TELOMERE STRUCTURE AND FUNCTION

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Telomeres or ends of chromosomes contain simple repeat sequences that serve as buffer to, preserve integrity of genetic information coded in the chromosome. Several recent reports linked the shortening of telomere lengths to aging (Nature 345:458; Cell 61:193). However, it is not clear whether telomere shortening is the cause or the effect of aging. Activities such as synthesis of repeat sequences and recombination by unequal exchanges can contribute to the lengthening of telomeres. Incomplete replication of the lagging daughter strand and exonucleolytic degradation result in shortening of telomeres. To maintain an optimal balance between these many metabolic processes that take place at telomeres, many proteins must interact directly with telomeric sequences. It has been shown in human and in Tetrahymena that replication of telomeres is carried out by a specialized reverse transcriptase-termed telomerase. Alteration of telomerase activity in Tetrahymena resulted in cells that exhibited abnormal morphology and that became senescent. In yeast, telomerase activity has not yet been identified. Our laboratory has partially purified two novel yeast telomere-binding activities, TBFalpha and TBFbeta. TBFalpha binds to the junction between the subtelomeric X sequence and the polyC(1-3)A telomeric sequence in a cloned yeast telomere. Under certain conditions, TBFalpha also interacts with the end of the chromosome, and specifically, with the GT strand of the terminus. Examination of the junctions of known X sequences indicate that they all contain one or more repeats of CCCTAA, a sequence which is repeated in vertebrate telomeres. Such heterologous telomeric sequences, positioned as far as several hundred base pairs from the termini of linear molecules, allow the addition of yeast telomeric sequences from the nontelomeric termini in vivo. We propose that TBFalpha serves as an anchor for the yeast telomerase by binding to the conserved junction sequence at a distance from the terminus to allow addition of an irregular repeating sequence to the GT-rich strand at the chromosome end. We will test this hypothesis by assaying for telomerase activity associated with TBFalpha. We will also try to clone the genes that encode TBFalpha and TBFbeta by screening an expression library of yeast genes, either with DNA substrates of TBFalpha and TBFbeta, or with antibodies raised against these proteins. Further biochemical characterizations of TBFalpha and TBFbeta coupled with genetic analysis of the genes that encode these proteins should reveal the biological functions of these telomere-binding proteins in yeast.