329 research outputs found
Limited TTP supply affects telomere length regulation in a telomerase-independent fashion
An adequate supply of nucleotides is essential for DNA replication and DNA repair. Moreover, inhibition of TTP synthesis can cause cell death by a poorly characterized mechanism called thymine-less death. In the yeast Saccharomyces cerevisiae, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (CDC8) are both essential for de novo TTP synthesis. The effects of temperature-sensitive mutations in these genes have been characterized and, curiously, the phenotypes displayed by cells harboring them include shortened telomeric repeat tracts. This finding raised the possibility that the enzyme telomerase is very sensitive to TTP-pools. We tested this possibility in vivo by assessing telomerase-dependent extension in situations of lowered TTP supply. The results show that the above-mentioned short telomere phenotype is not a consequence of an inability of telomerase to elongate telomeres when TTP synthesis is impaired. Moreover, this telomere shortening was abolished in cells harboring a mutation in DNA polymerase α. Previously, this same mutation was shown to affect the coordination between conventional replication and telomerase-mediated extension. These results thus re-emphasize the importance of the interplay between conventional replication and telomerase-mediated addition of telomeric repeats in telomere replication
Free uptake of cell-penetrating peptides by fission yeast
AbstractAn increasing number of peptides translocate the plasma membrane of mammalian cells promising new avenues for drug delivery. However, only a few examples are known to penetrate the fungal cell wall. We compared the capacity of different fluorophore-labelled peptides to translocate into fission yeast and human cells and determined their intracellular distribution. Most of the 20 peptides tested were able to enter human cells, but only one, transportan 10 (TP10), efficiently penetrated fission yeast and was distributed uniformly inside the cells. The results show that the fungal cell wall may reduce, but does not block peptide uptake
Human heterochromatin protein 1 isoforms HP1(Hsα) and HP1(Hsβ) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation
Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsα) or HP1(Hsβ), but not HP1(Hsγ), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsα) or HP1(Hsβ) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-ΔHP1(Hsα), GFP-ΔHP1(Hsβ), or GFP-ΔHP1(Hsγ) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsα) or HP1(Hsβ). In contrast, cells expressing mutant GFP-ΔHP1(Hsα) or GFP-ΔHP1(Hsβ) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsα) or HP1(Hsβ) suppressed tumorigenicity, whereas expression of mutant HP1(Hsα) or HP1(Hsβ) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsα) or HP1(Hsβ) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state
Two Pathways Recruit Telomerase to Saccharomyces cerevisiae Telomeres
The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1) and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1Ä, tlc1-SD, and tlc1-BD), Est2p telomere binding was normal in G1/early S phase but reduced to about 40–50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2Ä), Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells
Genome-Wide Mapping of DNA Strand Breaks
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed “damaged DNA immunoprecipitation” (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage
Methylated H3K4, a Transcription-Associated Histone Modification, Is Involved in the DNA Damage Response Pathway
Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells
Silenced yeast chromatin is maintained by Sir2 in preference to permitting histone acetylations for efficient NER
Very little is currently known about how nucleotide excision repair (NER) functions at the ends of chromosomes. To examine this, we introduced the URA3 gene into either transcriptionally active or repressed subtelomeric regions of the yeast genome. This enabled us to examine the repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in identical sequences under both circumstances. We found that NER is significantly more efficient in the non-repressed subtelomere than the repressed one. At the non-repressed subtelomere, UV radiation stimulates both histones H3 and H4 acetylation in a similar fashion to that seen at other regions of the yeast genome. These modifications occur regardless of the presence of the Sir2 histone deacetylase. On the other hand, at the repressed subtelomere, where repair is much less efficient, UV radiation is unable to stimulate histone H4 or H3 acetylation in the presence of Sir2. In the absence of Sir2 both of these UV-induced modifications are detected, resulting in a significant increase in NER efficiency in the region. Our experiments reveal that there are instances in the yeast genome where the maintenance of the existing chromatin structures dominates over the action of chromatin modifications associated with efficient NER
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Novel Phosphorylation Sites in the S. cerevisiae Cdc13 Protein Reveal New Targets for Telomere Length Regulation
The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics. [Image: see text
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