chromosome healing: spontaneous and programmedde novo telomere formation by telomerase

Meni Melek and Dorothy E. Shippen

Summary

Telomeres are protective caps for chromosome ends that are essential for genome stability. Broken chromosomes missing a telomere will not be maintained unless the chromosome is ‘healed’ with the formation of a new telomere. Chromosome healing can be a programmed event following developmentally regulated chromosome fragmentation, or it may occur spontaneously when a chromosome is accidentally broken. In this article we discuss the consequences of telomere loss and the possible mechanisms that the enzyme telomerase employs to form telomeres de novo on broken Accepted

12 January 1996 chromosome ends.

Introduction The essential function of the telomere, the DNA-protein structure that comprises the physical terminus of the eukary- otic chromosome, has been established for more than 60 years. However, the molecular basis for telomere behavior is still a mystery. In the late 1930s and early ’ ~ O S , Hermann Muller and Barbara McClintock demonstrated that broken chromosomes were highly unstable(lS2), cycling through repeated breakage-fusion-bridging eventd2). On occasion a broken chromosome became stabilized and could be propa- gated normally. Subsequent work has shown that these ’healed’ chromosomes had acquired telomeres. Research in many laboratories over the ensuing years confirmed that the natural ends of chromosomes provide a protective cap that imparts stability to chromosomes by protecting against end- to-end fusion, recombination and exonucleolytic degrada- tion. The importance of the telomere is further highlighted by recent studies in yeast demonstrating that the loss of even a single telomere causes cell-cycle arrest(3).

Human chromosomes lose a small amount of telomeric DNA following each cell division. This phenomenon is thought to result from the inability of the conventional DNA replication machinery to replicate the extreme 5’terminus of each DNA strand(4). Consequently, terminal DNA sequences are lost with each round of replication, and over time chromosomes gradually shorten at their ends. The striking correlation between telomere shortening and cellular aging led to the telomere hypothesis of aging, which pro- poses that telomeres represent a biological clock(5). Accord- ing to this hypothesis, once telomeres shorten below a critical threshold, they lose the capacity to cap chromo-

somes effectively and activate a damaged DNA response pathway that causes cell-cycle arrest@).

The loss of telomeric DNA does not always culminate in cell death. Telomere function may be restored if the terminal DNA sequences are replenished by the action of telomerase. Telomerase is the specialized DNA polymerase that synthesizes telomeric DNA. This enzyme not only maintains pre-existing telomeres, but also adds telomeres directly onto broken chromosome ends in a process known as chromosome healing (Fig. 1). In this review we focus on the consequences and mechanisms of chromosome healing, beginning with an overview of telomere structure and synthesis. More extensive treatises on telomeres and their maintenance can be found el~ewhere(~1~).

Telomere formation by telomerase In the majority of organisms surveyed, the chromosome terminus consists of short repeated DNA sequence arrays, typified by a clustering and segregation of G-residues on one DNA strand(g). The terminal segments of mammalian telomeres, for example, comprise hundreds to thousands of consecutive TTAGGG repeats(l0). This DNA is complexed with specific telomere-binding proteins(ll).

The G-rich DNA strand of the telomere forms a 3’ single- stranded overhang and this protrusion is the substrate onto which telomerase synthesizes additional G-rich repeats. Telomerases represent a novel class of DNA polymerases in that they are ribonucleoproteins. The essential RNA subunit within telomerase carries a sequence complementary to the G-rich telomeric DNA strand. This domain functions as the template to specify the sequences added to chromo-

Developmentally Regulated Developmentally Regulated Chromosome Fragmentation

Fig. 1. Telomere addition by telomerase. (A) Telomerase elongates pre- existing telomeres. Complementarity of the telomeric DNA termini to the RNA templating domain within the telomerase RNP is adequate for enzyme recruitment and DNA polymerization (see text for details). Shaded boxes represent telomeres. (6) Chromosome healing by telomerase. Telorneres can be lost spontaneously in cases such as human a-thalassemia (upper panel) or through developmentally regulated chromosome fragmentation (lower panel). In the first case, chromosome breakage and healing are independent events. In the latter instance chromosome fragmentation and de novo telomere addition are temporally linked.

