Mechanisms for maintaining the telomere cap in eukaryotic cells

PhD ThesisTelomeres are found at the end of linear eukaryotic chromosome ends and contribute to genetic stability. It is crucial that telomeres are not perceived and treated as doublestrand breaks by checkpoint and DNA repair proteins in order to prevent potentially lethal consequences, such as cellular ageing and cancer formation in mammalian cells (Murnane, 2006; Shin et al., 2006; Gilley et al., 2005). Hence, eukaryotic chromosome ends are masked by various telomere-binding proteins. Two crucial telomere capping proteins in Saccharomyces cerevisiae are Cdc13 and Yku70. Cdc13 forms a complex with Stn1 and Ten1 and binds specifically to single-stranded telomeric DNA (Grandin et al., 2001). Yku70 is a subunit of the Yku complex that is found at double-stranded telomeric DNA (Tuteja and Tuteja, 2000). A temperature-sensitive allele of CDC13, known as cdc13-1, or a null-mutation of YKU70 is used in this study to induce telomere uncapping and to study its consequences in vivo. This work looks into the puzzling observation that components of the nonsensemediated mRNA decay pathway (Upf1, Upf2, Upf3) suppress one form of telomere capping defect (yku70Δ), but enhance another (cdc13-1) (Addinall et al., 2011). The same effect can be seen in Ebs1, which has previously been linked to NMD (Azzalin et al., 2007). Here it is shown that Ebs1, like components of nonsense-mediated decay (NMD), regulate transcript levels of the two telomere binding proteins Stn1 and Ten1. Interestingly, increased levels of Stn1, but not Ten1, suppress the yku70Δ capping defect, but enhance the cdc13-1 capping defect, indicating that NMD and Ebs1 influence Cdc13- and Yku70-dependent telomere capping through modification of Stn1 levels. It is also shown that increase in Stn1 levels alters stochiometry of the Cdc13/Stn1/Ten1 (CST) complex. Cdc13 association to telomeres is significantly reduced at the presence of increased levels of Stn1. Inefficient telomere protection allows resection of telomeric DNA and the generation of ssDNA tracts triggers cell cycle arrest (Booth et al., 2001; Garvik et al., 1995; Lydall and Weinert, 1995; Weinert and Hartwell, 1993). It is shown that Upf2 and Ebs1 do not affect checkpoint activation in response to cdc13-1 uncapped telomeres, but are required for efficient G1 to S phase progression. Furthermore, Upf2 contributes to ssDNA generation at cdc13-1 uncapped telomeres in a parallel pathway to the exonuclease Exo1 and the checkpoint protein Rad24. Remarkably, elimination of Upf2 and Exo1 or Upf2 and Rad24 allows viability of cells in the absence of the otherwise essential telomere capping protein Cdc13.BBSRC, ONDEX and the Wellcome Trus

Systematic analysis of the DNA damage response network in telomere defective budding yeast

Functional telomeres are critically important to eukaryotic genetic stability. Scores of proteins and pathways are known to affect telomere function. Here, we report a series of related genome-wide genetic interaction screens performed on budding yeast cells with acute or chronic telomere defects. Genetic interactions were examined in cells defective in Cdc13 and Stn1, affecting two components of CST, a single stranded DNA (ssDNA) binding complex that binds telomeric DNA. For comparison, genetic interactions were also examined in cells with defects in Rfa3, affecting the major ssDNA binding protein, RPA, which has overlapping functions with CST at telomeres. In more complex experiments, genetic interactions were measured in cells lacking EXO1 or RAD9, affecting different aspects of the DNA damage response, and containing a cdc13-1 induced telomere defect. Comparing fitness profiles across these data sets helps build a picture of the specific responses to different types of dysfunctional telomeres. The experiments show that each context reveals different genetic interactions, consistent with the idea that each genetic defect causes distinct molecular defects. To help others engage with the large volumes of data, the data are made available via two interactive web-based tools: Profilyzer and DIXY. One particularly striking genetic interaction observed was that the chk1∆ mutation improved fitness of cdc13-1 exo1∆ cells more than other checkpoint mutations (ddc1∆, rad9∆, rad17∆, and rad24∆), whereas, in cdc13-1 cells, the effects of all checkpoint mutations were similar. We show that this can be explained by Chk1 stimulating resection—a new function for Chk1 in the eukaryotic DNA damage response network

Study protocol for a randomised placebo-controlled trial of pramipexole in addition to mood stabilisers for patients with treatment resistant bipolar depression (the PAX-BD study)

Abstract Background Treatment Resistant Bipolar Depression (TRBD) is a major contributor to the burden of disease associated with Bipolar Disorder (BD). Treatment options for people experiencing bipolar depression are limited to three interventions listed by National Institute for Health and Care: lamotrigine, quetiapine and olanzapine, of which the latter two are often not well tolerated. The majority of depressed people with BD are therefore prescribed antidepressants despite limited efficacy. This demonstrates an unmet need for additional interventions. Pramipexole has been shown to improve mood symptoms in animal models of depression, in people with Parkinson’s Disease and two proof of principle trials of pramipexole for people with BD who are currently depressed. Methods The PAX-BD study, funded by the United Kingdom (UK) National Institute for Health Research, aims to extend previous findings by assessing the efficacy, safety and health economic impact of pramipexole in addition to mood stabilisers for patients with TRBD. A randomised, double-blind, placebo controlled design is conducted in a naturalistic UK National Health Service setting. An internal pilot study to examine feasibility and acceptability of the study design is included. Participants with TRBD are screened from National Health Service secondary care services in up to 40 mental health trusts in the UK, with the aim of recruiting approximately 414 participants into a pre-randomisation phase to achieve a target of 290 randomised participants. Primary safety and efficacy measures are at 12 weeks following randomisation, with follow up of participants to 52 weeks. The primary outcome is depressive symptoms as measured by Quick Inventory for Depressive Symptomatology – Self Report. Secondary outcomes include changes in anxiety, manic symptoms, tolerability, acceptability, quality of life and cost-effectiveness. Outcome measures are collected remotely using self-report tools implemented online, and observer-rated assessments conducted via telephone. ANCOVA will be used to examine the difference in rating scale scores between treatment arms, and dependent on compliance in completion of weekly self-report measures. A mixed effects linear regression model may also be used to account for repeated measures. Trial registration ISRCTN72151939. Registered on 28 August 2019, http://www.isrctn.com/ISRCTN72151939 Protocol Version: 04-FEB-2021, Version 9.0

Customizable views on semantically integrated networks for systems biology

Motivation: The rise of high-throughput technologies in the post-genomic era has led to the production of large amounts of biological data. Many of these datasets are freely available on the Internet. Making optimal use of these data is a significant challenge for bioinformaticians. Various strategies for integrating data have been proposed to address this challenge. One of the most promising approaches is the development of semantically rich integrated datasets. Although well suited to computational manipulation, such integrated datasets are typically too large and complex for easy visualization and interactive exploration

Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology

Interplay between Nonsense-Mediated mRNA Decay and DNA Damage Response Pathways Reveals that Stn1 and Ten1 Are the Key CST Telomere-Cap Components

A large and diverse set of proteins, including CST complex, nonsense mediated decay (NMD), and DNA damage response (DDR) proteins, play important roles at the telomere in mammals and yeast. Here, we report that NMD, like the DDR, affects single-stranded DNA (ssDNA) production at uncapped telomeres. Remarkably, we find that the requirement for Cdc13, one of the components of CST, can be efficiently bypassed when aspects of DDR and NMD pathways are inactivated. However, identical genetic interventions do not bypass the need for Stn1 and Ten1, the partners of Cdc13. We show that disabling NMD alters the stoichiometry of CST components at telomeres and permits Stn1 to bind telomeres in the absence of Cdc13. Our data support a model that Stn1 and Ten1 can function in a Cdc13-independent manner and have implications for the function of CST components across eukaryotes