Replication proteins influence the maintenance of telomere length and telomerase protein stability

We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.     

Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length.     

Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint.

A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the double-strand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G2, these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.     

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.     

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.     

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.     

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.     

New function of CDC13 in positive telomere length regulation

Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation. In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13. Although CDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells. Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level. Thus, in cdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres. Furthermore, chromosome end structure remains protected in cdc13-4 mutants. Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1. Strikingly, cdc13-4/tel1Delta double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y' elements after 225 generations. Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance. Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres. Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity.     

Hyper-phosphorylated retinoblastoma protein suppresses telomere elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase alpha (pol-alpha) is reported to be required for telomere maintenance, the critical role of pol-alpha in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-alpha in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-alpha or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-alpha might thus contribute to telomere maintenance, and might be regulated by ppRb.     

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.     

Hyper-Phosphorylated Retinoblastoma Protein Suppresses Telomere Elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase α (pol-α) is reported to be required for telomere maintenance, the critical role of pol-α in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-α in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-α or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-α might thus contribute to telomere maintenance, and might be regulated by ppRb.     

Interactions between telomerase and primase physically link the telomere and chromosome replication machinery

In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase alpha-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.     

Distinct roles of TRF1 in the regulation of telomere structure and lengthening

The telomere is a functional chromatin structure that consists of G-rich repetitive sequences and various associated proteins. Telomeres protect chromosomal ends from degradation, provide escape from the DNA damage response, and regulate telomere lengthening by telomerase. Multiple proteins that localize at telomeres form a complex called shelterin/telosome. One component, TRF1, is a double-stranded telomeric DNA binding protein. Inactivation of TRF1 disrupts telomeric localization of other shelterin components and induces chromosomal instability. Here, we examined how the telomeric localization of shelterin components is crucial for TRF1-mediated telomere-associated functions. We found that many of the mTRF1 deficient phenotypes, including chromosomal instability, growth defects, and dysfunctional telomere damage response, were suppressed by the telomere localization of shelterin components in the absence of functional mTRF1. However, abnormal telomere signals and telomere elongation phenotypes were either not rescued or only partially rescued, respectively. These data suggest that TRF1 regulates telomere length and function by at least two mechanisms; in one TRF1 acts through the recruiting/tethering of other shelterin components to telomeres, and in the other TRF1 seems to play a more direct role.