The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein

Saccharomyces telomeres consist of approximately 350 bp of C(1-3)A/TG(1-3) DNA. Most of this approximately 350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C(1-3)A strand is degraded to generate a long single strand TG(1-3) tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG(1-3) DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase alpha, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 or POL1 that reduced the Cdc13p-Pol1p interaction resulted in telomerase mediated telomere lengthening. Over-expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG(1-3) strand lengthening by telomerase and its interaction with Pol1p promotes C(1-3)A strand resynthesis by DNA polymerase alpha.     

Pol12, the B subunit of DNA polymerase alpha, functions in both telomere capping and length regulation

The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase alpha and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.     

Stn1–Ten1 is an Rpa2–Rpa3-like complex at telomeres

In budding yeast, Cdc13, Stn1, and Ten1 form a heterotrimeric complex (CST) that is essential for telomere protection and maintenance. Previous bioinformatics analysis revealed a putative oligonucleotide/oligosaccharide-binding (OB) fold at the N terminus of Stn1 (Stn1N) that shows limited sequence similarity to the OB fold of Rpa2, a subunit of the eukaryotic ssDNA-binding protein complex replication protein A (RPA). Here we present functional and structural analyses of Stn1 and Ten1 from multiple budding and fission yeast. The crystal structure of the Candida tropicalis Stn1N complexed with Ten1 demonstrates an Rpa2N–Rpa3-like complex. In both structures, the OB folds of the two components pack against each other through interactions between two C-terminal helices. The structure of the C-terminal domain of Saccharomyces cerevisiae Stn1 (Stn1C) was found to comprise two related winged helix–turn–helix (WH) motifs, one of which is most similar to the WH motif at the C terminus of Rpa2, again supporting the notion that Stn1 resembles Rpa2. The crystal structure of the fission yeast Schizosaccharomyces pombe Stn1N–Ten1 complex exhibits a virtually identical architecture as the C. tropicalis Stn1N–Ten1. Functional analyses of the Candida albicans Stn1 and Ten1 proteins revealed critical roles for these proteins in suppressing aberrant telomerase and recombination activities at telomeres. Mutations that disrupt the Stn1–Ten1 interaction induce telomere uncapping and abolish the telomere localization of Ten1. Collectively, our structural and functional studies illustrate that, instead of being confined to budding yeast telomeres, the CST complex may represent an evolutionarily conserved RPA-like telomeric complex at the 3′ overhangs that works in parallel with or instead of the well-characterized POT1–TPP1/TEBPα–β complex.     

De novo telomere formation is suppressed by the Mec1-dependent inhibition of Cdc13 accumulation at DNA breaks

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability, since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. Here we report that Mec1, the ATR ortholog, couples the detection of DNA ends with the inhibition of telomerase. Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, a phosphorylation event that suppresses Cdc13 accumulation at DSBs. Conversely, telomere addition at accidental breaks is promoted by Pph3, the yeast protein phosphatase 4 (PP4). Pph3 is itself modulated by Rrd1, an activator of PP2A family phosphatases. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DNA breaks. These studies therefore identify a mechanism by which the ATR family of kinases enforces genome integrity, and a process that underscores the contribution of Cdc13 to the fate of DNA ends.     

DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks by packaging them in a protective structure referred to as the telomere "cap." Here we investigate the nature of the telomere cap by examining events at DNA breaks generated adjacent to either natural telomeric sequences (TG repeats) or arrays of Rap1-binding sites that vary in length. Although DNA breaks adjacent to either short or long telomeric sequences are efficiently converted into stable telomeres, they elicit very different initial responses. Short telomeric sequences (80 base pair [bp]) are avidly bound by Mre11, as well as the telomere capping protein Cdc13 and telomerase enzyme, consistent with their rapid telomerase-dependent elongation. Surprisingly, little or no Mre11 binding is detected at long telomere tracts (250 bp), and this is correlated with reduced Cdc13 and telomerase binding. Consistent with these observations, ends with long telomere tracts undergo strongly reduced exonucleolytic resection and display limited binding by both Rpa1 and Mec1, suggesting that they fail to elicit a checkpoint response. Rap1 binding is required for end concealment at long tracts, but Rif proteins, yKu, and Cdc13 are not. These results shed light on the nature of the telomere cap and mechanisms that regulate telomerase access at chromosome ends.     

