The yeast Saccharomyces cerevisiae, like other eukaryotes, contains linear chromosomes. The long-term goal of the proposed research is to establish the structure chromosomes. The long-term goal of the proposed research is to establish the structure and mode of replication of the ends (telomeres) of the yeast chromosome. Since all known DNA polymerases elongate DNA in a 5' to 3' direction and require a primer to initiate replication, it is clear that the telomeres cannot be replicated by a conventional replication mechanism. In previous studies, it has been observed that yeast chromosomes terminate in a simple sequence that can be abbreviated polyC1-3A and that yeast cells have a mechanism that allows the untemplated addition of polyC1-3A sequences to certain linear substrates. Since it is likely that this mechanism is related to telomere replication, most of the proposed experiments are concerned with elucidating the details of the terminal addition reaction. Both genetic and physical studies of the reaction will be done. Yeast mutants that add tracts of polyC1-3A that are longer (or shorter) than normal will be isolated and characterized. The physiological conditions that influence tract length in the cell and the substrate sequences necessary to mediate the terminal addition of polyC1-3A in vivo will be examined. In addition, experiments designed to look for de novo formation of telomeres on the ends of broken chromosomes are discussed. A system that mediates the in vitro addition of terminal polyC1-3A sequences will be developed. Since all eukaryotes contain linear chromosomes, it is likely that the proposed studies will have general relevance. In addition, since the terminal addition of polyC1-3A residues to linear DNA molecules is a previously unobserved modification of DNA, the mechanism involved in the modification deserves further analysis.
