The long range goal of this grant is to understand processes that ensure the faithful maintenance of eukaryotic chromosomes, using Saccharomyces cerevisiae as a model organism. In most eukaryotes, the very ends of chromosomes, telomeres, consist of simple repetitive DNA. For example, in yeast, there are about 300 bps of C1-3A/TG1-3 DNA at each end of each chromosome. Many organisms, including yeast, also have internal tracts of telomeric sequence. Some of these internal tracts are near telomeres, whereas others are at more internal chromosomal sites. Telomeres are absolutely essential for the stable maintenance of yeast chromosomes (*). Although the function of internal tracts of telomeric sequence is unknown, internal tracts of C1-3A/TG1-3 repress transcription of nearby genes and have reduced recombination. The transcriptional repression and reduced recombination characteristic of internal1-3A/TG1-3 tracts are both accentuated when the tracts are near telomeres, suggesting that internal tracts and telomeres interact. The specific goals for the next funding period concern maintenance of both telomeres and internal tracts of C 1-3 A/TG1-3 DNA.
The first aim i s to develop a system to detect telomere- telomere recombination. Telomeres will be marked with a variant repeat encoded by a mutant telomerase RNA. The transfer of the variant to other telomeres will be monitored in wild type and mutant cells. The hypothesis that telomere-telomere recombination allows telomere maintenance when the primary replication pathway, telomerase, is impaired will be tested.
The second aim i s to identify genes that regulate recombination between internal tracts of C1-3A/TG1-3 DNA. These experiments will determine if there are specific regressors that limit recombination and if reduced recombination correlates with late replication.
The third aim i s to determine how the Piflp helicase limits the length of existing telomeres and reduces the rate and specificity of de novo telomere addition. Two major models, that Piflp is an inhibitor of telomerase or that Piflp is an inhibitor of recombination will be tested. Also, the functional relatedness of PIF-1-like genes from Saccharomyces and S. pombe will be determined, and a 2-hybrid approach will be used to identify Piflp- interacting proteins.
The fourth aim i s to determine if internal tracts of C1-3A/TG1-3 DNA, like telomeres, have a non-nucleosomal chromatin structure and if this chromatin structure is altered by proximity to a telomere. Aneuploidy and chromosomal rearrangements are associated with virtually all human cancers, with aging, and with birth defects. Loss of telomeric DNA and/or recombination between internal tracts of telomeric sequence can trigger the kinds of chromosomal abnormalities associated with these conditions. Since telomeric regions of chromosomes are similar from yeast to humans, understanding how telomeres and internal tracts of telomeric sequence are maintained in yeast is likely to be relevant to an understanding how telomeres and internal tracts of telomeric sequence are maintained in yeast is likely to be relevant to an understanding of the sources of genetic instability in humans.
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