Delivery of yeast telomerase to a DNA break depends on the recruitment functions of Cdc13 and Est1

The yeast single-strand TG-repeat telomere binding protein Cdc13 and the telomerase accessory protein Est1 play essential roles in chromosome end replication. To determine whether a proposed Cdc13-Est1 interaction recruits telomerase (Est2), we used a simplified system in which telomere formation was monitored at an HO-induced DNA double-strand break (DSB). Tethering of either Cdc13 or Est1 adjacent to a DSB promoted telomere formation, and tethering of Est1, even in the absence of a DSB, resulted in the recruitment of Est2. Est1 association with a DSB containing an adjacent short TG-repeat sequence depended on the Cdc13-Est1 interaction affected by cdc13-2 and est1-60 mutations, whereas Cdc13 association did not. Similarly, Est2 binding to the DSB also required the Cdc13-Est1 interaction, but not synthesis of new TG repeats at the break site. These data demonstrate a critical role for Est1 in recruiting telomerase to its site of action, in cooperation with the telomere binding protein Cdc13.     

Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects (‘caps'') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1–Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tel1 kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.     

Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13

In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA.     

Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p

Appropriate control of the chromosome end-replicating enzyme telomerase is crucial for maintaining telomere length and genomic stability. The essential telomeric DNA-binding protein Cdc13p both positively and negatively regulates telomere length in budding yeast. Here we test the effect of purified Cdc13p on telomerase action in vitro. We show that the full-length protein and its DNA-binding domain (DBD) inhibit primer extension by telomerase. This inhibition occurs by competitive blocking of telomerase access to DNA. To further understand the requirements for productive telomerase 3′-end access when Cdc13p or the DBD is bound to a telomerase substrate, we constrained protein binding at various distances from the 3′-end on two sets of increasingly longer oligonucleotides. We find that Cdc13p inhibits the action of telomerase through three distinct biochemical modes, including inhibiting telomerase even when a significant tail is available, representing a novel ‘action at a distance’ inhibitory activity. Thus, while yeast Cdc13p exhibits the same general activity as human POT1, providing an off switch for telomerase when bound near the 3′-end, there are significant mechanistic differences in the ways telomere end-binding proteins inhibit telomerase action.     

Chromosome end protection plasticity revealed by Stn1p and Ten1p bypass of Cdc13p

Genome stability necessitates a mechanism to protect the termini of linear chromosomes from inappropriate degradation or recombination. In many species this protection depends on 'capping' proteins that bind telomeric DNA. The budding yeast Cdc13p binds single-stranded telomeric sequences, prevents lethal degradation of chromosome ends and regulates telomere extension by telomerase. Two Cdc13-interacting proteins, Stn1p and Ten1p, are also required for viability and telomere length regulation. It has been proposed that Cdc13p DNA binding directs a Cdc13p-Stn1p-Ten1p complex to telomeres to mediate end protection. However, the functional significance of these protein interactions, and their respective roles in maintaining telomere integrity, remain undefined. Here, we show that co-overexpressing TEN1 with a truncated form of STN1 efficiently bypasses the essential role of CDC13. We further show that this truncated Stn1p binds directly to Pol12p, a polymerase alpha-primase regulatory subunit, and that Pol12 activity is required for CDC13 bypass. Thus, Stn1p and Ten1p control a Cdc13p-independent telomere capping mechanism that is coupled to the conventional DNA replication machinery.     

Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG_1–3 telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG_1–3 DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451–693) was sufficient to bind single-stranded TG_1–3 with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451–693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252–924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.     

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in?vitro and in?vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:     

CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca2+-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.     

Mpg2 interacts and cooperates with Mpg1 to maintain yeast glycosylation

Using a yeast two-hybrid screen, we isolated a gene from Schizosaccharomyces pombe, whose product interacts with Mpg1, a GDP-mannose-1-phosphate guanylyltransferase involved in the maintenance of cell wall integrity and glycosylation. We have designated this gene mpg2 based on its similarity to Mpg1. Mpg2 is evolutionarily conserved in higher eukaryotes. In the absence of Mpg2, defects in cell growth and sensitivity to hygromycin B are observed. When mpg1 is depleted, the lack of mpg2 causes a synthetic enhancement of the growth defect, the sensitivity to hygromycin B and the cell cycle phenotype previously reported for mpg1 mutant. Finally, Mpg1 overexpression complements the Δmpg2 mutant phenotypes. Taken together, these results indicate that mpg1 and mpg2 function together in glycosylation and septum formation.     

