Telomeric Dotaz Zobrazit nápovědu
More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.
- MeSH
- chlorid sodný farmakologie MeSH
- DNA chemie genetika metabolismus MeSH
- fluorescenční polarizace MeSH
- fluorescenční spektrometrie MeSH
- kinetika MeSH
- kompetitivní vazba účinky léků MeSH
- lidé MeSH
- povrchová plasmonová rezonance MeSH
- protein TRF2 chemie genetika metabolismus MeSH
- proteiny vázající telomery chemie genetika metabolismus MeSH
- statická elektřina MeSH
- telomery genetika metabolismus MeSH
- vazba proteinů účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
G-quadruplexes are types of secondary structures of nucleic acids, which are formed in guanine-rich regions. Their existence in vivo was confirmed in promoter regions of some genes and at the end of chromosomes in the telomeric regions. The specific structure of G-quadruplexes is a promising target in cancer treatment. There is a search for small ligands, which could stabilize a specific type of G-quadruplexes. Cancer cells are markedly different from normal cells in the telomeric region: the regions are profoundly shorter. Telomeric regions in cancer cells are prolonged by an enzyme telomerase, while this enzyme is inactive in almost all somatic cells. Stabilization of telomeric G-quadruplex by a specific small ligand induces disruption of a normal protective function of telomers leading to cell damage and its death. This approach could be successful in cancer treatment.
There are two basic mechanisms that are associated with the maintenance of the telomere length, which endows cancer cells with unlimited proliferative potential. One mechanism, referred to as alternative lengthening of telomeres (ALT), accounts for approximately 10-15% of all human cancers. Tumours engaged in the ALT pathway are characterised by the presence of the single stranded 5'-C-rich telomeric overhang (C-overhang). This recently identified hallmark of ALT cancers distinguishes them from healthy tissues and renders the C-overhang as a clear target for anticancer therapy. We analysed structures of the 5'-C-rich and 3'-G-rich telomeric overhangs from human and Caenorhabditis elegans, the recently established multicellular in vivo model of ALT tumours. We show that the telomeric DNA from C. elegans and humans forms fundamentally different secondary structures. The unique structural characteristics of C. elegans telomeric DNA that are distinct not only from those of humans but also from those of other multicellular eukaryotes allowed us to identify evolutionarily conserved properties of telomeric DNA. Differences in structural organisation of the telomeric DNA between the C. elegans and human impose limitations on the use of the C. elegans as an ALT tumour model.
- MeSH
- Caenorhabditis elegans genetika MeSH
- DNA chemie MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- telomery chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telomerase is an enzyme that adds repeats of DNA sequences to the ends of chromosomes, thereby preventing their shortening. Telomerase activity is associated with proliferative status of cells, organismal development, and aging. We report an analysis of telomerase activity and telomere length in the honeybee, Apis mellifera. Telomerase activity was found to be regulated in a development and caste-specific manner. During the development of somatic tissues of larval drones and workers, telomerase activity declined to 10 % of its level in embryos and remained low during pupal and adult stages but was upregulated in testes of late pupae, where it reached 70 % of the embryo level. Upregulation of telomerase activity was observed in the ovaries of late pupal queens, reaching 160 % of the level in embryos. Compared to workers and drones, queens displayed higher levels of telomerase activity. In the third larval instar of queens, telomerase activity reached the embryo level, and an enormous increase was observed in adult brains of queens, showing a 70-fold increase compared to a brain of an adult worker. Southern hybridization of terminal TTAGG fragments revealed a high variability of telomeric length between different individuals, although the same pattern of hybridization signals was observed in different tissues of each individual.
- MeSH
- chromozomy hmyzu genetika metabolismus MeSH
- hmyzí proteiny genetika metabolismus MeSH
- homeostáza telomer fyziologie MeSH
- telomerasa genetika metabolismus MeSH
- telomery genetika metabolismus MeSH
- včely genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although the impact of telomeres on physiology stands well established, a question remains: how do telomeres impact cellular functions at a molecular level? This is because current understanding limits the influence of telomeres to adjacent subtelomeric regions despite the wide-ranging impact of telomeres. Emerging work in two distinct aspects offers opportunities to bridge this gap. First, telomere-binding factors were found with non-telomeric functions. Second, locally induced DNA secondary structures called G-quadruplexes are notably abundant in telomeres, and gene regulatory regions genome wide. Many telomeric factors bind to G-quadruplexes for non-telomeric functions. Here we discuss a more general model of how telomeres impact the non-telomeric genome - through factors that associate at telomeres and genome wide - and influence cell-intrinsic functions, particularly aging, cancer, and pluripotency.