De novo Telomere Formation

some ends. For example, in human telomerase RNA the 9- nucleotide sequence 5’ CUAACCCUA 3‘ is the template for the addition of TTAGGG repeats(12). A templating function for the telomerase RNA subunit was demonstrated by site- directed mutagenesis. Mutations in telomere-complementary sequences within Tefrahymena, yeast and human telomerase RNAs result in altered telomeric DNA repeats in ~ i f r o ( ’ ~ - ~ ~ ) and in vid16-18). Thus, telomerase is a specialized form of reverse transcriptase that utilizes an internal RNA template to synthesize DNA.

Telomerase requires protein subunits as well as its RNA to generate telomeric DNA(19-22). Genes encoding two telomerase proteins have recently been cloned from the ciliate Tefrah~mena(~~) . One protein, p95, appears to be involved in DNA r e c o g n i t i ~ n ( ~ ~ , ~ ~ ) , while the other, p80, is more closely associated with the RNA subunit and may carry the catalytic a~tivity(~3).

Telomerase has been detected in a variety of different o r g a r ~ i s m s ( ~ ~ - ~ ~ ! ~ ~ - ~ ~ ) . Consequently, the enzymatic proper- ties of telomerase have been studied in vifro in some detail. Single-stranded oligonucleotides resembling the natural G- rich overhang at the chromosome terminus are efficient primers for telomerase. Primer DNA binds at two sites in the e n ~ y m e ( ~ ~ - ~ ~ ) : the anchor site resides in a protein subunit and the template site in the template domain of the RNA subunit. Acting in concert, these domains facilitate polymerization of long tracts of G-rich telomeric repeats in vifro. A discontinuous mechanism has been proposed for telomere synthesis that involves successive rounds of polymerization and translocation (Fig. 2). The S’terminus of the primer DNA aligns on the RNA template while upstream primer sequences bind in the anchor site. Polymerization is initiated by addition of a short stretch of DNA onto the primer 3’

terminus copied from nucleotides in the RNA template. Once the end of the templating region is reached, a translocation event realigns the new 3’ terminus of the primer back at the beginning of the RNA template, simultaneously feeding the primer 5‘terminus further into the anchor site. Primer association with the anchor site maintains DNA contact with the enzyme during translocation and permits another round of polymerization instead of primer dissociation.

In addition to polymerization, telomerase can cleave nucleotides from a primer if the primer aligns beyond the 5’ boundary of the telomerase RNA templating domain (refs 13,25,32; Melek, Greene and Shippen, manuscript submitted). Up to 15 non-telomeric nucleotides can be eliminated in an endonucleolytic reaction before polymerization begins (Melek, Greene and Shippen, manuscript submitted). Although the biological role of the endonuclease is unknown, such an activity could impart additional precision to the translocation event by ensuring that nucleotides beyond the RNA templating domain are not incorporated into telomeric DNA. Alterations in the telomere repeat sequence can be lethal(16). Cleavage could also function to improve enzyme processivity. By analogy, endonucleolytic cleavage activities associated with prokaryotic and eukary- otic RNA polymerases restore forward movement when the polymerase has been a r r e ~ t e d ( ~ ~ 1 ~ ~ ) . Many features of the mechanism proposed for telomerase closely resemble the inchworm model of transcription by RNA polymerase as it moves along the DNA template(35).

Telomerase is constitutively expressed in single-celled organisms such as yeast and ciliates. In humans, however, the activity is developmentally regulated and is found in germline, placenta, fetal and some normal adult tissues including liver and hematopoietic cells(28,36-38). Because telomerase levels are extremely low or non-detectable in most adult somatic tissue, it has been argued that telomerase inactivation during cellular differentiation accounts for the progressive loss of telomeric DNA in human tissues. This argument is strengthened by studies in yeast, which show that deletion of the telomerase RNA gene causes telomeres to shrink(l7!l8). Interestingly, telomerase is found at higher levels in many immortalized cell lines and t ~ m o r s ( ~ ~ 8 ~ ~ ) where telomere function is restored. The correlation between telomerase activation and long-term tumor survival suggests that telomerase inhibitors could be valuable anti-cancer therapeutics. This intriguing possibility is currently under intense investigation.

