Clues to catastrophic telomere loss in mammals from yeast ... - Nature

PERSPECTIVES 30. Heintz, N. BAC to the future: the use of bac transgenic mice for neuroscience research. Nature Rev. Neurosci. 2, 861–870 (2001). 31. Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003). 32. Pasyukova, E. G., Vieira, C. & Mackay, T. F. Deficiency mapping of quantitative trait loci affecting longevity in Drosophila melanogaster. Genetics 156, 1129–1146 (2000). 33. Ma, R. Z. et al. Identification of Bphs, an autoimmune disease locus, as histamine receptor H1. Science 297, 620–623 (2002). 34. Vivian, J. L., Chen, Y., Yee, D., Schneider, E. & Magnuson, T. An allelic series of mutations in Smad2 and Smad4 identified in a genotype-based screen of N-ethyl-N-nitrosourea-mutagenized mouse embryonic stem cells. Proc. Natl Acad. Sci. USA 99, 15542–15547 (2002). 35. Vogel, G. Scientists dream of 1001 complex mice. Science 301, 456–457 (2003).

Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink Mtap1a | Tub OMIM: http://www.ncbi.nlm.nih.gov/omim cystic fibrosis

FURTHER INFORMATION Complex Trait Consortium: http://www.complextrait.org Complex Trait Consortium 2003 Meeting: http://www.well.ox.ac.uk/~rmott/CTC Genetic Mapping Software: http://mapmgr.roswellpark.org/qtsoftware.html Genomics: a global resource: http://genomics.phrma.org/lexicon/r.html Mouse–Human Homologies: http://www.informatics.jax.org/reports/homologymap/mouse_ human.shtml Mouse Nomenclature: http://www.informatics.jax.org/mgihome/nomen Mouse Phenome Database: http://www.jax.org/phenome Mouse SNP Database: http://mousesnp.roche.com/cgibin/msnp.pl Neurogenetics at University of Tennessee Health Science Center: http://www.nervenet.org R/qtl: a QTL mapping environment: http://www.biostat.jhsph.edu/~kbroman/qtl Rat Genome Database: http://rat.lab.nig.ac.jp/qtls SNPview: SNPs, SSLPs, alleles and haplotypes: http://www.gnf.org/SNP Software for QTL data analysis: http://www.stat.wisc.edu/~yandell/qtl/software The Mouse Brain Library: http://www.mbl.org The WebQTL Project: http://www.webqtl.org/search.html University of Wisconsin-Madison Department of Statistics: http://www.stat.wisc.edu Access to this interactive links box is free online.

OPINION

Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion Arthur J. Lustig Catastrophic losses of telomeric sequences have recently been described during apoptosis, senescence and tumorigenesis in murine and human cells, in ataxia telangiectasia patients and in immortalized cells in which telomerase is inactive. A mechanism that underlies a single-step non-reciprocal telomere deletion called telomere rapid deletion in Saccharomyces cerevisiae might provide clues for future studies of catastrophic telomere loss in higher eukaryotes.

Eukaryotic chromosome stability relies on the presence of intact telomeres, which are the protein–DNA complexes that are present at chromosomal termini. Telomeres are replicated by telomerase. This unique ribonucleoprotein complex uses its RNA template to elongate the telomere by the addition of G-rich TELOMERIC REPEATS to the terminal 3′ overhang — a mechanism that is ubiquitous among eukaryotes1. Secondstrand synthesis depends on the standard

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DNA polymerase machinery, including DNA primase and polymerase I, but is nonetheless coordinated with telomerase addition2,3. Notably, although telomerase is ubiquitous in all single-celled organisms, telomerase activity in human cells is restricted primarily to the germ line and to highly proliferative cells4. Telomere stability is conferred by a protein–DNA structural cap that protects the terminus in many organisms (including yeast and humans) from nonspecific factors and allows the regulated access of specific enzymes that are necessary for chromosome function. Telomere size is controlled by mechanisms that produce an inverse relationship with telomerase activity, which leads to a steady state5. This balance is achieved in part through a mechanism that ‘counts’ telomere-binding proteins (Rap1 in yeast and TRF1 in human cells) and presumably affects changes in chromatin structure6 and accessibility to telomerase5,7–9. A variation on this theme occurs in somatic cells that

lack telomerase. These cells are characterized by a regulated ‘disequilibrium’ between the mechanisms of sequence loss owing to incomplete SEMI-CONSERVATIVE REPLICATION and the partial protection that is afforded by the telomere cap. This leads to a slow decrease in telomere size, loss of terminal DNA structures and the consequent DNA-DAMAGE CHECKPOINTS that put a stop to TELOMERIC TRACT 10 ATTRITION and cell growth . Telomere size is also affected by specific recombination activities. These include a unique recombinational single-step deletion process that was identified in our laboratory. This process, termed telomere rapid deletion (TRD), is another element of the telomeresizing machinery. Recent discoveries of telomere deletion in mammalian cells (TABLE 1) have sparked interest in the possible mechanistic and functional similarities between the two processes. This article is intended to stimulate investigators to explore new avenues into these deletion events, which were first described in single-celled eukaryotes. This represents a starting point and impetus for future research that will probably take us into unexpected and unanticipated areas of telomere regulation. Telomere rapid deletion in yeast

TRD was first discovered during our studies of the telomere-binding protein Rap1. Deletions of the 144 amino acids at the C-terminal (rap1t alleles) not only increased average telomere size but also rapidly gave rise to a highly heterogeneous distribution of telomere sizes11. Closer examination of individual marked telomeres showed that in the mutant, telomeres were both increasing in size and undergoing abrupt decreases ranging from 200 base pairs (bp) to several kilobases (kb). Deletion was observed for telomeres that were only 400-bp longer than the wild-type 300-bp tract. TRD did not depend on the C-terminus of Rap1, as elongated telomeres that were introduced into wild type and rap1t-mutant cells conferred identical deletion rates. Remarkably, both the precursor and the deleted product (sometimes in equal proportion) could be seen in cultures that were derived from an individual cell, which indicates that TRD represents a unique single-step non-reciprocal deletion in these haploid strains12 (FIG. 1). TRD is distinct from the average replicative loss of 4–5 nucleotides per population doubling (the equivalent process in human cells removes 100–200 bp13). Notably, unlike rap1t cells in which deletion size seems to be random, deletions of elongated telomeres in

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PERSPECTIVES wild-type cells shortened them to wild-type size or to the size of most of the telomeres. The precision of TRD is therefore governed by the sizes of the telomeres. Also, TRD is much more efficient during pre-meiotic DNA synthesis and HOMOLOGY SEARCHING — there is a 20% chance per telomere that a TRD event will occur during meiosis, which indicates that TRD might epigenetically reset telomere size during meiosis (D. Jia and A. J. Lustig, manuscript in preparation). This effect might be related to the requirement for the correct spatial organization of telomeres in meiotic cells for normal telomere pairing and telomeric recombination, as shown in both budding yeast and the fission yeast Schizosaccharomyces pombe (REFS 14–16 and J. Cooper, personal communication). Physical evidence for a TRD t-loop model. The analysis of telomeres that had been marked with single individual restriction sites (introduced into the telomere by transient expression of a mutant telomerase RNA that encoded an HaeIII site in its template sequence) provided a better mechanistic insight into TRD17. Using these telomeric markers, alterations in telomeric DNA can be analysed without disrupting telomere function. The study that followed the fate of single HaeIII sites between chromosomes and in a chromatid showed that interchromosomal recombination occurs rarely, if at all, and is not associated with TRD. Rather, deletion is initiated at the telomeric terminus and invades sequences that are proximal to the terminus (FIG. 1). The most parsimonious hypothesis to explain these results is that TRD is the consequence of intrachromatid deletion, which is initiated by strand invasion of the 3′ overhang into distal sequences, followed by the formation of an intermediate that is identical to the t-loop structures that have been identified in human cells (FIG. 1). I propose that in yeast, this transient t-loop is resolved to form the terminally deleted telomere. However, neither the intermediate nor the non-replicating linear or circular end product has been physically identified in yeast. The presence of t-loops has been confirmed in human and murine cells by electron microscopy and their role might be to protect the telomere18. This DNA structure is formed by the invasion of the 3′ overhang into proximal sequences, which leads to a small displaced single-stranded D-loop (FIG. 1). Similar structures have not yet been observed in yeast and it has been proposed that this is the result of potential topological constraints in the formation of small t-loops. However, the shorter size of yeast telomeres (300 bp)

NATURE REVIEWS | GENETICS

compared with human telomeres (3,000 bp) should not pose a structural constraint because 150–800-bp t-loops can be readily found in TRYPANOSOMES19. Also, replication studies have shown that circles as small as 80 bp can be replicated efficiently, at least in vitro 20. Genetic requirements for TRD. TRD depends on Rad52, which is a protein that is essential for most yeast mitotic and all meiotic recombination. Interestingly, TRD is enhanced in cells that lack hyper-recombination protein 1 (Hpr1) (REF. 12), the absence of which leads to high rates of intrachromosomal recombination21. This finding raised the possibility that TRD is mediated through intrachromosomal interactions. Curiously, the heterochromatin protein Sir3 is important for maintaining the precision of TRD, possibly by mediating clustering among telomeres12. Yeast meiotic recombination 11 homologue A (Mre11) and Rad50 are components of the Mre11/Rad50/Xrs2 (MRX) complex, which is an important regulator of homologous recombination, including telomeric

recombination 22. The finding that both proteins are required for TRD provides further support for a recombination model23. Importantly, the human orthologous MRE11/RAD50/NBS1 (MRN) complexes associate with telomeres in vivo through their interaction with the telomere-binding protein TRF2, which is a mediator of t-loop formation24. The absence of a yeast orthologue of TRF2 indicates that yeast t-loop structures might form only inefficiently and transiently. The yeast Ku70/Ku80 heterodimer acts as a gatekeeper against deleterious processes such as nuclease digestion and recombination, which allows TRD to occur, albeit at a lower rate25. A secondary deletion mechanism in yeast. A second inefficient and poorly understood RAD52-independent pathway for TRD has been described in yeast17. This pathway is probably the consequence of either specific or nonspecific nuclease activity at the chromosomal terminus. One characteristic of Rad52dependent TRD is the high level of precision of deletion size. Telomeres return to the size of

Table 1 | Examples of telomere rapid-loss events in mammals Gene

Mutation/ condition

Deletion

Telomeric fusions

Source

Other

Mre 11

Null, E

NA

NA

Murine

TRF2 association

22,54

Rad50

K22M(S)

+

+

Murine

53

Null, E

NA

NA

Murine

TRF2 association TRF2 association

NBS1

Non-null

(+/–)

+

Human

TRF2 association

53,54

ATM

Null

NT

+

Murine

22,42

Null

NT

+

Human

Circle production Circle production

p53

Null

NT

Suppress RAD50(S/S)

Murine

T-loop binding

References

50

22,42 56

RB

Rb94∆N

+++

++

Human

NR

55

Ku70

Null

+

NT

Murine

Telomere association

61

Ku86

Null

+ with Ku70–/–

++ with Ku70–/–

Murine

Telomere association

63

TRF2

Null, E

NT

NT

Murine

45

∆B, ∆M, DN

+

+

Human

T-loop association T-loop association

EST1A

Reduced abundance

+

NT

Human

Telomere SS binding

‡

RAP1

∆BCRT ∆Myb

H

–

Human

TRF2 association

48,49

47*

*Additional data from D. Brocolli and T. de Lange, personal communication. ‡Data from C. Azzalin and J. Lingner, personal communication. ATM, ataxia telangiectasia mutated; DN, dominant negative; E, essential; EST1A, telomerase subunit EST1A; H, extreme telomere heterogeneity; MRE11, meiotic recombination 11 homologue A; NA, not applicable; NBS1, Nijmegen breakage syndrome 1; NR, not reported; NT, not yet done; RAP1, repressor/activator protein 1; Rb, retinoblastoma 1; SS, single strand; TRF2, telomeric repeat binding factor 2.

VOLUME 4 | NOVEMBER 2003 | 9 1 7

PERSPECTIVES a

Strand invasion

b MRX

Branch migration Formation of Holliday junction

c TRD

Resolution of Holliday junction Nicking in the D-loop D-loop degradation

d

are responsible for the Rad52-independent pathway trim or cleave telomeric DNA near a nuclease-hypersensitive site, the location of which is governed by telomere clustering. This activity could explain the precision of both RAD52-dependent and independent TRD. Relationship between type II recombination and TRD. Both TRD and the rapid elongation that is conferred by telomere–telomere recombination (TYPE II RECOMBINATION), which occurs in the survivors of cells that lack telomerase, require the MRX complex. This indicates that the processes share common mechanistic elements 23,26–29. Interestingly, multiple rounds of replication through the t-loop (‘rolling loop’ replication) would predict the formation of the rapid elongation that is typical of type II recombination (FIG. 2). Indeed, such a mechanism for elongation might be similar to the proposed rolling-circle replication of small excised telomeric circles to elongate telomeres in yeast (M. McEachern, personal communication). Hence, in some circumstances, TRD and type II recombination might represent different outcomes of a common t-loop intermediate (FIGS 1,2).

Re-examining telomere loss

Recent studies have shown large-scale losses of telomeric DNA that are distinct from attrition owing to replicative loss (~150-bp per population doubling 34). So, do these mammalian deletion events share mechanistic features with TRD? The answer to this question depends on the

a

Strand invasion

b MRX

Branch migration Formation of Holliday junction Type II recombination

c

e

Figure 1 | A working model for telomere rapid deletion. a | Both strands of the telomeric double-stranded DNA are shown, including the 3′ overhang with the G+T-rich strand (red). b | Overhang sequences with or without further rescission invade proximal telomeric tract sequences; this results in a displacement of DNA to form a D-LOOP and leads to a structure that is identical to the T-LOOP. Both the telomerebinding protein Rap1 (grey ovals) and the Mre11/Rad50/Xrs2 (MRX) complex are shown. c | Following branch migration, the D-loop is nicked and the resulting HOLLIDAY JUNCTION is resolved by cleavage of the outer strands. d,e | The D-loop is subsequently degraded, which leads to a structure that resolves into two products: a deleted telomere and a circular or linear product (e). TRD, telomere rapid deletion.

most of the telomeres, which points to the presence of a ‘yardstick’ that is mediated through association among telomeres. Surprisingly, Rad52-independent deletions also produce wild-type-size products. Given the rarity of mechanisms for maintaining wild-type size, these data imply that the two pathways might share a similar yardstick mechanism. One unifying, albeit speculative, hypothesis is that the putative nucleases that

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Relationship between type II recombination and ALT. Before proceeding, we need to distinguish between some possible differences between recombination pathways for telomere elongation in yeast and humans30. Although superficially similar, yeast type II recombination is normally observed after telomeres become sufficiently short to provoke a G2/M ARREST (not death) in senescence31,32. However, type II recombination occurs in a fraction of cells even before senescence ensues — a probable checkpoint response, albeit inefficient — to induce recombination before cellular crisis23. Indeed, in some species, such as Candida albicans, both telomerase and type II recombination are robust in wild-type cells33. By contrast, cells that survive the chromosomal crisis that occurs in cultured human post-senescent cells do so after the formation of short non-functional telomeres. Cells that bypass senescence undergo many genomic rearrangements and, in a few cases, are able to use an alternative mechanism of telomere–telomere recombination to elongate telomeres. Hence, although it is probably mechanistically similar, the interpretation of the ALT pathway in immortalized cells and tumours — which involves recombination between telomeric sequences and leads to telomere elongation in the absence of active telomerase — must take into account the possibility of further mutations and/or rearrangements.

DNA replication Second-strand synthesis

d

e

Horizontal resolution of Holliday

f

Figure 2 | Type II recombinants formed through rolling-loop replication. a,b | These steps are identical to those in FIG. 1 and both strands of the double-stranded DNA are shown. c | Following strand invasion, semi-conservative DNA replication elongates the invading strand with the concomitant formation of a larger D-loop. d–f | Following DNA replication through the loop (d) and lagging-strand DNA replication using the D-loop as a template, a structure is formed (e) that resolves into a telomeric DNA that is extended by the length of the D-loop (f), which can then re-enter the cycle. Re-invasion of the 3′ end after one round of DNA replication could give rise to a rolling loop in which t-loop-sized extensions are formed in each round. MRX, MreII/Rad50/Xrs2 complex.


PERSPECTIVES definition of TRD. On the basis of studies in yeast, we can define TRD as an end-mediated intrachromatid homologous-recombination event that is mediated through a transient t-loop intermediate that, when resolved, forms the deleted telomere and a linear or circular by-product (FIG. 1). Given the recombinational nature of this process, we would expect that gene products such as RAD52 and X-ray cross complementation 2 (XRCC2) in human cells might be partially or completely required for the deletion. Of course, more than one mechanism is probably involved in mammalian telomere loss. Below, I propose that TRD explains many of the features of the sudden losses of telomeric sequences in mammals. Although no processes in mammals have been sufficiently investigated to evaluate these criteria, the above definition of TRD serves as a comparative standard for interpreting both the physical characteristics of spontaneous or induced deletions and the phenotypes that are conferred by mutant gene products, which are predicted to have a strong influence on TRD mechanisms.

“…TRD explains many of the features of the sudden losses of telomeric sequences in mammals.” Catastrophic telomere loss. One characteristic feature of TRD is the presence of deleted telomeres that are derived from cloned yeast or human cells that contained a longer telomeric tract at an individual telomere. Indeed, the phenotypic similarity between the clonal deletion patterns that were initially observed in human ALT cell lines35 and yeast TRD11,17 is notable. TRD precision is lost in both rap1t mutant cells and ALT cells — both form deletion products over a broad range of sizes (see below). Similar losses of telomeric sequences from individual telomeres have been observed in cultured human somatic cells, which lack telomerase, as a function of population growth before senescence36, at the point of p53- and retinoblastoma 1 (Rb)checkpoint mediated growth arrest10. Interestingly, the telomere-loss data fit the theoretical prediction of several mechanisms (for example, replicative loss and rapid telomere loss)37,38. Similarly,‘ultra-short’ telomeres have been observed in primary human cells that are in REPLICATIVE SENESCENCE39. Further


telomere deletions have been recovered from among the elongated telomeres that form after the fusion of ALT and cells that contain active telomerase40. Recent data have indicated that a catastrophic loss of telomeric sequences might, in some cases, precede the apparent onset of DNA damage-induced apoptosis in the absence of both telomerase and the checkpoint regulator p53 (REF. 34). Telomeric loss in the range of 6 kb preceded two early events during the induction of apoptosis: depolarization of the mitochondrial membrane and the induction of caspase-mediated degradation. These data provoked speculation that rapid large-scale loss of telomeric sequences might be a trigger for apoptosis. However, the possibility that an early susceptibility of telomeres to apoptotic nucleases is responsible cannot yet be excluded41. One of the expected products of TRD is an excised circle or linear fragment that contains telomeric repeats (FIG. 1). It is therefore intriguing that extra-chromosomal telomeric DNA, as detected by two-dimensional gel electrophoresis and Q-FISH, is present in ALT cells, in cells that are derived from murine Atm–/– mutants and ataxia telangiectasia (AT) patients, and in a human hepatoma cell line42–44. These data are also consistent with the formation of the circular telomeric species that are among type II recombinants (M. McEachern, personal communication). However, the possibility that these DNA species might be the products of a more generalized genomic instability cannot be excluded. Although they have not been observed in yeast, the presence of other internal intrachromatid deletion events remains a viable possibility. Proteins that influence telomeric deletion. An array of genes has been implicated in mammalian deletions under a range of conditions, some of which might be mechanistically related to TRD. Human TRF2 facilitates the formation of t-loops in vitro45. The t-loop structure seems to have a protective function in vivo46, but might also be intimately associated with the deletion process18,45,47. The lack of an obvious yeast orthologue of TRF2 might explain the absence of stable t-loops in yeast, but does not eliminate the possibility that more transient t-loop structures could be recombination intermediates in TRD or in intrachromatid rolling-loop replication (FIG. 2). TRF2 might have a more direct role in a rapid telomere-loss process. Tethering of an amino-terminal truncated variant of TRF2 to individual human telomeres results in a tenfold increase in the loss of telomere

a Human

Cell-cycle regulation

Stable MRN

b Yeast Transient MRX

Yeast Rap1

Yeast Xrs2

Yeast/human RAD50

Human RAP1

Human NBS1

Yeast/human MRE11

TRF1

TRF2

Human POT1

Rad52

Rad59

Cdc13

Figure 3 | Proposed equilibrium states for yeast and human t-loop formation. This speculative view, which is not drawn to scale, shows both the equilibrium between the formation of the t-loop and open states in mammalian cells (a), and the putative t-loop equilibrium that we propose might be present in yeast (b). Both DNA strands of the duplex telomeric DNA are shown. In the case of the mammalian telomere, the t-loop structure is strongly favoured at specific points outside cell-cycle stages that would block the activity or loading of telomerase to the 3′ overhang. The telomere-binding protein TRF2 promotes the formation of the terminal t-loop structure. Further associations that are of importance to t-loop stability and formation are the binding of protection of telomeres1 ( POT1) to the single-stranded D-loop, and the recruitment of Nijmegen breakage syndrome 1 (NBS1) (and presumably the MRE11/RAD50/NBS1 (MRN) complex) and the repressor/activator protein 1 RAP1 by TRF2. By contrast, we envision that a yeast t-loop forms transiently and is associated with the recruitment of the Mre11/Rad50/Xrs2 (MRX) complex at the site of recombination, possibly acting as a sensor for the resolution of the t-loop through the Rad52 pathway. The equilibrium of this transient intermediate is likely to be strongly shifted towards other chromatin states in mitotic cells.


PERSPECTIVES

a Telomere attrition

b

Strand invasion

c

Rolling loop

d

Resolution

e T-loop

TRD

Figure 4 | A recombinational balancing act in telomerase negative cells. This highly speculative view, which is not drawn to scale, shows a crude mechanism of size control that might be maintained in human ALT cells or in yeast cells using the type II recombination pathway. Both strands of the duplex telomeric DNA are shown. For simplicity the dynamics of only the right telomere are shown (not drawn to scale). a | The elongated telomere, in the absence of other mechanisms of elongation, undergoes several rounds of replicative loss. b,c | Shorter telomeres are more recombinogenic, so they might be more likely to carry out the internal strand invasion that is necessary for t-loop formation (c). The shorter telomeres are then able to carry out strand invasion followed by semi-conservative DNA replication of one or more cycles. d,e | Ultimately, the molecule will be resolved to form the product of this rolling-loop mechanism: a hyper-elongated telomere (e). At a certain frequency, the identical t-loop structure will form the deletion product (TRD), together with circular or linear by-products that are not shown here. In this way, cells are prevented from carrying either telomeres that are overly long or, importantly, short telomeres that lack cap function. Small arrows refer to the direction of resolution either before or after replication.

sequences in the absence of telomerase. The increase leads to a loss of 400-bp per mean population doubling — an effect that is further accentuated by the overproduction of TRF2. This provides strong evidence for a direct role for TRF2 in a deletion process7. The TRF2-binding protein RAP1 acts as a negative regulator of telomere length, as does the C-terminus of its DNA-binding yeast orthologue48. RAP1, through its association with TRF2, is probably present at the t-loop (FIG. 3). Furthermore, deletion of either or both of the BCRT and myb domains of RAP1

920


decreases the heterogeneity of the telomeric tract only in the presence of telomerase. The authors of the latter study propose that, as in yeast11, human telomerase activity is balanced by TRD. A large decrease in TRD would be expected to lead to a less diffuse distribution, as observed in this study49. If mammalian deletion events have several roles in human cells, then the t-loop is probably a more dynamic state. This viewpoint is supported by the requirement for an accessible chromosome end in late S phase and the S phase-dependent association of

Nijmegen breakage syndrome 1 (NBS1) with TRF2 (REF. 24). Indeed, TRF2 might well modulate the equilibrium between the t-loops with different stabilities (FIG. 3). An interdependent functional relationship between TRF2 and the MRN telomerebinding complex is supported by the loss of MRE11 and RAD50 subnuclear localization in the absence of NBS1 (REF. 50), and the delocalization of telomeric RAD50 in human cells in which TRF2 is not bound to the telomere24. Furthermore, the orthologues of all of the components (RAD52, MRE11, RAD50, NBS1 and TRF2) that are required for TRD50–52 are localized in APBs — one of several promyelotic leukaemia (PML) bodies that correlate with instances of the ALT pathway of telomeric recombination50. Interestingly, the MRN complex also localizes to the telomeres of human meiotic cells51, which is an intriguing observation in light of the high rate of TRD in yeast meiotic cells. Strong evidence for telomere loss in the murine hypomorphic RAD50(S/S) HOMOZYGOUS MUTANT is shown by the frequent formation of fusions between chromosomes with short telomeres, which is consistent with a rapid loss of the originally elongated telomere53. Consistent with this viewpoint, a rapid rate of telomere loss in cell lines from Nijmegen breakage syndrome (NBS) patients (who carry a non-null NBS1 allele), might be caused by a combination of replicative loss, other mechanisms of attrition and an elevated rate of telomeric fusions (REF. 54 and Tauchi, unpublished data). Of course, given the many pathways in which the MRN complex is involved, telomeric-loss defects might represent a direct or indirect effect of the primary defect. An intriguing recent report found that a dominant-negative amino-terminal-truncated version of the checkpoint-control protein Rb94 enhances tumour-suppressor activity55. Remarkably, the introduction of this dominant-negative Rb into bladder cancer cells in which telomerase is active leads to rampant deletion, losing on average 3 kb of telomeric tract in 48 hours, and provokes genomic rearrangement, apoptosis and cell death55. Also, the checkpoint protein p53 associates with t-loops in vitro56. Taken together, these data highlight the importance of checkpoint control over the mechanisms of telomere loss. The yeast telomere-elongation protein Est1 binds to the terminal 3′ overhang, where it functions with cell-division control protein 13 (Cdc13) as a telomerase recruiter and/or modifier of telomerase activity57,58. Two laboratories have recently identified human orthologues of Est1 (REFS 59,60). Repression of one of these orthologues,


PERSPECTIVES EST1A, by small interfering (si) RNAs confers an increased frequency of rapid loss. Nonetheless, these results show that disruption of the extreme 3′ terminal end complex (the site of the putative invading strand) enhances mammalian telomere-loss events (REF. 59 and C. Azzalin and J. Lingner, personal communication).

“…the main question is whether deletion that is based on a recombination pathway is responsible for some or all of the deletions that have been documented in mammalian cells.” Interestingly, Ku70 deficiency in mouse cells confers a sudden loss of telomeric DNA as if TRD has been unleashed61, which is consistent with a role for telomeric Ku as an inhibitor of TRD. This conclusion is indicated by the increase in apparent deletions of ~20 kb in Ku70 (G22p1)–/– ES cells and in the first litter derived from mouse Ku70 –/– knockout strains61, as determined by Q-FISH telomeresize analysis. This result contrasts with what is seen in wild-type strains and with the slower 2–7-kb loss of sequences per generation that is seen in the murine telomerase RNA-knockout strains34,62. This finding is consistent with the vastly increased rate of TRD in yeast ku70null mutants. Although still controversial, these mammalian studies indicate that the murine Ku70/Ku86 heterodimer restricts rapid telomere attrition. In the combined absence of murine Ku86 and telomerase, telomere loss is further accentuated63 with ensuing apoptosis and telomeric fusions. These observations are remarkably similar to the phenotype of yeast cells that lack both Ku70 and telomerase64. These severe telomere losses, coupled with telomere fusion and apoptosis, point to a loss of redundant protective mechanism(s) in strains that lack both Ku and telomerase. TABLE 1 shows a summary of the proteins that are involved in telomere loss and their basic characteristics.

First, replication alone can result in a slow continual loss of telomeric sequences at any given telomere. In human cells, this loss occurs at ~150-bp per population doubling in vitro13. Of course, the rate of loss will then be directly linked to the number of population doublings. Second, exonucleases or endonucleases at individual telomeres of subclones of elongated progenitors would be expected to show a heterogeneous distribution of sizes, ranging from the inherited telomere size to shorter sizes. Such an effect has not yet been observed in mammalian cells. However, the loss of yeast Ku70 results in both increases in TRD and nuclease activity25. Although some of the events presented here (such as apoptotic telomere loss) are consistent with this possibility, the deletion of progenitor telomeres to subclones of smaller discrete telomere sizes points to other explanations. The third and most parsimonious model to fit much of the mammalian data is a variation on the TRD mechanistic theme that was developed in yeast and is analogous to some models that explain how ALT cells are generated30 (FIG. 1). Specifically, I propose that the


Potential implications of deletions

Mutations in terminal deletion components. Mutations in the human orthologues that are required for TRD, such as MRE11, RAD50 and the checkpoint regulator AT mutated (ATM), result in notable chromosomal rearrangements, telomere fusions and a greater propensity towards malignancy. In normal cells, ATM seems to functionally interact with the MRN complex at both the

Glossary D-LOOP

REPLICATIVE SENESCENCE

A displaced DNA single strand that is created after strand invasion.

The cessation of growth after several rounds of DNA replication, which leads to the slow loss of telomeric sequences owing to the lack of an RNA primer in the absence of compensatory mechanisms such as telomerase.

DNA-DAMAGE CHECKPOINTS

The stages of the cell cycle that monitor double-strand breaks and other forms of DNA damage. In wild-type cells, damage results in the arrest and repair of the defect. G2/M ARREST

The cessation of growth owing to the DNA-damage checkpoint at the boundary between G2 and mitosis in response to perceived DNA damage defects, such as blunt-ended telomeres. HOLLIDAY JUNCTION

A point at which the strands of two double-stranded DNA molecules exchange partners, which occurs as an intermediate in genetic recombination.

SEMI-CONSERVATIVE REPLICATION

The main form of DNA replication, which uses both strands as templates for DNA synthesis in a coordinated process that results in one strand deriving from the original duplex and the other from the newly synthesized strand. T-LOOP

The TRF2-dependent structure that has been observed by electron microscopy of fixed cells and in vitro, which is formed by invasion of the terminal G-rich single strand into proximal telomere sequences. TELOMERIC REPEATS

Refers to a stage after pre-meiotic DNA replication and before synapses when DNA is free to ‘pair’ with short regions of homology and, consequentially, to undergo gene conversion.

The G/C-rich simple sequences that are present at the ends of eukaryotic chromosomes. The G-rich strain always proceeds 5′ to 3′ towards the telomeric terminus. Yeast has an irregular sequence, which is abbreviated as (TG)1–3, whereas in most vertebrates (TT2AGGG)n is the predominant repeated sequence.

Q-FISH

TELOMERIC TRACT ATTRITION

A modification of the FISH procedure that allows the quantification of signals at specific chromosomal sites.

The slow loss of telomeric simple sequences both through replicative attrition and nuclease activities.

HOMOLOGY SEARCH

Models for catastrophic telomere loss

In considering these data, I sought a unifying theme, although it remains possible that the events described above represent several mechanisms that act at the telomere. Three models might explain these data, as discussed below30.

first step of the TRD process, intrachromatid deletion, is maintained in mammals. In this model, TRF2- and MRN-complex-mediated t-loops are in a dynamic equilibrium with t-loop structures of lower stability that lack TRF2 or MRN complexes, with the less stable form acting as a transient recombination intermediate (FIG. 3). By contrast, in the second step of TRD, the precision of deletion is not maintained in the studies that have been carried out so far. This might be the consequence of a lack of an efficient mechanism for comparing tract sizes among heterologous telomeres of different sizes, or owing to the lack of clustering among mammalian telomeres65 .

TRYPANOSOMES RAD50/RAD50(S/S)

The missense allele of human RAD50 that carries a mutation at the site of the rad52 (S) site in yeast. Mutations at this site confer meiosis I arrest, which terminates in unprocessed DNA double-stranded breaks.

Single-celled parasitic protozoa. TYPE II RECOMBINATION

A specific form of MRX-dependent telomere–telomere recombination that results in elongated telomeres that bypass the need for telomerase.


