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T-loops and the origin of telomeres
Author: T. deLange
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"PERSPECTIVES eukaryotes evolved. The presence of t-loops at present-day telomeres and their association with proteins that have evolved from RDR factors might be remnants of the original telomere system. Furthermore, the relative ease with which many eukaryotes can main- tain telomeres without telomerase might reflect this ancient system of chromosome-end replication. This proposal ends with a dis- cussion of the advantages of the telom- erase-based system that could explain the emergence of this almost universal mecha- nism for telomere maintenance. 66. Steinman, R. M., Mellman, I. S., Muller, W. A. & Cohn, Z. A. Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96, 1?27 (1983). 67. Lafont, F., Lecat, S., Verkade, P. & Simons, K. Annexin XIIIb associates with lipid microdomains to function in apical delivery. J. Cell Biol. 142, 1413?1427 (1998). 68. Aniento, F., Gu, F., Parton, R. & Gruenberg, J. An endosomal ?cop is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J. Cell Biol. 133, 29?41 (1996). 69. Gu, F., Aniento, F., Parton, R. & Gruenberg, J. Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes. J. Cell Biol. 139, 1183?1195 (1997). 70. Gu, F. & Gruenberg, J. ARF1 regulates pH-dependent COP functions in the early endocytic pathway. J. Biol. Chem. 275, 8154?8160 (2000). 71. Daro, E., Sheff, D., Gomez, M., Kreis, T. & Mellman, I. Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component ?COP. J. Cell Biol. 139, 1747?1759 (1997). 72. Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E. & Mellman, I. Cytoplasmic coat proteins involved in endosome function. Cell 83, 703?713 (1995). 73. Press, B., Feng, Y., Hoflack, B. & Wandinger-Ness, A. Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment. J. Cell Biol. 140, 1075?1089 (1998). 74. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107?117 (2001). Acknowledgments H.S. is supported by the Norwegian Cancer Society and the Research Council of Norway, and J.G. by the Swiss National Science Foundation and the Human Frontier Science Programme Organization. Competing interests statement The authors declare that they have no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Interpro: http://www.ebi.ac.uk/interpro/ FYVE domain | PX domain LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ ANXA2 | VPS34 Saccharomyces genome database: http://www.yeastgenome.org/ Fab1 | Vps4 | Vps10 | Vps23 | Vps36 | Vps37 Swiss-Prot: http://us.expasy.org/sprot/ EGFR | HRS | LAMP1 | LAMP2 | PIKFYVE | TSG101 FURTHER INFORMATION Jean Gruenberg?s laboratory: http://www.unige.ch/sciences/biochimie/website/pages/gruen berg.html Harald Stenmark?s laboratory: http://folk.uio.no/stenmark/ Access to this interactive links box is free online. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | APRIL 2004 | 323 Most eukaryotes stabilize the ends of their linear chromosomes with a telomerase- based system. Telomerase maintains specific repetitive sequences, which protect chromosome ends with the help of telomere-binding proteins. How did this elaborate system evolve? Here, I propose that telomere function was originally mediated by t-loops, which could have been generated by prokaryotic DNA- replication factors. These early telomeres would have required only the presence of a few repeats at chromosome ends. Telomerase could have been a later innovation with specific advantages for telomere function and regulation. At the time of their switch to linear genomes, eukaryotes must have evolved a mechanism to manage their chromosome ends. Chromosome ends create two major prob- lems. The DNA-replication machineries of all cells, regardless of their genome organization, use short RNA primers to initiate DNA syn- thesis. Removal of the terminal primer at the end of lagging-strand synthesis leaves a small gap that cannot be filled in. If left unfixed, this gap leads to the loss of terminal sequences. This is known as the end-replication problem. The second problem is that cells must distin- guish their natural chromosome ends from sites of DNA damage, so that checkpoint acti- vation and inappropriate repair are avoided. Present-day telomeres solve both problems. The protection and replication functions are currently provided by a telomere system that comprises the telomere-specific reverse transcriptase known as telomerase, an array of telomeric repeats that are created by telomerase, and telomere-specific proteins that bind to the telomeric repeats. The telomeric proteins protect chromosome ends from being recognized as sites of DNA damage and also