Many Saccharomyces telomeres bear one or more copies of the repetitive Y' element followed by approximately 350 bp of telomerase-generated C(1-3)A/TG(1-3) repeats. Although most cells lacking a gene required for the telomerase pathway die after 50 to 100 cell divisions, survivors arise spontaneously in such cultures. These survivors have one of two distinct patterns of telomeric DNA (V. Lundblad and E. H. Blackburn, Cell 73:347-360, 1993). The more common of the two patterns, seen in type I survivors, is tandem amplification of Y' followed by very short tracts of C(1-3)A/TG(1-3) DNA. By determining the structure of singly tagged telomeres, chromosomes in type II survivors were shown to end in very long and heterogeneous-length tracts of C(1-3)A/TG(1-3) DNA, with some telomeres having 12 kb or more of C(1-3)A/TG(1-3) repeats. Maintenance of these long telomeres required the continuous presence of Rad52p. Whereas type I survivors often converted to the type II structure of telomeric DNA, the type II pattern was maintained for at least 250 cell divisions. However, during outgrowth, the structure of type II telomeres was dynamic, displaying gradual shortening as well as other structural changes that could be explained by continuous gene conversion events with other telomeres. Although most type II survivors had a growth rate similar to that of telomerase-proficient cells, their telomeres slowly returned to wild-type lengths when telomerase was reintroduced. The very long and heterogeneous-length telomeres characteristic of type II survivors in Saccharomyces are reminiscent of the telomeres in immortal human cell lines and tumors that maintain telomeric DNA in the absence of telomerase.
Expansion of DNA trinucleotide repeats (TNRs) is the causative mutation in a growing number of human genetic diseases. Large expansions of a CTG tract were obtained and shown by genetic and physical assays to be length-dependent sites of chromosome breakage in Saccharomyces cerevisiae. Deletion of RAD27, which encodes a nuclease involved in Okazaki fragment processing, caused length-dependent destabilization of CTG tracts and a substantial increase in expansion frequency. The genetic assay described here can be used to evaluate other factors that induce TNR expansion or chromosome fragility in humans.
Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.
In Saccharomyces cerevisiae, proximity to a telomere affects both transcription and replication of adjacent DNA. In this study, we show that telomeres also impose a position effect on mitotic recombination. The rate of recombination between directly repeated tracts of telomeric C1-3A/TG1-3 DNA was reduced severely by proximity to a telomere. In contrast, recombination of two control substrates was not affected by telomere proximity. Thus, unlike position effects on transcription or replication, inhibition of recombination was sequence specific. Moreover, the repression of recombination was not under the same control as transcriptional repression (telomere position effect; TPE), as mutations in genes essential for TPE did not alleviate telomeric repression of recombination. The reduction in recombination between C1-3A/TG1-3 tracts near the telomere was caused by an absence of Rad52p-dependent events as well as a reduction in Rad1p-dependent events. The sequence-specific repression of recombination near the telomere was eliminated in cells that overexpressed the telomere-binding protein Rap1p, a condition that also increased recombination between C1-3A/TG1-3 tracts at internal positions on the chromosome. We propose that the specific inhibition between C1-3A/TG1-3 tracts near the telomere occurs through the action of a telomere-specific end-binding protein that binds to the single-strand TG1-3 tail generated during the processing of recombination intermediates. The recombination inhibitor protein may also block recombination between endogenous telomeres.
Trinucleotide repeat expansion is the causative mutation for a growing number of diseases including myotonic dystrophy, Huntington's disease, and fragile X syndrome. A (CTG/CAG)130 tract cloned from a myotonic dystrophy patient was inserted in both orientations into the genome of Saccharomyces cerevisiae. This insertion was made either very close to the 5' end or very close to the 3' end of a URA3 transcription unit. Regardless of its orientation, no evidence was found for triplet-mediated transcriptional repression of the nearby gene. However, the stability of the tract correlated with its orientation on the chromosome. In one orientation, the (CTG/CAG)130 tract was very unstable and prone to deletions. In the other orientation, the tract was stable, with fewer deletions and two possible cases of expansion detected. Analysis of the direction of replication through the region showed that in the unstable orientation the CTG tract was on the lagging-strand template and that in the stable orientation the CAG tract was on the lagging-strand template. The orientation dependence of CTG/CAG tract instability seen in this yeast system supports models involving hairpin-mediated polymerase slippage previously proposed for trinucleotide repeat expansion.
Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997) Genes Dev. 11, 345-357]. Although cac1 delta cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual cac1 delta cells switch from transcriptionally "off" to transcriptionally "on" more often per cell cycle than wild-type cells. In addition, cac1 delta cells were defective for transcriptional silencing near internal tracts of C(1-3)A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription-PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C(1-3)A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the Escherichia coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.
The Werner syndrome (WS) is characterized by the premature onset and accelerated rate of development of major geriatric disorders, including atherosclerosis, diabetes mellitus, osteoporosis, ocular cataracts, and various neoplasms. Cultures of WS skin-fibroblastlike cells have been previously shown to undergo accelerated rates of decline of the replicative potentials and to exhibit variegated chromosomal translocations and deletions. Since the replicative decline of normal somatic cells is associated with a loss of telomeric repeats, we investigated the kinetics of telomeric repeat loss in WS cells. The mean length of telomere restriction fragments (TRF) from the earliest passages of WS cells studied was not shorter than those of controls, possibly reflecting selective pressure for subsets of cells with relatively high residual replicative capacity. Statistical evidence indicated an accelerated shortening of TRF length in serially passaged WS cultures, but the mean TRF lengths of WS cultures that had ceased replicating were significantly longer than those of senescent controls. Thus, while accelerated loss of telomeric repeats could potentially explain the rapid decline in proliferation of WS cells, it is possible that WS cells exit the cell cycle via mechanisms that differ from those of replicatively senescent cells from control subjects.
The strand of telomeric DNA that runs 5'-3' toward a chromosome end is typically G rich. Telomerase-generated G tails are expected at one end of individual DNA molecules. Saccharomyces telomeres acquire TG1-3 tails late in S phase. Moreover, the telomeres of linear plasmids can interact when the TG1-3 tails are present. Molecules that mimic the structures predicted for telomere replication intermediates were generated in vitro. These in vitro generated molecules formed telomere-telomere interactions similar to those on molecules isolated from yeast, but only if both ends that interacted had a TG1-3 tail. Moreover, TG1-3 tails were generated in vivo in cells lacking telomerase. These data suggest a new step in telomere maintenance, cell cycle-regulated degradation of the C1-3A strand, which can generate a potential substrate for telomerase and telomere-binding proteins at every telomere.
A combination of classical genetic, biochemical, and molecular biological approaches have generated a rather detailed understanding of the structure and function of Saccharomyces telomeres. Yeast telomeres are essential to allow the cell to distinguish intact from broken chromosomes, to protect the end of the chromosome from degradation, and to facilitate the replication of the very end of the chromosome. In addition, yeast telomeres are a specialized site for gene expression in that the transcription of genes placed near them is reversibly repressed. A surprisingly large number of genes have been identified that influence either telomere structure or telomere function (or both), although in many cases the mechanism of action of these genes is poorly understood. This article reviews the recent literature on telomere biology and highlights areas for future research.
Telomeres are specialized DNA protein structures that form the ends of eukaryotic chromosomes. In yeast, loss of even a single telomere causes a prolonged, but transitory, cell-cycle arrest. During this arrest, many broken chromosomes acquire a new telomere by one of three pathways, although at the cost of a partial loss of heterozygosity. In addition, a substantial fraction of the chromosomes lacking a telomere is lost, which generates an aneuploid cell. In these cases, the broken chromosome is usually replicated and segregated for ten or more cell divisions in unstable form. Extrapolation from yeast suggests that the gradual loss of telomeric DNA that accompanies ageing in humans may initiate the kinds of chromosomal rearrangements and genetic changes that are associated with tumorigenesis.
Saccharomyces telomeres consist of approximately 300 bp of C1-3A/TG1-3 DNA. Cells lacking the activity of the essential gene CDC13 display a cell cycle arrest mediated by the DNA damage sensing, RAD9 cell cycle checkpoint, presumably because they exhibit strand-specific loss of telomeric and telomere-adjacent DNA [Garvik, B., Carson, M. & Hartwell, L. (1995) Mol. Celi. Biol. 15,6128-6138]. Cdc13p expressed in Escherichia coli or overexpressed in yeast bound specifically to single-strand TG1-3 DNA. The specificity of binding displayed by Cdc13p in vitro indicates that in vivo it could bind to both the short, constitutive single-strand TG1-3 tails thought to be present at telomeres at most times in the cell cycle as well as to the long single-strand TG1-3 tails that are intermediates in telomere replication. Genes located near yeast telomeres are transcriptionally repressed, a phenomenon known as telomere position effect. Cells overexpressing a mutant form of Cdc13p had reduced telomere position effect at high temperatures. These data suggest that Cdc13p functions by binding directly to telomeric DNA, thereby limiting its accessibility to degradation and transcription as well as masking it from factors that detect damaged DNA.
