The long term goal of this research is to understand the mechanism of telomere function. Although the biological importance of telomeres is well established, much remains to be determined about fundamental aspects of telomere biochemistry (eg. their complete structure, how they are synthesized during chromosome healing, and how pre-existing telomeres are replicated). We will use the ciliates Euplotes and Oxytricha to study telomere biochemistry because these organisms have a huge number of telomeres. This trait has facilitated isolation of telomere components and is why studies of Euplotes and Oxytricha telomeres are relatively well advanced. As Euplotes will undergo synchronous de novo telomere synthesis, this species provides a particularly useful model system for studying chromosome healing. During the previous grant period we identified two unique telomere proteins; the 51 kDa telomere-binding-protein and a related telomere protein homolog. Our studies suggested that these two proteins, together with telomerase and a DNA primase, are required for specific aspects of telomere synthesis and maintenance. The overall objectives of this proposal are: to learn more about how ciliate telomere-binding proteins bind telomeric DNA; to determine how the Euplotes telomere-binding protein and the telomere protein homolog participate in telomere replication and de novo telomere synthesis; and to better understand how synthesis or replication of the telomeric C-strand is initiated.
The specific aims are as follows. First, we will characterize the features of the Euplotes telomere-binding protein that enable it to bind tightly to telomeric DNA as a monomer. In particular, we will explore how the C- and N-terminal domains provide binding specificity and stability. Second, we will determine the role of the telomere-binding protein during de novo telomere synthesis. Since the protein may regulate (G- or C- strand synthesis, we will determine whether the protein binds to the newly synthesized (G-strands. Third, we will investigate the role of the telomere protein homolog in telomere replication. We will isolate the homolog and determine whether it has any of the properties expected of a replication protein. We will also generate recombinant homolog protein and use deletion mutagenesis to define functional domains within the protein. Finally, we will characterize the primase activity that is involved in telomere replication and synthesis. We will isolate primase from Oxytricha and examine the specificity of the activity for telomeric DNA in the presence and absence of telomere-binding protein.
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