- MeSH
- DNA metabolismus MeSH
- G-kvadruplexy * MeSH
- heterochromatin MeSH
- telomery * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Telomeres, which form the protective ends of eukaryotic chromosomes, are a ubiquitous and conserved structure of eukaryotic genomes but the basic structural unit of most telomeres, a repeated minisatellite motif with the general consensus sequence T(n)A(m)G(o), may vary between eukaryotic groups. Previous studies on several species of green algae revealed that this group exhibits at least two types of telomeric sequences, a presumably ancestral type shared with land plants (Arabidopsis type, TTTAGGG) and conserved in, for example, Ostreococcus and Chlorella species, and a novel type (Chlamydomonas type, TTTTAGGG) identified in Chlamydomonas reinhardtii. We have employed several methodical approaches to survey the diversity of telomeric sequences in a phylogenetically wide array of green algal species, focusing on the order Chlamydomonadales. Our results support the view that the Arabidopsis-type telomeric sequence is ancestral for green algae and has been conserved in most lineages, including Mamiellophyceae, Chlorodendrophyceae, Trebouxiophyceae, Sphaeropleales, and most Chlamydomonadales. However, within the Chlamydomonadales, at least two independent evolutionary changes to the Chlamydomonas type occurred, specifically in a subgroup of the Reinhardtinia clade (including C. reinhardtii and Volvox carteri) and in the Chloromonadinia clade. Furthermore, a complex structure of telomeric repeats, including a mix of the ancestral Arabidopsis-type motifs and derived motifs identical to the human-type telomeric repeats (TTAGGG), was found in the chlamydomonadalean clades Dunaliellinia and Stephanosphaeria. Our results indicate that telomere evolution in green algae, particularly in the order Chlamydomonadales, is far more dynamic and complex than thought before. General implications of our findings for the mode of telomere evolution are discussed.
- MeSH
- Chlorophyta genetika MeSH
- molekulární evoluce MeSH
- telomery genetika MeSH
- Volvocida genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Up to 15% of human cancers maintain their telomeres through a telomerase-independent mechanism, termed "alternative lengthening of telomeres" (ALT) that relies on homologous recombination between telomeric sequences. Emerging evidence suggests that the recombinogenic nature of ALT telomeres results from the formation of RNA:DNA hybrids (R-loops) between telomeric DNA and the long-noncoding telomeric repeat-containing RNA (TERRA). Here, we show that the mismatch repair protein MutSβ, a heterodimer of MSH2 and MSH3 subunits, is enriched at telomeres in ALT cancer cells, where it prevents the accumulation of telomeric G-quadruplex (G4) structures and R-loops. Cells depleted of MSH3 display increased incidence of R-loop-dependent telomere fragility and accumulation of telomeric C-circles. We also demonstrate that purified MutSβ recognizes and destabilizes G4 structures in vitro. These data suggest that MutSβ destabilizes G4 structures in ALT telomeres to regulate TERRA R-loops, which is a prerequisite for maintenance of telomere integrity during ALT.
- MeSH
- DNA metabolismus MeSH
- homeostáza telomer MeSH
- lidé MeSH
- nádory * genetika MeSH
- R-smyčka MeSH
- RNA dlouhá nekódující * metabolismus MeSH
- telomery metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA metabolismus MeSH
- G-kvadruplexy MeSH
- homeostáza telomer fyziologie MeSH
- jednovláknová DNA metabolismus MeSH
- kinetika MeSH
- konformace nukleové kyseliny MeSH
- oligonukleotidy genetika MeSH
- proteiny vázající telomery metabolismus MeSH
- retardační test MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- telomerasa genetika MeSH
- telomery metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
It has been proposed that oxidative stress, elicited by high levels of reactive oxygen species, accelerates telomere shortening by erosion of telomeric DNA repeats. While most eukaryotes counteract telomere shortening by telomerase-driven addition of these repeats, telomeric loss in Drosophila is compensated by retrotransposition of the telomeric retroelements HeT-A, TART and TAHRE to chromosome ends. In this study we tested the effect of chronic exposure of flies to non-/sub-lethal doses of paraquat, which is a redox cycling compound widely used to induce oxidative stress in various experimental paradigms including telomere length analyses. Indeed, chronic paraquat exposure for five generations resulted in elevated transcriptional activity of both telomeric and non-telomeric transposable elements, and extended telomeric length in the tested fly lines. We propose that low oxidative stress leads to increased telomere length within Drosophila populations. For a mechanistic understanding of the observed phenomenon we discuss two scenarios: adaption, acting through a direct stimulation of telomere extension, or positive selection favoring individuals with longer telomeres within the population.
- MeSH
- Drosophila melanogaster účinky léků genetika MeSH
- genetická transkripce účinky léků MeSH
- homeostáza telomer účinky léků MeSH
- hormeze * MeSH
- paraquat farmakologie MeSH
- reaktivní formy kyslíku farmakologie MeSH
- retroelementy účinky léků MeSH
- telomery účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zkracování telomer účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
