Isolation and Characterisation of Genes Encoding Malate Synthesis and Transport Determinants in the Aluminum-Tolerant Australian Weeping-Grass (\u3cem\u3eMicrolaena Stipoides\u3c/em\u3e)

Acid soils cover some 40% of the Earth’s arable land where they represent a major limitation to plant production. Plant growth on acid soils is primarily limited due to aluminium (Al) solubilized by acidity into toxic Al3+ cations which will inhibit root growth resulting in poor uptake of water and nutrients. Many important pasture species lack sufficient Al tolerance within their germplasm to allow effective breeding for this character

Isolation and Characterisation of Genes Encoding Ice Recrystallisation Inhibition Proteins (IRIPs) in the Cryophilic Antarctic Hair-Grass (\u3ci\u3eDeschampsia Antarctica\u3c/i\u3e) and the Temperate Perennial Ryegrass (\u3ci\u3eLolium Perenne\u3c/i\u3e)

Antarctic hairgrass (D. antarctica Desv.), the only grass species indigenous to Antarctica, has a well developed tolerance of freezing, strongly induced by cold-acclimation. In response to low temperatures D. antarctica exhibits recrystallisation inhibition (RI) activity, localised to the apoplasm, that prevents further growth of ice crystals following freezing

Modulation of telomere terminal structure by telomerase components in Candida albicans

The telomerase ribonucleoprotein in Candida albicans is presumed to contain at least three Est proteins: CaEst1p, CaEst2p/TERT and CaEst3p. We constructed mutants missing each of the protein subunit of telomerase and analyzed overall telomere dynamics and single-stranded telomere overhangs over the course of many generations. The est1-ΔΔ mutant manifested abrupt telomere loss and recovery, consistent with heightened recombination. Both the est2-ΔΔ and est3-ΔΔ mutant exhibited progressive telomere loss, followed by the gradual emergence of survivors with long telomeres. In no case was telomere loss accompanied by severe growth defects, suggesting that cells with short telomeres can continue to proliferate. Furthermore, the amount of G-strand terminal overhangs was greatly increased in the est2-ΔΔ mutant, but not others. Our results suggest that in addition to their well-characterized function in telomere elongation, both CaEst1p and CaEst2p mediate some aspects of telomere protection in Candida, with the former suppressing excessive recombination, and the latter preventing excessive C-strand degradation

Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13

Budding yeast telomerase is mainly activated by Tel1/Mec1 (yeast ATM/ATR) on Cdc13 from late S to G2 phase of the cell cycle. Here, we demonstrated that the telomerase-recruitment domain of Cdc13 is also phosphorylated by Cdk1 at the same cell cycle stage as the Tel1/Mec1-dependent regulation. Phosphor-specific gel analysis demonstrated that Cdk1 phosphorylates residues 308 and 336 of Cdc13. The residue T308 of Cdc13 is critical for efficient Mec1-mediated S306 phosphorylation in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13 S/TP motifs phosphorylated by Cdk1 caused cell cycle delay and telomere shortening and these phenotypes could be partially restored by the replacement with a negative charge residue. In the absence of Ku or Tel1, Cdk1-mediated phosphorylation of Cdc13 showed no effect on telomere length maintenance. Moreover, this Cdk1-mediated phosphorylation was required to promote the regular turnover of Cdc13. Together these results demonstrate that Cdk1 phosphorylates the telomerase recruitment domain of Cdc13, thereby preserves optimal function and expression level of Cdc13 for precise telomere replication and cell cycle progression

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

Shelterin-Like Proteins and Yku Inhibit Nucleolytic Processing of Saccharomyces cerevisiae Telomeres

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Δ than in rif2Δ G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Δ G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases

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

Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair

Rif1 Supports the Function of the CST Complex in Yeast Telomere Capping

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex