Studies of molecular mechanisms of telomere maintenance in Naumovozyma castellii
Abstract: Telomeres are special DNA-protein structures that protect ends of chromosomes from being recognized as double-strand breaks. Telomeres consist of tandemly repeated units of TG-rich DNA and associate with telomere-specific proteins.When a cell replicates its chromosomes, a certain piece of DNA is lost from chromosomal ends due to the ´´ end replication problem´´. This progressive shortening of telomeric sequences leads to inability of the chromosomal ends to distinguish themselves from double-stranded breaks. When a cell reaches to this crisis point, its proliferation will stop, leading to replicative senescence and eventually apoptosis. The most common way to counteract telomere loss is utilization of telomerase enzyme. Telomerase extends telomeres by using its intrinsic RNA template. Telomerase is active in human germ cells, cancer cells and certain eukaryotic species including the budding yeast Naumovozyma castellii. Most cancers cells (85-90 %) gain cellular immortality by re-activating the telomerase enzyme. The remaining cancer cells use alternative ways to restore lengths of their telomeres.In my doctoral studies I investigated molecular mechanisms of telomere maintenance in N. castellii. We reviewed the historical background of its establishment as a model organism and summarized its significant contributions to understanding of various molecular biological pathways. We developed stable haploid strains, which are amenable for genetic studies, to study telomerase-independent telomere maintenance. We characterized N. castellii telomerase regarding to its substrate specificity and priming capacity. We proposed a model where four different interactions occur, influencing the initiation of its priming capacity. We have showed the anchoring DNA regions that influence the initiation of telomere extension. Lastly, we investigated how telomeres are maintained when telomerase is disabled. Surprisingly, telomerase-negative cells proliferate without a detectable growth crisis. We showed that they maintain a short stretch of telomeric seqeunce at the very ends of chromosomes and also wild type structural organization at the chromosomal ends.In conclusion, I investigated the telomere maintenance from two different perspectives. These parallel studies contributed to understanding of the dynamics of telomere maintenance. Moreover, they emphasized conserved features of telomere biology, helping visualizing the evolutionary origins of telomerase and maintenance of linear chromosomes.
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