Telomeres are essential for the integrity of eukaryotic chromosomes: they cap the end protecting it from fusion event and degradation. In order to confer this protection, telomeric DNA sequence repeats must be maintained at the end of the chromosome, along with a plethora of interacting factors that are essential for providing capping function. The inability to maintain the cap results in genomic instability, a genetic basis of cancer. How telomeres provide this protection is not well understood, nor is it clear how the capping structure is (re)assembled after chromosomes are duplicated. We have developed a very sensitive genetic assay in S. cerevisiae that can detect when a telomere loses its capping function and propose to use this assay to identify the capping components to understand the steps required for cap assembly. Telomerase is required in most eukaryotic cells to maintain the telomeric DNA sequences. A lack of its activity has been correlated with aging in many cell types. It is well documented that telomerase synthesizes telomeric DNA sequences onto the 3' end of telomeric DNA oligonucleotides in vitro. However, it is much less clear how the full duplex of telomeric DNA at the end of the chromosome is added in vivo. We have developed an assay in S. cerevisiae that allows us to monitor the addition of telomeric DNA onto a de novo telomere in vivo. In preliminary results the investigator finds that telomere addition not only requires telomerase, but also the DNA polymerases involved in lagging strand synthesis. This tight coordination between telomerase and lagging strand synthesis helps them understand the nature of telomeric DNA synthesis in vivo and provides a framework for further experiments with this assay. In S. cerevisiae, the telomere initiates a chromatin structure that appears to be like heterochromatin in other eukaryotes. In addition, telomere proximal sequences replicate late in S phase, just as heterochromatic regions of eukaryotes do. They found that telomeric chromatin causes late replication of telomere proximal DNA. It can delay initiation at nearby origins of replication, or prevent initiation from occurring at all. Based on these results he proposes a genetic assay to monitor late replication of telomeric origins, and to use this assay to isolate mutants. He will identify components, and their regulators, in the replication initiation process within a heterochromatic domain. This system may serve as a model for how heterochromatic induced late replication occurs, such as on the inactive X in female mammals.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cell Development and Function Integrated Review Group (CDF)
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Carter, Anthony D
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Fred Hutchinson Cancer Research Center
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