Chromosome healing by telomerase The majority of telomerase studies have focused on the enzyme’s ability to act on pre-existing tracts of telomeric DNA. Even in most transformed cells, a fringe of telomeric DNA remains at chromosome ends prior to telomerase activation. Recent evidence suggests that the entire telomere may be lost in some tumor cells(39). In order for the chromosomes in such cells to be maintained, a new telomere must

form de novo. Chromosome healing by de novo telomere formation represents a different type of telomere synthesis whereby telomeric repeats are added directly onto non- telomeric DNA. In 1991, Yu and Blackburn demonstrated

Fig. 2. Telomere synthesis by the Euplofes telomerase. Telomerase is postulated to contain three functional domains: the anchor site, template site and cleavage site. The single stranded 3’ overhang of a chromosome end aligns on the RNA template while distal G-rich sequences interact with the anchor site. In vitro single-stranded DNA oligonucleotides are used to prime telomere synthesis (as shown here). The 3’ terminus of the DNA (blue) is extended in a polymerization reaction in which nucleotides in the RNA template are copied into DNA. When the end of the template is reached, a translocation event repositions the newly extended primer 3’ terminus back at the beginning of the template while simultaneously feeding the distal portion of the DNA molecule through the anchor site. The endonucleolytic cleavage activity described for telomerase may serve a type of proof-reading role (see text for details).

that telomerase is responsible for chromosome healing by transforming Tetrahymena with a telomerase RNA gene carrying a mutation in the templating domain. The telomeric sequence added to a fragmented chromosome end in vivo corresponded exactly to the mutant RNA template(40).

This important observation confirmed telomerase’s involvement in chromosome healing. However, two seminal questions remained unanswered. How is the enzyme recruited to a broken end, and what is the mechanism of de novo telomere addition? ln vitro studies of the interaction of telomerase with DNA primers provide some insight in this regard (Fig. 3). Telomerase has the highest affinity for single- stranded primers corresponding to the G-rich strand of the natural chromosome terminus. The primer need not display perfect complementarity to the telomerase RNA template for a given organism, however. Tetrahymena telomerase, for example, recognizes and extends G-rich telomeric DNA from the yeast Saccharomyces cere~isiae(~’), despite .the inability of the Tefrahymena telomerase RNA to form extended Watson-Crick base-paired duplexes with this sequence(42). This finding is consistent with an earlier observation in yeast demonstrating that yeast telomeric DNA can be added directly onto Tefrahymena telomeric sequences in V ~ V O ( ~ ~ ) . The available evidence indicates that primers carrying multiple clusters of G-residues are optimal for enzyme elongation. These ‘G-rich’ primers are predicted to bind at both the anchor and templates sites (Fig. 3).

As few as 2 to 4 residues at the primer 3’ terminus, which precisely match the RNA template, convert an otherwise unreactive primer into one that can be utilized by telomerase(30) (Fig. 3). Such a primer is predicted to bind in the template site, but not the anchor site. Nevertheless, the ability of the primer 3’terminus to form a few base-pairs with the RNA template is apparently sufficient to initiate elongation. Telomerase will also add telomeric repeats to a 3’ terminus lacking any complementarity to the telomerase RNA template, providing a tract of G residues is present somewhere within the primer to recruit t e l o m e r a ~ e ( * ~ ~ ~ ~ ) (Fig. 3). The Euplofes telomerase can be efficiently recruited to and extend a non-telomeric DNA sequence carrying a single cluster of three dG residues located more than 30 nucleotides away from the primer 3’ terminus (Melek, Greene and Shippen, manuscript submitted). Presumably, binding of the upstream dG cluster by the anchor site deliv- ers the non-telomeric 3’ terminus into the template site for polymerization. Primer elongation is initiated in the absence of Watson-Crick base-paired alignment between the primer 3’ terminus and the RNA template (see below).

Taken together these findings demonstrate that telomerase has relatively relaxed sequence requirements for DNA in vitro. Work in several laboratories indicates that telomerase will extend random single-stranded DNA oligonucleotides only under very high primer concentrations (ref. 29; Melek, Greene and Shippen, manuscript submitted). How- ever, recent kinetic analyses revealed that the Tefrahymena

Primer:

5 3 I >

I I I

Predicted Interaction Extension by with Telomerase: Telomerase:

Anchor siteflemplate site + Template site + Anchor site +

* -

Anchor site(?)Kemplate site +

Fig. 3. DNA recognition by telomerase. Primer interactions with the telomerase anchor and RNA template sites determine the efficiency of recognition and extension. Open boxes depict G-rich telomeric sequences, narrow lines represent non-telomeric sequences and arrowheads represent primer 3’ termini. Blunt ended double-stranded primers are not extended by telomerase in vitro. ‘Under some conditions, telomerase will extend a DNA primer lacking any telomeric sequence (see text for details).