Genetic interactions between CDC7 and CDC28: growth inhibition of cdc28-1N by Cdc7 point mutants

Background: Cdc7 kinase of Saccharomyces cerevisiae , a nuclear phosphoprotein, regulates initiation of chromosomal DNA replication. Overexpression of kinase-negative Cdc7 point mutants (T281E, D182N and D163N) arrests the cell cycle of the wild-type Saccharomyces cerevisiae cells at the G1/S boundary. This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity.
Results: We report here that kinase-negative Cdc7 mutants, when overexpressed in cdc28-1N (ts) at a permissive temperature, not only inhibit DNA replication by inactivating the wild-type Cdc7 but may also disturb coordination between DNA replication and cell division. Suppression of growth inhibition under this condition requires co-expression of both Dbf4 and Cdc28, whereas Dbf4 alone can counteract the growth inhibition in the wild-type cells. In cdc28-1N (ts), co-expression of the wild-type Dbf4 rescues only the G1/S defect and results in accumulation of those cells with less than 1C DNA as well as 2C DNA. On the other hand, co-expression of Cdc28 alone leads to increase of those cells arrested at the G1/S boundary, as found typically in the wild-type. We also report that overexpression of T281A, a 'weak' allele of Cdc7, causes growth arrest in cdc28-1N (ts) cells, but not in the CDC28 wild-type cells. This suggests that T281A is inactive in cdc28-1N (ts) and is consistent with the idea that Cdc28 activates Cdc7 by phosphorylation.
Conclusion: We conclude that two essential serine-threonine kinases, Cdc28 and Cdc7, genetically inter-act for initiation of the S phase and possibly for G2/M progression and/or S phase checkpoint control.     

Fission yeast Cdc23 interactions with DNA replication initiation proteins

Schizosaccharomyces pombe Cdc23 is an essential DNA replication protein, conserved in eukaryotes and functionally homologous with Saccharomyces cerevisiae Dna43 (Mcm10). We sought evidence for interactions between Cdc23 and the MCM2-7 complex, a component of both the pre-replicative complex and the replication fork. Cdc23 shows genetic interactions with four MCM subunits: cdc23-M36 and cdc23-1E2 alleles both show synthetic phenotypes with mcm2 (cdc19-P1) and mcm6 (mis5-268), and cdc23-M36 is synthetically lethal with mcm4 (cdc21-K46) and with mcm5 (nda4-108). The wild-type cdc23 gene on multicopy plasmids can partially suppress temperature-dependent defects in mcm5 (nda4-108). Two-hybrid analysis demonstrates interactions at the protein-protein level between Cdc23 and Mcm4, Mcm5 and Mcm6. Cdc23 also interacts with four subunits of the Schizosaccharomyces pombe origin recognition complex (ORC) in yeast two-hybrid assay: Orc1, Orc2, Orc5 and Orc6. We found no evidence for interaction between Cdc23 and the MCM recruitment factor Cdc18 (the homologue of Saccharomyces cerevisiae Cdc6). Unlike Cdc18, Cdc23 mRNA shows no significant fluctuation in level through the cell cycle. These data suggest that fission yeast Cdc23 is an MCM-associated factor which has a role in the initiation of DNA replication.     

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

SUMO修饰与端粒维持

端粒(telomere)长度维持在一定的范围内是细胞正常生理功能的一个重要的基础,端粒长度的变化可导致两个截然相反的病理生理过程-癌变和衰老,端粒过长可引起细胞永生化而癌变,端粒进行性缩短则有丝分裂能力下降导致细胞衰老[1].端粒的长度和结构依赖端粒酶的活性及端粒蛋白复合体(shelterin)的调节[2],端粒酶的激活可引起端粒DNA序列增加而延长细胞的寿命.泛素样小分子修饰(small ubiquin-like modifier,SUMO修饰)在不同的端粒维持机制中的作用途径不同,SUMO修饰可激活端粒酶的活性,促进依赖端粒酶合成端粒的途径,SUMO修饰也可促进同源重组途径合成端粒的能力[3],增加端粒的长度,在保持端粒的长度上发挥重要的调节作用.     

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1^CP) in two siblings with CP. POT1^CP induced a proliferative arrest that could be bypassed by telomerase. POT1^CP was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1^CP was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1^CP was also defective in the maintenance of the telomeric C strand, causing extended 3′ overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).     

Normal mammalian cells negatively regulate telomere length by telomere trimming

In human cancer cells with telomeres that have been over-lengthened by exogenous telomerase activity, telomere shortening can occur by a process that generates circles of double-stranded telomeric DNA (t-circles). Here, we demonstrate that this telomeretrimming process occurs in cells of the male germline and in normal lymphocytes following mitogen-stimulated upregulation of telomerase activity. Mouse tissues also contain abundant t-circles, suggesting that telomere trimming also contributes to telomere length regulation in mice. In cancer cells and stimulated lymphocytes, the mechanism involves the XRCC3 homologous recombination (HR) protein and generates single-stranded C-rich telomeric DNA. This suggests that, in addition to the well-documented gradual telomere attrition that accompanies cellular replication, there is also a more rapid form of negative telomere length control in normal mammalian cells, which most likely involves HR-mediated removal of telomere loops in the form of t-circles. We therefore propose that this telomere trimming mechanism is an additional factor in the balance between telomere lengthening and telomere shortening in normal human germline and somatic cells that may prevent excessive lengthening by processes such as telomerase activity.     

Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.     

Verrocchio,a Drosophila OB fold-containing protein,is a component of the terminin telomere-capping complex

Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. Drosophila telomeres are protected by terminin, a complex that includes the HOAP (Heterochromatin Protein 1/origin recognition complex-associated protein) and Moi (Modigliani) proteins and shares the properties of human shelterin. Here we show that Verrocchio (Ver), an oligonucleotide/oligosaccharide-binding (OB) fold-containing protein related to Rpa2/Stn1, interacts physically with HOAP and Moi, is enriched only at telomeres, and prevents telomere fusion. These results indicate that Ver is a new terminin component; we speculate that, concomitant with telomerase loss, Drosophila evolved terminin to bind chromosome ends independently of the DNA sequence.     

Molecular basis of telomere syndrome caused by CTC1 mutations

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase α-primase (polα-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA polα-primase to promote faithful telomere DNA replication.     

Telomeres in evolution and evolution of telomeres

This paper examines telomeres from an evolutionary perspective. In the monocot plant order Asparagales two evolutionary switch-points in telomere sequence are known. The first occurred when the Arabidopsis-type telomere was replaced by a telomere based on a repeat motif more typical of vertebrates. The replacement is associated with telomerase activity, but the telomerase has low fidelity and this may have implications for the binding of telomeric proteins. At the second evolutionary switch-point, the telomere and its mode of synthesis are replaced by an unknown mechanism. Elsewhere in plants (Sessia, Vestia, Cestrum) and in arthropods, the telomere “typical” of the group is lost. Probably many other groups with “unusual” telomeres will be found. We question whether telomerase is indeed the original end-maintenance system and point to other candidate processes involving t-loops, t-circles, rolling circle replication and recombination. Possible evolutionary outcomes arising from the loss of telomerase activity in alternative lengthening of telomere (ALT) systems are discussed. We propose that elongation of minisatellite repeats using recombination/replication processes initially substitutes for the loss of telomerase function. Then in more established ALT groups, subtelomeric satellite repeats may replace the telomeric minisatellite repeat whilst maintaining the recombination/replication mechanisms for telomere elongation. Thereafter a retrotransposition-based end-maintenance system may become established. The influence of changing sequence motifs on the properties of the telomere cap is discussed. The DNA and protein components of telomeres should be regarded – as with any other chromosome elements – as evolving and co-evolving over time and responding to changes in the genome and to environmental stresses. We describe how telomere dysfunction, resulting in end-to-end chromosome fusions, can have a profound effect on chromosome evolution and perhaps even speciation.     

Role of the CDC8 gene in the repair of single strand breaks in DNA of the yeast Saccharomyces cerevisiae

Summary It has been found that the repair of single strand breaks is defective in the DNA replication mutants cdc8-1 and cdc8-3 of Saccharomyces cerevisiae both in permissive (23°C) and restrictive conditions (36°C). In permissive conditions we observed a significant delay in single strand break repair in a diploid strain HB7 (cdc8-1/cdc8-1), as compared with the wild-type strain. Under restrictive conditions no repair was observed, but rather degradation of MMS-damaged DNA occurred. It has been also found that the repair of single strand breaks in yeast is inhibited by cycloheximide but not by hydroxyurea.     

A mouse cdc25 homolog is differentially and developmentally expressed.

The timing and activation of the p34cdc2 kinase in mammals is associated with dephosphorylation of phosphotyrosine and phosphothreonine residues on the p34cdc2 kinase. For fission yeast, the timing of mitosis is regulated by cyclic accumulation of cdc25, which promotes dephosphorylation of p34cdc2 and concomitant protein kinase activation. We report the identification and characterization of a structural and functional mouse homolog, Cdc25M2, of the cdc25 phosphatase. Cdc25M2 shows high sequence identity to the previously reported human homolog cdc25Hu2. Cdc25M2 can functionally complement for a Schizosaccharomyces pombe cdc25ts mutation, and when expressed in Escherichia coli and purified, Cdc25M2 is an active phosphatase. cdc25M2 mRNA shows variation in expression in different tissues in the mouse embryo and is expressed in a developmental and cell-cycle-dependent fashion. We suggest that the expression and accumulation of the cdc25 mitotic inducer may play a critical role in the regulation of mouse development.     

Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1

Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein. MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCF^Met30 complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity.