Dissection of the telomere complex CST in Arabidopsis thaliana

Abstract

Telomeres are the ends of linear chromosomes tasked with preventing their recognition by the DNA damage machinery and providing a mechanism to solve the end replication problem. The telomeric DNA is mostly double-stranded, but it terminates in a 3’ protrusion termed the G-overhang. Telomeres utilize telomerase, a reverse transcriptase, to elongate the telomere, and thus, solve the end replication problem. Both the double strand region and the G-overhang are bound by specific proteins to facilitate the objectives of the telomere. First discovered in budding yeast, the CST (Cdc13(CTC1)/Stn1/Ten1) complex binds to the G-overhang and is important for both chromosome end protection and telomere replication. Work reported in this dissertation provided the first evidence that CST was present outside of yeast, which led to its subsequent identification in a number of vertebrates.

Here I present the identification and characterization of the three components of CST in Arabidopsis thaliana. Similar to yeast, Arabidopsis CST is required for telomere length maintenance, for preventing telomere recombination and chromosome end-to-end fusions. Mutations in the CST complex result in severe genomic instability and stem cells defects. My research also shows that CST and telomerase act synergistically to maintain telomere length. Together these data provide evidence for an essential role for CST in maintaining telomere integrity.

Unexpectedly, I discovered that the TEN1 component of CST may have a more complex role than other members of the heterotrimer. The majority of telomere-related functions we can assay using molecular and cytological approaches are shared by CTC1, STN1 and TEN1, though TEN1 has additional roles in maintaining genome stability, modulating telomerase activity and possibly non-telomeric functions in the chloroplast.

I also present genetic evidence that TEN1 and STN1 act in the same pathway for the maintenance of telomere length and chromosome end protection. Interestingly, however, disrupting the STN1/TEN1 interaction reveals a separation of STN1 function for chromosome end protection versus telomere length maintenance.

Finally, I describe the design and creation of a library of STN1 and TEN1 mutants that will be used to further characterize their functions and their interaction partners. By disrupting such interactions, it will be possible to elucidate the functional significance of these interactions, and thus, provide new insight into how CST functions in Arabidopsis.