telomerase can efficiently add telomeric G4T2 repeats to DNA primers lacking any G-residue stretch, but which corre- spond to the target site for de novo telomere addition in vivo (H. Wang and E. Blackburn, personal communication). This observation suggests that telomerase has an intrinsic sequence-independent DNA binding property that allows it to accomplish de novo telomere addition. Additional studies are required to understand how telomerase recognizes DNA and to elucidate the mechanism and functional contribution of DNA interaction at the template and anchor sites.

Developmentally programmed chromosome healing The bulk of our knowledge concerning de novo telomere formation comes from studying organisms that undergo developmentally programmed chromosome fragmentation and healing. At the molecular level, the best characterized examples are the ciliated protozoa. Ciliates figure very prominently in the telomere field because of their unusual genomic arrangement and exceptionally large number of telomeres. For example, hypotrichous ciliates such as Euplotes contain approximately 10 million telomeres, the majority of which are formed de novo during the sexual process of ~onjugation(~4).

The genome of ciliates comprises two distinct nuclei within each cell, the transcriptionally silent germline micronucleus and the transcriptionally active somatic macronucleus. During conjugation, a copy of the micronu- clew is processed to generate a new macronucleus in a series of developmentally controlled events that include massive site-specific chromosome fragmentation and DNA elimination, telomere formation and many rounds of DNA replication(44). The resulting macronucleus comprises thousands, and in some cases, millions of linear DNA molecules that are capped by telomeres. Cis-acting determinants of chromosome breakage have been proposed for

Euplotes(45) and precisely defined in Te t rah~mend~~) . Chromosome breakage sequences (CBS) are typically very A/T rich and they, and their surrounding DNA, bear no resemblance to telomeres. Moreover, oligonucleotides corresponding to the CBS are not elongated by ciliate telomerases in vitro (M. Lee and E. H. Blackburn, personal communication; M. Melek and D. Shippen, unpublished).

The mechanism of telomerase recruitment to new chromosome ends generated from developmentally programmed chromosome fragmentation is unknown. Studies in Tetrahymena showed that the two processes, chromosome breakage and de novo telomere formation, are temporally linked(46). Given telomerase’s relatively lax sequence specificity, it is conceivable that a newly generated chromosome terminus placed in the vicinity of the enzyme active site could become a substrate for polymerization (see below). One possibility is that telomerase is part of a multi-subunit complex, one component of which binds the CBS element and cleaves the DNA. Telomerase, the second component, elongates the broken chromosome end with telomeric repeats. In a variation of this model, telomerase could be directly involved in chromosome breakage through its inher- ent endonuclease activity. If telomerase cut DNA at the CBS, the newly formed 3’ terminus would automatically be placed within the enzyme active site for telomere elongation (Melek, Greene and Shippen, manuscript submitted). Since regulated chromosome fragmentation has not yet been achieved in vitro, both of these models remain highly speculative.

De novo telomere addition in ciliates is extremely precise. Euplotes macronuclear telomeres, formed following developmentally programmed chromosome fragmentation and healing, always initiate with the sequence GGGGTTTT, only one of the eight possible permutations of the TTTTGGGG telomeric repeats found in this organi~m(~7). How is such precision in telomere synthesis achieved when the chromosome end cannot base-pair with the telomerase RNA template? A priorione might predict that a non-telomeric 3’ end would be randomly positioned within the enzyme active site for the first round of elongation. In vitro experiments indicate that this is not the case. Recall that telomerase can elongate a non-telomeric %terminus in vitro (Fig. 3). Recent studies indicate that the Oxytricha and Euplotes telomerases add a ‘default’ register of telomeric repeats to non-telomeric 3’ ends (Melek, Greene and Shippen, manuscript submitted)(48) (Fig. 4). The permutation of repeats synthesized matches exactly the sequence added to fragmented chromosomes in vivo. This ‘default’ positioning of a primer 3’ terminus on the RNA template effectively bypasses the requirement for Watson-Crick base-paired template alignment and may increase the efficiency of the initial round of DNA elongation (Fig. 4).

