Survival and Growth of Yeast without Telomere Capping by Cdc13 in the Absence of Sgs1, Exo1, and Rad9

Maintenance of telomere capping is absolutely essential to the survival of eukaryotic cells. Telomere capping proteins, such as Cdc13 and POT1, are essential for the viability of budding yeast and mammalian cells, respectively. Here we identify, for the first time, three genetic modifications that allow budding yeast cells to survive without telomere capping by Cdc13. We found that simultaneous inactivation of Sgs1, Exo1, and Rad9, three DNA damage response (DDR) proteins, is sufficient to allow cell division in the absence of Cdc13. Quantitative amplification of ssDNA (QAOS) was used to show that the RecQ helicase Sgs1 plays an important role in the resection of uncapped telomeres, especially in the absence of checkpoint protein Rad9. Strikingly, simultaneous deletion of SGS1 and the nuclease EXO1, further reduces resection at uncapped telomeres and together with deletion of RAD9 permits cell survival without CDC13. Pulsed-field gel electrophoresis studies show that cdc13-1 rad9Δ sgs1Δ exo1Δ strains can maintain linear chromosomes despite the absence of telomere capping by Cdc13. However, with continued passage, the telomeres of such strains eventually become short and are maintained by recombination-based mechanisms. Remarkably, cdc13Δ rad9Δ sgs1Δ exo1Δ strains, lacking any Cdc13 gene product, are viable and can grow indefinitely. Our work has uncovered a critical role for RecQ helicases in limiting the division of cells with uncapped telomeres, and this may provide one explanation for increased tumorigenesis in human diseases associated with mutations of RecQ helicases. Our results reveal the plasticity of the telomere cap and indicate that the essential role of telomere capping is to counteract specific aspects of the DDR

Essential and checkpoint functions of budding yeast ATM and ATR during meiotic prophase are facilitated by differential phosphorylation of a meiotic adaptor protein, Hop1

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage

Ipl1/aurora kinase suppresses S-CDK-driven spindle formation during prophase I to ensure chromosome integrity during meiosis

Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics

Deletion of the gabra2 gene results in hypersensitivity to the acute effects of ethanol but does not alter ethanol self administration

Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABA(A) α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABA(A) α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol's rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchange

Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks

A Novel Checkpoint and RPA Inhibitory Pathway Regulated by Rif1

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular “band-aid” for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA–dependent DNA–damage responses in eukaryotic cells

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

Taming the tiger by the tail: modulation of DNA damage responses by telomeres

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell-cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell-cycle division are discussed