Eukaryotic chromosomes are replicated and segregated with extremely high fidelity. The long range goal of this grant is to identify the mechanisms that ensure the accuracy of these processes, using S. cerevisiae as a model system.
The specific aims for this funding period concern telomeric and sub-telomeric regions of the chromosome. Telomeres were long thought to be essential for the stable maintenance and complete replication of eukaryotic chromosome. However, studies in Drosophila demonstrate that chromosome without telomeres are not degraded nor do they fuse with other broken ends (8,68). In contrast, indirect experiments in other systems suggest that chromosomes without telomeres are unstable. To determine the precise role of telomeres in yeast, we will eliminate the telomeric repeats from an authentic, but totally dispensable, chromosome in a strain where broken chromosomes cannot be repaired by conventional recombination. In addition to the simple repeats found at the very ends of chromosomes, sub-telomeric regions of most eukaryotic chromosomes have middle repetitive DNAs called telomere-associated (TA) sequences. To determine the function of TA sequences, derivatives of chromosome VII that lack TA sequences on both arms have been constructed. We will determine the stability of these deleted chromosomes in mitosis, meiosis, and after X-ray induced breakage. In virtually all eukaryotes, telomeres are dynamic structures. Almost surely recombination in telomeric and sub-telomeric regions contributes to variability. work from our lab provided the first evidence for telomere- telomere recombination by demonstrating that foreign telomeric sequences recombine in yeast by a novel mechanism that requires very little homology (93,128). To determine the function of this recombination, we will use a system where telomere-telomere recombination can be detected between """"""""natural"""""""" yeast telomeres on plasmids and chromosomes and have designed schemes to identify mutants in telomere-telomere recombination. A possible role for telomere recombination is to assist in the replication of chromosome ends. Using two dimensional gel electrophoresis of plasmid DNA from synchronized cells, we have identified a presumptive intermediate in telomere replication, the first in vivo intermediate identified in any system. We describe experiments to continue analysis of its structure, to determine the gene products on which its synthesis depends, to determine if replication of chromosomal telomeres proceeds by the same mechanism, and to understand how its interactions in vivo contribute to processes like telomere recombination. Aneuploidy and chromosome rearrangements are associated with virtually human cancers, with aging, and with birth defects. Changes in telomeric and sub-telomeric regions can generate the kinds of chromosome abnormalities that are associated with these diseases. Given that the telomeric regions of chromosomes in yeast and humans are similarly organized, a molecular and genetic understanding of the mechanisms that underly these events in yeast will contribute to the understanding of the genesis of chromosome abnormalities in higher cells.
Showing the most recent 10 out of 49 publications
