Browsing by Subject "telomeres"
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Item Molecular dissection of telomere dysfunction and analysis of G-overhangs in Arabidopsis thaliana(2009-05-15) Heacock, Michelle L.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.Item Telomere-associated proteins in Arabidopsis thaliana(2009-05-15) Surovtseva, Yulia V.Telomeres comprise the physical ends of chromosomes. Essential functions of telomeres include protecting the terminus from being recognized as a DNA doublestrand break and facilitating the complete replication of the physical end of the DNA. Telomere functions are mediated by a large array of telomere-associated proteins. Mutations in telomere-related genes cause immediate telomere dysfunction, activation of DNA damage response, and accumulation of end-to-end chromosome fusions. In addition, changes in telomere complex composition may affect the ability of the telomerase enzyme to maintain telomeres in vivo. Here, we describe the characterization of telomere-associated proteins in the flowering plant, Arabidopsis thaliana. Using a bioinformatics approach, we identified twelve proteins with sequence similarity to vertebrate duplex telomere DNA binding proteins TRF1 and TRF2. We showed that, like their vertebrate counterparts, some of the Arabidopsis TRFL (TRF-LIKE) proteins can homodimerize and bind telomeric DNA in vitro, indicating that Arabidopsis encodes a large family of double-strand telomeric DNA binding proteins. We have also characterized three Arabidopsis POT1 proteins whose homologs in yeast and vertebrates associate with the single-stranded portion of telomeric DNA. Unexpectedly, we found that unlike POT1 protein in other organisms, Arabidopsis AtPOT1a protein associates with telomeres only in the S phase of the cell cycle and is a physical component of the active telomerase RNP complex, providing positive telomere length regulation. Our data implicated AtPOT1b, another Arabidopsis POT1 protein, in chromosome end protection. Finally, we showed that Arabidopsis thaliana has evolved a third POT1 protein, AtPOT1c, which contributes to both telomere length regulation and telomerase activity, and maintenance of the structure of the chromosome terminus. Thus, Arabidopsis has evolved a set of POT1 proteins that make distinct and novel contributions to telomere biology. Finally, we describe the identification and characterization of a novel Arabidopsis protein CIT1 (Critical for Integrity of Telomeres 1), and show that CIT1 deficiency leads to an immediate and profound telomere dysfunction and chromosome end deprotection. Altogether, these data provide new insight into plant telomereassociated factors and significantly improve our understanding of the overall architecture and evolution of telomeric complex in Arabidopsis.