telomere formation (Aim 4). These studies will generate new insight into the structure and function of telomerase and provide needed information regarding telomerase regulation.