BIK1 cooperates with BAK1 to regulate constitutive immunity and cell death in Arabidopsiss

In Arabidopsis,both the membrane-anchored receptor-like kinase(RLK) BAK1 and the receptor-like cytoplasmic kinase(RLCK) BIK1 are important mediators of transmembrane signal transduction that regulate plant development and immunity.However,little attention has been paid to their genetic association.This study found the bak1 bik1 double mutant of Arabidopsis displayed a severe dwarfism phenotype due to constitutive immunity and cell death in developing plants.These data suggest that BIK1 cooperates with BAK1 to regulate constitutive immunity and cell death.     

The role of Stn1p in Saccharomyces cerevisiae telomere capping can be separated from its interaction with Cdc13p

The function of telomeres is twofold: to facilitate complete chromosome replication and to protect chromosome ends against fusions and illegitimate recombination. In the budding yeast Saccharomyces cerevisiae, interactions among Cdc13p, Stn1p, and Ten1p are thought to be critical for promoting these processes. We have identified distinct Stn1p domains that mediate interaction with either Ten1p or Cdc13p, allowing analysis of whether the interaction between Cdc13p and Stn1p is indeed essential for telomere capping or length regulation. Consistent with the model that the Stn1p essential function is to promote telomere end protection through Cdc13p, stn1 alleles that truncate the C-terminal 123 residues fail to interact with Cdc13p and do not support viability when expressed at endogenous levels. Remarkably, more extensive deletions that remove an additional 185 C-terminal residues from Stn1p now allow cell growth at endogenous expression levels. The viability of these stn1-t alleles improves with increasing expression level, indicating that increased stn1-t dosage can compensate for the loss of Cdc13p-Stn1p interaction. However, telomere length is misregulated at all expression levels. Thus, an amino-terminal region of Stn1p is sufficient for its essential function, while a central region of Stn1p either negatively regulates the STN1 essential function or destabilizes the mutant Stn1 protein.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homolog of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not significantly in an mcm2 or polε mutant. These results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.Key words: Cdc7, Cdc45, checkpoint, DNA replication, Mrc1     

Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.     

Separase cooperates with Zds1 and Zds2 to activate Cdc14 phosphatase in early anaphase

Completion of mitotic exit and cytokinesis requires the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. A key enzyme that counteracts Cdk during budding yeast mitotic exit is the Cdc14 phosphatase. Cdc14 is inactive for much of the cell cycle, sequestered by its inhibitor Net1 in the nucleolus. At anaphase onset, separase-dependent down-regulation of PP2A^Cdc55 allows phosphorylation of Net1 and consequent Cdc14 release. How separase causes PP2A^Cdc55 down-regulation is not known. Here, we show that two Cdc55-interacting proteins, Zds1 and Zds2, contribute to timely Cdc14 activation during mitotic exit. Zds1 and Zds2 are required downstream of separase to facilitate nucleolar Cdc14 release. Ectopic Zds1 expression in turn is sufficient to down-regulate PP2A^Cdc55 and promote Net1 phosphorylation. These findings identify Zds1 and Zds2 as new components of the mitotic exit machinery, involved in activation of the Cdc14 phosphatase at anaphase onset. Our results suggest that these proteins may act as separase-regulated PP2A^Cdc55 inhibitors.     

PrP cooperates with STI1 to regulate SOD activity in PrP-deficient neuronal cell line

Cellular prion protein (PrP(C)) plays anti-apoptotic and anti-oxidative roles in apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line. The octapeptide repeat region (OR) and N-terminal half of the hydrophobic region (HR) of PrP(C) are indispensable for PrP(C) activity, but the mechanisms remain unclear. In the present study, elucidation of the mechanisms by which PrP(C) elicits the anti-oxidative activities was facilitated by evidence of stress-inducible protein 1 (STI1) mediating PrP(C)-dependent superoxide dismutase (SOD) activation. Immunoprecipitation revealed that PrP(C) was associated with STI1. The inhibitory peptides against PrP(C)-STI1 binding [STI1 pep.1 and PrP(113-132)] indicated toxic activity in PrP(C)-expressing cells by inhibiting SOD activity but not in Prnp(-/-) cells. Furthermore, OR and N-terminal half of the HR were required for the inhibitory effect of PrP(113-132) but not STI1 pep.1. These data are consistent with results established with a model where OR and N-terminal half of the HR mediate the action of STI1 upon cell survival and upregulation of SOD activity.