Although telomerase enzymatic activity and RNA levels peak in macronuclear development at the time of chromosome fragmentation and de novo telomere formation(49850), the enzyme is expressed at all stages of the ciliate life cycle.

This observation may explain why telomere length in ciliates is maintained over hundreds of cell divisions, while humans undergo progressive telomere loss. The constitutive expression of ciliate telomerases implies that chromosome healing might be possible during asexual as well as sexual stages of cell growth. Experiments in Paramecium support this prediction. Linear DNA molecules, comprising prokaryotic vector sequences microinjected into the macronucleus of Paramecium, acquire telomeres and are stably maintained(51). These results not only imply that de novotelomere formation occurs during vegetative growth, but also that no cis-acting signals are required to initiate telomere synthesis. Paramecium may be unusual in its ability to add telomeres so promiscuously, however. Linear DNA molecules do not acquire telomeres when microinjected into Tetrahymena unless they contain a CBS sequence and are fragmented by the cellular machinery(46). Clearly even in ciliates, the best characterized systems for studying chromosome healing, further experimentation is required to determine how telomerase becomes localized to a break site in vivo.

Ciliates are not unique in their ability to undergo develop-

Fig. 4. Default positioning of DNA by telomerase. In vitro, a distal telomeric sequence (shaded blue box) can efficiently recruit telomerase to a non- telomeric 3' end (grey line), presumably through DNA anchor site interactions. The non-telomeric 3' end is delivered into the enzyme active site to a default position (underlined). Default positioning on the Euplotes RNA template results in the addition of the sequence dGGGGTnT to the primer. The distance between the upstream telomeric sequence and the 3' terminus can be substantial with no effect on primer positioning within the RNA template (Melek, Greene and Shippen, manuscript submitted).

mentally programmed chromosome fragmentation and healing. Other examples include nematodes, crustaceans and insects(52). In the presomatic cells of the early Ascaris embryo, chromosomes are fragmented and approximately 25% of the germline DNA is eliminated. The broken chromosomes are stabilized by the acquisition of TTAGGC telomeric repeats(53). Unlike the situation in many ciliates, DNA cleavage in Ascaris initiates within a region of approximately 3-4 kb. This region does not contain any interstitial telomeric sequences nor can any discernible CBS be detected. Mole- cular analysis of one break site revealed that telomeric DNA had been added to a site containing two dG residues(53). These residues may have been sufficient to align the chromosome terminus onto the telomerase RNA template for chromosome healing (see below).

Spontaneous chromosome breakage and healing When a double-strand break occurs in a genome, it must be repaired or the cell dies. In most organisms, the only available evidence for terminal chromosome deletions is through detection of aberrant karyotypes that are successfully prop- agated, primarily as a result of de novo telomere formation. In such cases, chromosome breakage and healing are uncoupled. Thus, in contrast to developmentally programmed chromosome healing, these breakage events are spontaneous and are captured only by rare chromosome healing. Nevertheless, chromosomes carrying terminal deletions are relatively common in some plants. Wheat and barley stocks bearing a multitude of terminally deleted chromosomes have been successfully maintained for many genera t i on~(~~1~~) . In sifu hybridization studies using probes directed against the plant telomere sequence TTTAGGG revealed that the broken chromosomes have been capped by t e l ~ m e r e s ( ~ ~ J ~ ) . Thus, chromosome healing accounts for the long-term maintenance of the aberrant chromosomes.

De novo telomere formation is associated with only a subset of tissues in higher eukaryotes. Presumably this is because telomerase expression is developmentally regulated. As mentioned earlier, studies with human telomerase show an expression pattern confined primarily to the germline. Numerous reports in plants, dating back to Bar- bara McClintock's early work, are consistent with telomerase expression being limited to the germline and early develop- mental stages. Broken maize chromosomes can be healed in the embryo, but not in the endosperm(*). More recent plant studies indicate that de novo telomere formation occurs only during gametogenesis and early zygote development(54). When telomerase activity is identified in plants, it will be inter- esting to determine whether it's expression pattern corre- lates with the apparent inability to heal broken chromosomes at all stages in the plant life cycle.

