Telomere length regulation: coupling DNA end processing to feedback regulation of telomerase

The conventional DNA polymerase machinery is unable to fully replicate the ends of linear chromosomes. To surmount this problem, nearly all eukaryotes use the telomerase enzyme, a specialized reverse transcriptase that utizes its own RNA template to add short TG-rich repeats to chromosome ends, thus reversing their gradual erosion occurring at each round of replication. This unique, non-DNA templated mode of telomere replication requires a regulatory mechanism to ensure that telomerase acts at telomeres whose TG tracts are too short, but not at those with long tracts, thus maintaining the protective TG repeat cap at an appropriate average length. The prevailing notion in the field is that telomere length regulation is brought about through a negative feedback mechanism that counts TG repeat-bound protein complexes to generate a signal that regulates telomerase action. This review summarizes experiments leading up to this model and then focuses on more recent experiments, primarily from yeast, that begin to suggest how this counting mechanism might work. The emerging picture is that of a complex interplay between the conventional DNA replication machinery, DNA damage response factors, and a specialized set of proteins that help to recruit and regulate the telomerase enzyme

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^(scFEN1), encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^(ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset

The heritability of telomere length among the elderly and oldest-old

A tight link exists between telomere length and both population doublings of a cell culture and age of a given organism. The more population doublings of the cell culture or the higher the age of the organism, the shorter the telomeres. The proposed model for telomere shortening, called the end replication problem, explains why the telomere erodes at each cellular turnover. Telomere length is regulated by a number of associated proteins through a number of different signaling pathways. The determinants of telomere length were studied using whole blood samples from 287 twin pairs aged 73 to 95 years. Structural equation models revealed that a model including additive genetic effects and non-shared environment was the best fitting model and that telomere length was moderately heritable, with an estimate that was sensitive to the telomere length standardization procedure. Sex-specific analyses showed lower heritability in males, although not statistically significant, which is in line with our earlier finding of a sex difference in telomere dynamics among the elderly and oldest-old

Effect of surgery on the long-term use of opioids in patients with degenerative back disorders:a retrospective register-based study

PURPOSE: This study based exclusively on register-data provides a scientific basis for further research on the use of opioids in patients with degenerative back disorder. The main objective of this study is to investigate whether surgically treated back pain patients have the same risk of being long-term opioid users as back pain patients who did not have surgery.METHODS: We performed a retrospective register-based cohort study based on all patients diagnosed with a degenerative back disorder at the Spine Center of Southern Denmark from 2011 to 2017. The primary outcome of the study was the use of opioids two years after the patient's first hospital contact with a degenerative back condition. Fisher exact tests were used for descriptive analyses. The effect of the surgery was estimated using adjusted logistic regression analyses.RESULTS: For patients who used opioids before the first hospital contact, the ratio for long-term opioid use for surgically treated patients is significantly lower than for non-surgically treated patients (OR = 0.75, 95%CI (0.66; 0.86)). For patients who did not use opioids before, the ratio for long-term opioid use for surgically treated patients does not differ from that of non-surgically treated patients (OR = 1.01, 95%CI (0.84; 1.22)).CONCLUSIONS: Patients with a degenerative back disorder who used opioids before their first visit to a specialized spine center have a lower risk of becoming long-term opioid users if they were surgically treated. Whereas for patients who did not use opioids before the first visit, surgical treatment does not influence the risk of becoming long-term opioid users.</p

Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo

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

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

In the absence of the telomere capping protein Cdc13, budding yeast telomeres erode, resulting in checkpoint arrest. This study shows that the helicase Pif1, known as a telomerase inhibitor, also has a direct role in the resection of uncapped telomeres, acting in parallel to the nuclease Exo1

C-reactive protein and albumin kinetics before community-acquired bloodstream infections- A Danish population-based cohort study

aEarly changes in biomarker levels probably occur before bloodstream infection (BSI) is diagnosed. However, this issue has not been fully addressed. We aimed at evaluating the kinetics of C-reactive protein (CRP) and plasma albumin (PA) in the 30 days before community-acquired (CA) BSI diagnosis. From a population-based BSI database we identified 658 patients with at least one measurement of CRP or PA from day-30 (D-30) through day-1 (D-1) before the day of CA-BSI (D0) and a measurement of the same biomarker at D0 or D1. Amongst these, 502 had both CRP and PA measurements which fitted these criteria. CRP and PA concentrations began to change inversely some days before CA-BSI diagnosis, CRP increasing by day-3.1 and PA decreasing by day-1.3. From D-30 to D-4, CRP kinetics (expressed as slopes-rate of concentration change per day) was-1.5 mg/l/day. From D-3 to D1, the CRP slope increased to 36.3 mg/l/day. For albumin, the slope between D-30 to D-2 was 0.1 g/l/day and changed to-1.8 g/l/day between D-1 and D1. We showed that biomarker levels begin to change some days before the CA-BSI diagnosis, CRP 3.1 days and PA 1.3 days before.publishersversionepub_ahead_of_prin

TERRA Promotes Telomere Shortening through Exonuclease 1–Mediated Resection of Chromosome Ends

The long noncoding telomeric repeat containing RNA (TERRA) is expressed at chromosome ends. TERRA upregulation upon experimental manipulation or in ICF (immunodeficiency, centromeric instability, facial anomalies) patients correlates with short telomeres. To study the mechanism of telomere length control by TERRA in Saccharomyces cerevisiae, we mapped the transcriptional start site of TERRA at telomere 1L and inserted a doxycycline regulatable promoter upstream. Induction of TERRA transcription led to telomere shortening of 1L but not of other chromosome ends. TERRA interacts with the Exo1-inhibiting Ku70/80 complex, and deletion of EXO1 but not MRE11 fully suppressed the TERRA–mediated short telomere phenotype in presence and absence of telomerase. Thus TERRA transcription facilitates the 5′-3′ nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate. Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities