Cell, Vol.

77, 621-623,

June

3, 1994, Copyright

0 1994 by Cell Press

Telomeres:

No End in Sight

Minireview

Elizabeth H. Blackburn Department of Microbiology and immunology and Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143-0414

Why do eukaryotic chromosomes end the way they do? One basic function of teiomeres, the ends of chromosomes, is to preserve themselves; they allow replenishment of the chromosomai DNA termini by an independent mechanism of DNA synthesis, to make up for the incomplete replication of the 5 end of each chromosomal DNA strand by the primary cellular DNA replication machinery. But why is a special DNA sequence needed at teiomeres? Obsetvations of teiomere behavior indicate that teiomeres play multiple roles, some new and surprising. This minireview highlights recent work that throws into sharp relief the question of why a specific DNA sequence is required to stabilize chromosomes. The Correct Telomeric DNA Sequence Is Necessary to Stabilize Chromosomes In a great phyiogenetic variety of eukaryotes, teiomeric DNA consists of a terminal stretch of tandem, speciesspecific repeats of very short (typically 5-8 bp) simple sequences, characterized by clusters of G residues on the strand that forms the 3 end of each chromosomai DNA strand. This strand is synthesized by the ribonucieoprotein enzyme telomerase (reviewed by Blackburn, 1992) an essential cellular reverse transcriptase that uses a short sequence within its RNA moiety as the template for addition of the G-cluster teiomeric DNA strand to chromosome ends. Without replenishment of telomeric sequences, which normally requires telomerase to make up for losses caused by incomplete replication, the chromosome ends gradually recede (Figure 1). Usually, chromosomes with broken ends lacking their species-specific teiomeric DNA sequences or that fail to maintain preexisting teiomeric DNA on the end of chromosomes are not stably maintained (reviewed by Blackburn, 1991). This instability has been emphasized by recent studies in the yeast Saccharomyces cerevisiae. Sandeil and Zakian (1993) removed a telomere in a controlled way from a dispensable chromosome and watched the fate of the chromosome immediately thereafter. The short-term response of the ceil was very similar to that elicited by introducing a single break into a circular, nonessential plasmid, producing two teiomereiess ends; this response included temporary arrest of the ceil cycle (Bennett et al., 1993; Sandeii and Zakian, 1993). Subsequently, the linear chromosome lacking one teiomere was lost, although sometimes only after several ceil divisions, unless it was stabilized by acquiring ateiomere by some means (Sandeii and Zakian, 1993). Another line of evidence highlighting the need for the telomeric DNA sequence itself to maintain chromosomes stably in yeast has come from asking how cells can survive

the normally lethal estl- mutation. estl- yeast fail to replenish teiomeric DNA in the normal fashion, but a small fraction of ceils continues to grow, even after the teiomeric DNA fails below a critical length in most ceils, leading to chromosome loss and cessation of ceil division. Surviving cells have patched together teiomeres onto the ends of the chromosomes by a process involving recombination. They use the subteiomeric Y elements that bear tracts of teiomeric GIe3T repeat DNA at both ends of the elements to bring additional G13T sequences to the chromosomai termini (Lundbiad and Blackburn, 1993). Thus, even under strong selection for alternative ways to maintain chromosomes, no evidence was found for another sequence in the genome that could substitute for the normal yeast telomerit repeat sequences. Experimentally altering the sequence at the very terminus of the chromosome, as opposed to removing it, has provided hints as to the nature of the teiomeric interaction needed to stabilize chromosomes. In the ciliate Tetrahymena, when certain sequence changes were made in the teiomerase RNA template region, teiomeric repeats with novel sequences were added efficiently in vivo, and teiomeres remained long (Yu et al., 1990). If replenishment were ail that was necessary, and the precise sequence itself was unimportant, there should have been no effect on the ceil. instead, there were dramatic, DNA sequencespecific effects: addition of certain mutant teiomeric DNA sequences prevented or impaired nuclear division (vu et al., 1990; Lee et al., 1994). Yet harboring these mutant sequences in more internal portions of the teiomeres caused no adverse effects, so long as wild-type repeat sequences were at the very termini c/u and Blackburn, 1991). These results suggest that teiomere-mediated chromosome stabilization involves correct binding between the very terminus and sequence-specific teiomerebinding protein(s), which in turn is necessary for an as yet unknown teiomere-nucleus interaction. This sequence specific binding could be to the single-stranded overhanging strand, the distal duplex portion of the telomeric DNA, or both. A class of teiomere structural proteins, of which the best characterized is the a8 heterodimer of the ciliate Oxytricha, binds the protruding G-cluster strand with strong DNA sequence specificity (for references, see Fang and Cech, 1993).

Figure

1. Telomeric

DNA Replenishment

Cell 622

A Novel Role for Telomeres in Premeiotic Nuclei Insight into how telomeres stabilize chromosomes may come from an unexpected source: a newly uncovered, intriguing role of telomeres in premeiotic nuclei in the fission yeast Schizosaccharomyces pombe. Early in meiotic prophase, in many organisms, the telomeres cluster under the nuclear envelope (the bouquet stage; Dawe et al., 1994, and references therein), in some animal cells near the centrosome. Chikashige et al. (1994) combined molecular localization with modern imaging and recording techniques tostudy nuclei in action in lives. pombecellsduring karyogamy and meiosis. Unexpectedly, in karyogamy (fusion of haploid nuclei), telomeres form a single condensed spot at the spindle pole body (the yeast version of the centrosome) at the leading edge of each haploid nucleus as it approached the other. Then, after nuclear fusion and during early prophase, before the chromosome segregations of the meiotic divisions commence, a remarkable nuclear movement occurs for a few hours: the elongated nucleus (called the horsetail nucleus at this stage because of its shape) moves around and around the cell, led by the spindle pole body. Again, the telomeres are attached to the spindle pole body in a single cluster, with the centromeres trailing far away at the opposite end of the moving nucleus. Later, when the nuclear divisions of meiosis begin, the telomeres become dissociated from the spindle pole body, and the centromeres associate with it, as in mitotic cells. Is the purpose of this striking telomere-led movement of the premeiotic nucleus to facilitate interaction between chromosome homologs for recombination and pairing, as suggested by Chikashige et al. (1994)? Is this developmentally controlled telomere association with the spindle pole body in S. pombe a specialized form of the sequencespecific interaction of the terminal DNA that stabilizes chromosomes? For example, are the same DNA sequences required? Clearly, in S. pombe and quite possibly other eukaryotes, telomeres mediate a specific attachment to a part of the nucleus not previously associated with telomeric function, suggesting a direct role of telomeres in nuclear movement at certain stages in the life cycle. Telomeric Clustering and Heterochromatin Can Be Uncoupled from Mitotic Chromosome Stabilization Clustering of telomeres with each other under the nuclear envelope and condensed chromatin (heterochromatin) at telomeres have also been observed in nonmeiotic cells of many species. Surprisingly, apparently complete disruption of these facets of telomere behavior in S. cerevisiae has only slight effects on mitotic chromosome stability. Heterochromatic properties and peripheral localization of telomeres in the nuclei of mitotic S. cerevisiae center on the ability of its telomeric DNA to bind Rap1 p protein, an essential transcriptional activator and a specific repressor of expression of the silent mating type loci. The DNA consensussequence to which Rap1 p binds is also found at telomeres roughly every 20-40 bp. Thus, the few hundred base pair tracts of telomeric DNA at S. cerevisiae chromosome ends provide extensive platforms along which Raplp arrays can bind (Palladino et al., 1993, and refer-

ences therein). Rap1 p in turn attracts most components of the heterochromatin-assembling machinery, which has been shown genetically to silence the silent mating type loci by position effect-mediated repression (Renauld et al., 1993: Chien et al., 1993). When a gene is engineered to lie very close to a chromosome end in S. cerevisiae, it comes under this repression. Disrupting SIR3 or SIR4, two genes required for silent mating type and telomeric position effects, led to complete loss of the peripheral clustered localization of Raplp (and therefore, it is inferred, of the telomeric ends) (Palladino et al., 1993). But the effectson chromosome stability were minor; chromosome loss rates increased by 4-fold or less, being still only - 10m5 loss events per cell division for a natural chromosome. There was no destabilization of a smaller (150 kb) artificial chromosome, whose loss rate remained at 1Oe3to 1Om4 events per cell division (Palladino et al., 1993). In contrast, removal of a telomere caused over 25% of cells to lose the telomere- chromosome within ten cell divisions (Sandell and Zakian, 1993). Thus, in S. cerevisiae, neither assembling telomeric heterochromatin nor Raplp-dependent clustering of telomeres in the nuclear periphery is necessary for the primary telomeremediated stability of chromosomes. Replenishment of Telomeric DNA: In Large as Well as Small Increments It appears unlikely that telomeres need the correct DNA sequence because telomere replenishment is mechanistically restricted to elongating, or synthesizing, a specific DNA sequence in any given organism. Telomerases elongate a variety of telomeric sequences efficiently and at some developmental stages can even heal broken ends lacking telomeric repeats (for reviews, see Greider, 1991; Blackburn, 1992). The synthesis of different sequences by telomerase when its RNA template sequence is altered shows that this enzyme is not inherently constrained to synthesis of its usual sequence (Vu et al., 1990; Yu and Blackburn, 1991). And at least one species, Drosophila melanogaster, appears to bypass the need for any conventional telomerase at all. In Drosophila, there has been no sign of simple Gclustercontaining telomeric repeats at chromosome ends. Instead, at least two specific families of non-long terminal repeat type retroelements (the Het and TART families) are found at chromosome ends (Levis et al., 1993, and references therein). In principle, to counterbalance attrition through incomplete replication, terminal DNA could be replenished by frequent additions of short tracts of telomerit DNA by telomerase, or by infrequent additions of large tracts of DNA. There is good evidence that Drosophila uses the latter strategy, sporadically adding a large (up to several kilobases) Het or TART retroelement to the receding end by retroposition (Figure 1). These terminal retropositions exhibit no obvious specificity with respect to the terminal sequence of the chromosome that is elongated, and can occur on broken ends lacking telomeric retrotransposons. Drosophila appears unusual not only in its telomeric DNA sequences but also in the length of time it can propagate a chromosome lacking a telomere; such a chromo-

&ireview

some can be maintained for many fly generations (see Levis et al., 1993, for references). Whether these unusual properties are related is unknown. The ability of S. cerevisiae to propagate a chromosome lacking one telomere for several cell divisions before loss (Sandell and Zakian, 1993) may put the apparent disregard of Drosophila for a broken end into a useful context: is Drosophila simply able to maintain such a chromosome for a longer period than yeast? Or is some chromosome loss tolerated by the whole organism? Certain Drosophila mutants remain viable even when they produce chromosome breaks in 25% of cells (Baker et al., 1997). Yet even in this species, a broken end eventually becomes capped by tefomeric retrotransposons. Thus, whether Drosophila can bypass a requirement for the conventional type of telomeric sequence, or whether this requirement is still present to some degree, but now satisfied by the tetomeric retroposons, is unknown. G-Guarfefs at Te/omeres? The widespread conservation of simple-repeat, G-rich telomeric sequences has suggested that telomeres need a specific DNA sequence in order to assume a specific structure. The protruding G-cluster strand of telomeric DNA at the chromosome end could in theory take up various non-Watson-Crick paired structures, the most stable being four-stranded DNA forms termed Gquartet structures (reviewed by Schultze et al., 1994). However, cracks continue to appear in the bastion of evolutionary conservation of telomeric sequences. In several budding yeasts, the telomeric repeat units are surprisingly divergent in length (5-25 bp), sequence, and composition between these relatively closely related species (McEachern and Blackburn, 1994). The longest telomeric repeat units are not G-rich overall and do not bear much obvious resemblance to the short telomeric repeat units with prominent G-clusters in many other eukaryotes. Yet all these yeast telomeric repeat units contain a short TGZqTG motif. Whether it is conserved for DNA structural reasons is unknown. Some recent work has focused on the possibility of a role for Gquartet structures at telomeres. The a5 heterodimer telomere protein from the ciliate Oxytricha binds the single-stranded G-rich strand overhang at the chromosome end (reviewed by Fang and Cech, 1993). In vitro, the bases of the bound telomeric oligonucleotide are available to act as a substrate for telomerase, AMV reverse transcriptase, and DNA polymerase, with the oligonucleotide backbone apparently being protected by the ap heterodimer (Shippen et al., 1994). In the absence of the a subunit, the 5 subunit does not stably bind telomeric DNA, but a basic (23% lysine) domain of this subunit kinetically promotes formation of intermolecular Gquartets between telomeric DNA oligonucleotides in vitro (Fang and Cech, 1993) perhaps by shielding the backbone charges and exposing the bases favorably for such interactions; the basic proteins histone Hl, cytochrome c, and lysozyme also promote Gquartet formation, although more slowly (Fang and Cech, 1993). As Gquartet structures do not bind the a5 heterodimer or serve as a substrate for telomerase in vitro, Fang and Cech (1993) suggest that unfolding of a thermodynamically stable Gquartet structure may be a function

of the j3 subunit in vivo. Recently, other proteinsthat recog nize or bind Gquartet structures in vitro have been identified (e.g., Schierer and Henderson, 1994, and references therein). However, until the in vlvo roles of all these activities can be defined, the biological role of Gquartets remains enigmatic. A Current Outlook Our thinking about telomere function continues to evolve as new facts emerge. New results show that at least some of the multiple functions of telomeres are separable. What had been thought was an intrinsic need for telomeric DNA to consist of very simple repeats with prominent G-clusters is challenged by the unexpected complexity and diversity of telomeric DNA sequences that have surfaced recently in Drosophila melanogaster and certain budding yeasts. Intriguingly, reverse transcription appears to be the shared theme in the replenishment of these diverse types of telomerit DNA. Even when replenishment is adequate, the correct sequence of DNA at the extreme terminus is important for chromosome stability in most species, and in Tetrahymena at least, for nuclear division. But the mechanism of this stabilization is still a major unanswered question. Obviously we have a long way to go before telomeres cease to challenge and surprise us.
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