The chromosomes of the malaria parasite, Plasmodium falciparum, are exceptionally fragile. Spontaneous chromosome breakage happens frequently in clinical, asexually maintained isolates of this organism. In contrast, there is no

evidence for chromosome fragmentation and healing in field isolates of Plasmodium, which rely on both sexual and asexual multiplication(57). As with plants, the broken chromosomes are retained because telomeres have been added(58). Although telomerase activity has not yet been reported for Plasmodium, these observations strongly suggest that telomeres are maintained by telomerase and that the enzymatic activity is expressed during asexual parasite growth. Molecular analysis of chromosome breaks in this organism uncovered two classes of chromosome ends: the first do not exhibit any complementarity to the predicted telomerase RNA template(57) and the second do(59) (Fig. 5A). We predict that the Plasmodium telomerase can accom- modate both types of breaks. The first class of chromosome healing events were uncovered in a study of the P f l l - l gene(57). In 15 examples, telomeres were added to sites that lacked any complementary to the predicted Plasmodium telomerase RNA template. Interestingly, in all but one case, the telomere initiated with the same permutation of telomeric repeats(57) (Fig. 5A). The precision of these healing events is similar to that observed during developmentally regulated de novo telomere formation in ciliates. Perhaps the Plasmod- ium telomerase utilizes a ‘default’ mechanism to initiate the first round of telomere synthesis on DNA molecules lacking complementarity to the RNA template. In the second instance, observed at sites of de novo telomere formation in the Pf332 and P f87gene~(~~) , the register of repeats added to the break site varied. However, in all cases a few nucleotides of complementarity to the Plasmodium RNA template were found at these junctions. Given telomerase’s ability to extend primers bearing a few 3’ nucleotides of complementarity to the RNA template(30), it seems likely that these residues may be sufficient to prime telomere synthesis. For example, a broken chromosome ending in 5’ TCAG 3’ is predicted to hybridize to the complementary 5’ CUGA 3’ in the RNA template and be extended by dGGTTCA to initiate synthesis of the GGGTT(T/C)A sequence (Fig. 5A).

Several human genetic disorders arise as a result of terminal chromosomal deletions and concomitant loss of telomeres(60-62). The most notorious case involves telomere healing of a break in the distal region of chromosome 16q near the a-globin locus. This truncation results in a-thalassemia. The first molecular analysis of the break site was conducted by Wilkie and co-workers, who found that telomeric repeats had been added directly adjacent to the broken end(62). The telomere addition site displayed sequence similarity to human telomeres, containing a terminal GTT and a longer G- rich stretch located approximately 10-25 nucleotides internal to the break. Subsequent examination of chromosome 16q truncations uncovered additional examples of break sites. In all but one case, the break site contained 3-4 nucleotides of complementarity to the human telomerase RNA templating domain(63) (Fig. 5B). To test whether the break site would be a suitable primer for the human telomerase, a single- stranded DNA oligonucleotide corresponding to the telomere

Fig. 5. Spontaneous chromosome healing by telomerase. (A) Chromosome fragmentation in Plasmodium results in two types of chromosome ends. (i) Break sites with no sequence complementarity to the RNA template acquire the sequence dAGGGTlT/C(5g). Delivery into the enzyme active site may occur via the default mode. (ii) Broken ends with complementarity to the RNA template can be aligned onto the RNA and elongated as shown. DNA sequences at the broken chromosome ends are shown in blue uppercase lettering, the RNA template in bold uppercase and nucleotides added to the broken end are shown in lowercase italicized blue lettering. Plasmodium telomeres are a mixture of AGGGTTT and AGGGTTC repeatd73). It is unknown whether this IS due to primer slippage on the RNA template or misincorporation of T/C nucleotides. A templating domain for Plasmodium is shown to account for both possibilities. (B) Break sites on human chromosome 1 6(62,63) and their predicted alignment on the telomerase RNA template(12) are shown. As few as one or two nucleotides of complementarity to the RNA template are sufficient to initiate synthesis of TTAGGG repeats.

addition site was assayed for elongation by telomerase in vitro. The human enzyme extended this and similar oligonucleotides, requiring only 2 to 4 nucleotides of %terminal complementarity to the RNA template(30).

It has been estimated that short stretches (2-4 bp) of complementarity to some subset of the human telomerase RNA template occur every 10 nucleotides in the human genome(63). Thus, an appropriate substrate for telomerase may be readily uncovered upon chromosome breakage, with de novo telomere formation limited solely by the presence or absence of telomerase. This model is probably too simplistic. In vitro telomerase extends only 3’ single-stranded over- hangs and is not able to elongate blunt-ended duplex DNA(64). Therefore, in addition to complementarity to the telomerase RNA templating domain, the site of new telomere formation must exist as a 3’ overhang. It has been suggested that a 5-3’ exonuclease eliminates DNA from a chromosome

end to generate an appropriate o ~ e r h a n g ( ~ ~ 3 ~ ~ ) . However, for truly efficient recruitment of telomerase to broken ends, frans- acting factors are undoubtedly r e q ~ i r e d ( ~ ~ , ~ ~ - ~ ~ ) . Chromo- some healing at the human a-globin locus probably reflects an extremely rare event in the germline that was maintained only because telomerase is present at high levels there.

Artificial chromosome fragmentation and healing Spontaneous chromosome healing can be artificially induced in mammals and by introducing DNA containing a selectable marker and a telomere ‘seed’ sequence (multiple copies of a telomere repeat sequence). Recombination of the exogenous DNA into the genome results in DNA integration and chromosome fragmentation. In this reaction, the terminal portion of the resident chromosome is eliminated so that a new chromosome end forms and is stabilized by chromosome healing.

Experiments in yeast and humans indicate that induced telomere formation is strictly dependent upon the presence of the internal telomere seed sequence, although this sequence may be located more than 100 base pairs upstream from the healed site(66,67s70). The telomere seed sequence in yeast need not be a perfect match to yeast telomeres. By contrast, the requirements for new telomere formation in human cells are much more stringent. In this case, the telomere seed must consist of the naturally occur- ring telomere repeat sequence (TTAGGG)n; other telomerelike sequences do not substitute(67). Thus, de novo telomere formation in human cells requires interactions of exquisite sequence-specificity. While the human telomerase does not display such strict sequence requirement^(^^^^^), the mammalian telomere repeat binding factor, TRF, does(’ 1,67).

Binding of trans-acting factors such as TRF may be needed to recruit telomerase efficiently to nascent chromosome ends. Alternatively, TRF binding to the seed sequence may prevent complete integration of the incoming DNA at an internal site in the chromosome. Blocking the second cross- over event may provide telomerase with enough time to heal the new end. It should be noted that artificial chromosome healing events such as these have only been successful in transformed cells where telomerase activity is high.

Examination of artificial chromosome healing sites shows that the telomere is added to DNA bearing 1-4 nucleotides of complementarity to the RNA template(66s71,72). In S. cerevisiae, as with ciliated protozoa, new telomeres initiate with a precisely defined register of telomeric sequence(66). This finding proved extremely valuable in understanding the mechanism of telomere synthesis in yeast. Yeast telomeric DNA comprises irregular repeats of (TGI-~)~, sequences not readily generated via current models for telomerase-medi- ated telomere synthesis. Thus, prediction of an RNA templating domain for a yeast telomerase RNA was difficult. Kramer and Haber’s observation that yeast telomeres formed de novo initiate with a specific 11-nucleotide

sequence suggested a possible sequence for the RNA template@). One year later, the S. cerevisiae telomerase RNA subunit was cloned and shown to carry this 11 -nucleotide sequence core within the templating domain(17).

Although artificially induced chromosome healing events are relatively rare, they can be selected by genetic means. Therefore, this technique will be useful in unraveling the mechanism of spontaneous chromosome healing and determining the role that trans-acting factors play in recruit- ing telomerase to chromosome ends in vivo.

Conclusion Chromosome healing occurs both spontaneously and as part of developmentally regulated programs of DNA rearrangement. Recent studies of the telomerase enzyme activities and substrate utilization provide us with insight into the mechanism of telomere addition onto broken chromosome ends. However, precisely how the telomerase enzyme is recruited to these sites in vivo awaits further investigation.

Acknowledgements We thank Titia de Lange and Jeff Kapler for critically reading the manuscript and for insightful comments. The work car- ried out in my laboratory is supported by the National Insti- tutes of Health (GM49157), the American Cancer Society (JFRA-468) and a grant from GlaxoWellcome, Inc.

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8861-8865.

USA 88,7006-701 0.

Meni Melek and Dorothy E. Shippen* are at the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA. E-mail: dshippen Q bioch.tamu.edu *Corresponding author.

chromosome healing: spontaneous and programmedde novo telomere formation by telomerase

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