About event
The seminars are devoted to topics that develop directions of the research groups of the Mendel Center for Plant Genomics and Proteomics. The speakers are primarily prominent foreign and domestic experts. In addition, some seminars are dedicated to the presentation of research results by internal speakers, mainly postdoctoral fellows and PhD candidates. Seminars are thus a platform for sharing knowledge, establishing cooperation and developing communication skills.
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Telomeres are essential for genome stability since they serve as a protective cap at the ends of eukaryotic chromosomes. The ends of telomeric DNA resemble bona fide DNA double-strand breaks and must be protected from inappropriate DNA damage response sensing and aberrant DNA repair. The human shelterin complex, composed of six subunits (TRF1, TRF2, TIN2, TPP1, POT1, and RAP1) represents the major protein complex that specifically binds to telomeric DNA and counteracts the recognition of chromosome ends by DNA repair machinery. Moreover, shelterin recruits telomerase to telomeres to compensate for incomplete DNA replication. How the shelterin complex is able to simultaneously protect telomeres and allow telomerase access to chromosome ends is a critical unaddressed question in the field. Previous studies have suggested that shelterin proteins are not present in cells in equal numbers, which implies that a variety of subcomplexes must exist that can fulfill distinct functions. Additionally, in vitro reconstitutions showed that shelterin can assemble into various subcomplexes, but it is unclear how shelterin composition is dynamically regulated in cells to coordinate its disparate functions in telomere maintenance. To answer this outstanding question, we employed CRISPR/Cas9 genome editing to introduce the HaloTag at the endogenous loci of all shelterin components in telomerase-positive HeLa cells and U2OS cells, which maintain their telomeres using an alternative lengthening of telomeres (ALT) – a mechanism specific only to cancer cells. With generated HaloTagged shelterin cell lines, we measured the shelterin abundance and stoichiometry directly at telomeres in living cells. Strikingly, we found that shelterin stoichiometry at telomeres is altered in cells utilizing ALT compared to telomerase-positive cells, suggesting distinct functional requirements for shelterin in these cells. Furthermore, live-cell single-molecule imaging reveals that TRF2 and RAP1 subunits are highly dynamic, while the rest of the shelterin components are tightly bound to telomeres. These observations suggest that shelterin exists primarily in two subcomplexes that fulfil distinct functions. Specifically, TRF1-TIN2-TPP1-POT1 subcomplex controls telomerase access to chromosome ends, while the dynamic binding of TRF2-RAP1 likely facilitates the recruitment of a variety of factors involved in telomere protection from the DNA repair machinery. Overall, we define the biochemical properties of shelterin in living cells and we provide critical insights into the molecular mechanism of how shelterin can orchestrate multiple essential functions in achieving telomere homeostasis.