PERSPECTIVES checkpoint and nuclease level. Indeed, patients who carry mutations in NBS1 have a high rate of telomere loss, as noted above. However, these diseases are not phenotypically identical, and ATM and NBS1 act at least in part through different pathways on the same substrate66. Given the many functions of MRN and ATM in DNA damage control and telomere maintenance, the disease state might well be the indirect consequence of other defects in these cells. A better understanding of how these proteins function at telomeres in yeast and human cells will ultimately lead to more focused directions for medical intervention. Deletion and cancer. The ability to regulate telomeric deletion, regardless of the mechanistic relationship to yeast TRD, could be exploited to treat pre-oncogenic and oncogenic states. The behaviour of Rb94 is an interesting example. When introduced into human bladder-cancer cells in which telomerase is active, a drastic shortening of telomeres leads to inviability after the overexpression of Rb94 (REF. 55). Similarly, if we were able to delineate the MRE11 domains that control the equilibrium between recombinational elongation and deletion, we might selectively promote the deletion of telomeres in cancer cells, thereby ameliorating growth. There are many molecular targets in this system; these include the vast array of proteins that are thought to be associated with the human t-loop, such as RAP1, TRF2 and MRN, and the TRF1-interacting factor POT1, which has been proposed to associate with the singlestranded D-loop of the t-loop structure8,9 (FIG. 3). The regulation of TRD and these putative t-loop-associating proteins looks promising as a future therapeutic approach. There is an important associated clinical issue. Innumerable clinical and developmental studies use large changes in telomere size as the sole indicator of proliferation67,68. Given the presence of telomere deletions in mammalian cells, it might be precarious to equate telomere length with the number of cell divisions without extra criteria — further caution is required.

Clonal analyses of individual marked telomeres will be instrumental for a more direct comparison between yeast TRD and mammalian telomere deletions. Multiple tract sizes in a single subclone that carries the elongated and deleted forms of the individual telomeres are a strong indication of deletion. Such evidence is lacking from most investigations. It is crucial that the candidate genes that are thought to be important for the mammalian deletion process(es) are tested using this more defined assay. Are the observed deletions the true products of recombination? RAD52-depletion studies in human cells might be useful to address this issue. However, a large component of homologous recombination is RAD52independent in mammalian cells, so similar experiments with the second main homologous recombination protein XRCC2 will be required. Depletion experiments should also be pursued to investigate the role of mammalian RAD50 and MRE11 in deletion. Specific alleles of the essential gene RAD50 have already been constructed53. An examination of deletion in these strains should help to explain the pathway of mammalian deletions and its relationship to TRD. Ultimately, the central question that faces us is the biological function of telomere loss in mammalian cells. One possibility is that these are aberrant recombination events that occur only when the cell is under selective pressure for viability. The deletion process might also be a part of the normal sizing mechanism in human cells, although no tests have analysed this hypothesis directly. Alternatively, a related possibility is that it serves as part of a telomerebalancing act in ALT cells (FIG. 4). In this model, deletion would be balanced by the higher recombination activity of short telomeres69, which in turn leads to a more robust ALT system. This might explain the presence of a crude, but real, size-control mechanism even in cells that lack telomerase. Further research will be required to determine the role of deletion in telomere-size control and many surprises are likely to emerge after these crucial analyses. Arthur J. Lustig is at the Department of Biochemistry in the Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. e-mail: [email protected]

Future directions

The construction of a model is intended to provide experimental directions for investigating deletion events, including the relationship between mammalian and yeast deletion, recombination and t-loop dynamics. At present, the main question is whether deletion that is based on a recombination pathway is responsible for some or all of the deletions that have been documented in mammalian cells.

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Acknowledgements I appreciate the comments of the reviewers, which improved the quality of the manuscript. Also, I thank C. Azzalin, J. Lingner, J. Cooper and T. de Lange, who shared data before publication, and E. B. Hoffman for continual critical advice. This Perspective is dedicated to the memory of Anat Krauskopf.

Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink Atm | G22p1 | NBS1 OMIM: http://www.ncbi.nlm.nih.gov/omim ataxia telangiectasia | Nijmegen breakage syndrome Saccharomyces Genome Database: http://www.yeastgenome.org RAD52 SwissProt: http://us.expasy.org/sprot Cdc13 | Est1 | EST1A | Hpr1 | Ku70 | Ku80 | Ku86 | Mre11 | NBS1 | POT1 | Rad50 | Rad52 | Rap1 | Sir3 | TRF1 | TRF2 | XRCC2 | Xrs2 FURTHER INFORMATION Arthur J. Lustig’s Laboratory: http://www.tulane.edu/~biochem/lustig_home.html GenLink Multimedia Telomere Resource: http://www.genlink.wustl.edu/teldb/index.html Access to this interactive links box is free online.

ONLINE CORRESPONDENCE

Nature Reviews Genetics publishes items of correspondence online. Such contributions are published at the discretion of the Editors and can be subject to peer review. Correspondence should be a scholarly attempt to comment on a specific Review or Perspective article that has been published in the journal. The following correspondence has been recently published:

Teaching genetics in primary care through a transatlantic videoconference by Sean P. David and Robert Gramling This correspondence relates to the article:

GENETICS EDUCATION FOR PRIMARY-CARE PROVIDERS by Wylie Burke and Jon Emery Nature Rev. Genet. 3, 561–566 (2002)

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