The DNA-protein complexes at the ends of linear eukaryotic chromosomes are called the telomeres. In Saccharomyces cerevisiae, telomeric DNA consists of a variable length of the short repeated sequence C1-3A. The length of yeast telomeres can be altered by mutation, by changing the levels of telomere binding proteins, or by increasing the amount of C1-3A DNA sequences. Cells bearing the tel1-1 or tel2-1 mutations, known previously to have short telomeres, did not respond to perturbations that caused telomere lengthening in wild-type cells. The transcription of genes placed near yeast telomeres is reversibly repressed, a phenomenon called the telomere position effect. The tel2-1 mutation reduced the position effect but did not affect transcriptional repression at the silent mating type cassettes, HMRa and HML alpha. The TEL2 gene was cloned, sequenced, and disrupted. Cells lacking TEL2 function died, with some cells arresting as large cells with three or four small protrusions or "blebs."
Yeast telomeric DNA is assembled into a nonnucleosomal chromatin structure known as the telosome, which is thought to influence the transcriptional repression of genes placed in its vicinity, a phenomenon called telomere position effect (TPE). The product of the RAP1 gene, Rap1p, is a component of the telosome. We show that the fraction of cells exhibiting TPE can be substantially reduced by expressing large amounts of a deletion derivative of Rap1p that is unable to bind DNA, called Rap1 delta BBp, or by introducing extra telomeres on a linear plasmid, presumably because both compete in trans with telomeric chromatin for factor(s) important for TPE. This reduction in TPE, observed in three different strains, was demonstrated for two different genes, each assayed at a different telomere. In contrast, the addition of internal tracts of telomeric DNA on a circular plasmid had very little effect on TPE. The product of the SIR3 gene, Sir3p, appears to be limiting for TPE. Overexpression of Sir3p completely suppressed the reduction in TPE observed with expression of Rap1 delta BBp, but did not restore high levels of TPE to cells with extra telomeres. These results suggest that extra telomeres must titrate a factor other than Sir3p that is important for TPE. These results also provide evidence for a terminus-specific binding factor that is a factor with a higher affinity for DNA termini than for nonterminal tracts of telomeric DNA and indicate that this factor is important for TPE.
Telomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for approximately 30 or more cell divisions in the absence of the EST1 product, Est1p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1 delta cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Est1p is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.
Telomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210). The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays. Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes. The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes. Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes. These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt. In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210; Conrad, M. N., Wright, J. H., Wolf, A. J. and Zakian, V. A. (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA.
Telomeres are the protein-DNA structures at the ends of eukaryotic chromosomes. In yeast, and probably most other eukaryotes, telomeres are essential. They allow the cell to distinguish intact from broken chromosomes, protect chromosomes from degradation, and are substrates for novel replication mechanisms. Telomeres are usually replicated by telomerase, a telomere-specific reverse transcriptase, although telomerase-independent mechanisms of telomere maintenance exist. Telomere replication is both cell cycle- and developmentally regulated, and its control is likely to be complex. Because telomere loss causes the kinds of chromosomal changes associated with cancer and aging, an understanding of telomere biology has medical relevance.
Telomeric position effect (TPE) refers to the ability of telomeres to repress the transcription of genes in their vicinity. Internal stretches of C1-3A DNA, the sequence found at Saccharomyces telomeres, also repressed transcription of nearby genes. This repression, hereafter called C1-3A-based silencing, was observed at several chromosomal loci, including on a circular chromosome. The magnitude of C1-3A-based silencing was increased by both proximity to a telomere and increased length of the C1-3A tracts. C1-3A-based silencing was affected by many of the same genes and conditions that influence TPE and acted in an orientation-independent manner. Thus, in yeast, an expanded array of a simple repetitive DNA, C1-3A, is sufficient to promote the assembly of a transcriptionally silent chromosomal domain.