Molecular dissection of telomere dysfunction and analysis of G-overhangs in Arabidopsis thaliana

Telomeres comprise the physical ends of chromosomes. In the absence of telomerase, the enzyme responsible for replenishing telomeric DNA, telomeres progressively shorten due to the end replication problem. Eventually telomeres reach a length where they are recruited into end-to-end chromosome fusions. Through the use of novel PCR strategies, I followed the fate of telomeres in plants lacking telomerase as they progressed into dysfunction. I uncovered two distinct structural/functional length transitions. The first transition (~1 kb) marks the onset of telomere dysfunction, where telomeres are transiently uncapped and a subset of them engage in end-to-end fusions. The second transition (~300 bp) defines complete telomere dysfunction as telomeres below this length lack G-overhangs and the vast majority of the chromosome ends fuse. Thus, these two telomere lengths define architectural transitions that link structure and function. In addition, I uncovered a hierarchy of end-joining pathways that join dysfunctional telomeres in which the non-homologous end-joining (NHEJ) protein, KU predominates. In the absence of KU, telomeres are joined by a microhomologymediated end-joining pathway (MMEJ) that is dependent on Mre11. I also show that DNA ligase IV (LIG4) is the predominant enzyme that ligates dysfunctional telomeres as fusions are reduced in its absence. These studies highlight the importance of repairing DSBs and demonstrate that Arabidopsis possesses highly redundant means for processing dysfunctional telomeres. The G-overhang is an essential feature of the telomere that is required for proper telomere function. I employed methods to examine G-overhang status in various mutants known to contribute to telomere maintenance in Arabidopsis. My analysis revealed that the putative G-overhang binding proteins POT1a, POT1b and POT1c, make modest, but distinct contributions to the G-overhangs. Additionally, I uncovered a major role for the putative telomere capping protein, CIT1 in maintenance of the Goverhang. G-overhang signals obtained from cit1 mutants were grossly increased indicating that CIT1 is involved in either protecting the C-rich strand of the telomere from nuclease attack, or in controlling telomerase extension of the G-strand. Together, these data have provided new insight into factors that contribute to telomere integrity and have further developed Arabidopsis as a model for telomere biology.