Healing of Broken Eucaryotic Chromosomes

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Telomeres, specialized structures at chromosome ends, are essential to maintain chromosome stability. Broken chromosomes are unstable, but may become "healed" by recombination with an intact homologue, or by more novel means to produce terminally deficient chromosomes. These may arise either by de novo telomere formation or by translocations. A detailed analysis of chromosome repair will be carried out in the yeast Saccharomyces Cerevisiae, using several methods to introduce chromosome breaksat one chromosomal location. Strains containing de novo telemeres will be analyzed to determine the molecular mechaniswm by which these ends are added to broken chromosomes. Translocations will be monitored by an improved method of detection. We have discovered a novel form of repair of broken chromosomes, by recombination between homologous regions well-separated from, but flanking the break-point. This intra-chromosomal repair system will be analyzed in detail, to determine how far homologous sites can be separated and how much homology is required. This event can be induced synchronously and followed in "real time" by DNA analysis. Special emphasis will be given to understanding the mechanism by which broken chromosomes become shorter. The structure of normal telomeres will also be examined to determine the origin of a high degree of polymorphism and rearrangement in the regions immediately adjacent to telomeres. Finally, we will extend our study of the repair of broken chromosomes to another, evolutionarily divergent yeast, Schizosaccharomyces cerevisiae. Linear dicentric chromosomes will be generated by meiotic recombination between a linear chromosome and a circular chromosome constructed by recombinant DNA techniques. The differences in chromosome structure and DNA repair mechanisms between S. pombe and S. cerevisiae are expected to reveal different types of healing events. Little is known of chromosome structure and how it is maintained. The stability of chromosomes which allows faithful segregation must be inherent in the structure and this work will provide insight into that structure.