regulate telomere-length maintenance by telomerase. The complexity of the telomerase-based telomere system has raised the question of how it evolved. Here, I discuss the possibility that eukaryotes originally existed without the benefits of telomerase or telomeric-DNA- binding proteins. I propose that these first eukaryotes could have stabilized their chro- mosome ends using the t-loop structure. If the ends contained a few terminal repeats, t-loops could have been generated by factors that were involved in recombination-depen- dent replication (RDR), a form of DNA repli- cation that was already in existence before T-loops and the origin of telomeres Titia de Lange OPINION Figure 1 | Solutions to the end-replication problem. a | In eukaryotic chromosomes, the maintenance of terminal sequences is primarily facilitated by telomerase, which extends the 3? terminus using reverse transcriptase (RT in the figure) and an RNA template. b | Dipteran insects solve the end-replication problem by retrotransposition, a pathway that is analogous to the telomerase pathway in that a reverse transcriptase uses the 3? end of the chromosome as a primer for DNA synthesis using an RNA template. c | Experiments in telomerase-deficient Kluyveromyces lactis have provided evidence for rolling-circle replication in which the 3? end is extended on an extrachromosomal circular template as a means of extension of chromosome ends. d | In Saccharomyces cerevisiae strains that lack telomerase, telomeric sequences can be maintained with a break-induced replication (BIR)/recombination-dependent replication (RDR)- like pathway in which one telomere uses another telomere as a template for extension. e | T-loop formation using terminal repeats and extension of the invaded 3? end might be another mechanism of telomere maintenance. The figure only shows the elongation of the 3? ends. For each pathway, elongation of the 5? end will require further (lagging-strand) DNA synthesis, which could take place concomitantly with 3?-end extension or in the next round of DNA replication. The blue boxes in parts a and b signify the sequence in the telomerase RNA and in the retrotransposon RNA, respectively, that will be added to the chromosome end by reverse transcription. RNA template Genomic RNA 5? 3? 5? 5? 5? 3? 3? 3? 5? 3? 5? 3? 5? 3? 5? 5? 3? 3? 3? 3? AAAAA RT RT a b c d e � 2004 Nature Publishing Group 324 | APRIL 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES without telomerase has been possible, if not good, for at least 200 million years. The fruitfly uses retrotransposition to keep its chromosome ends intact. D. melanogaster chromosome ends carry a medley of two retrotransposons, HetA and TART. Occasionally, a new HetA or TART element will jump onto a chromosome end, using the 3? chromosome terminus as a primer to reverse transcribe its RNA genome. While haphazard, this system of balancing the loss of terminal sequences clearly works well. Interestingly, telomerase loss has also occurred in several species of Coleoptera (beetles) 8 , an order of insects that is closely related to the diptera. Genetic disruption of the telomerase path- way has revealed the ease with which eukary- otes get by without this enzyme. S. cerevisiae strains lacking telomerase lose their telomeres gradually and eventually perish due to chro- mosome instability 9 .But survivors arise read- ily and are so frequent that spontaneous mutations cannot explain their occurrence 10 . They can use two homologous-recombina- tion pathways to elongate their telomeres. One pathway is dependent on Rad50 (a com- ponent of the Mre11 complex, see below) and involves recombination between tracts of telomeric repeats; the second pathway is gov- erned by the recombination protein Rad51 and creates arrays of sub-telomeric tandem repeats (reviewed in REF. 11). Analogous experiments in another bud- ding yeast, Kluyveromyces lactis,have revealed yet another mode of telomerase-independent telomere maintenance. Here, small extrachro- mosomal circles can provide the template for DNA-repeat addition to the 3? chromosome end. Through a rolling-circle mode of DNA replication, long arrays of terminal repeats can be added in a single step 12 . Even human cells are quite good at main- taining their telomeres without telomerase. Many human cell lines that were isolated by in vitro immortalization have escaped from the telomere crisis without the use of telomerase. The mechanism of telomere maintenance in such cell lines is referred to as alternative lengthening of telomeres (ALT) 13 .ALT is also observed in a subset of human tumours (reviewed in REF. 14). The molecular mecha- nism(s) of ALT is not yet fully understood, but possible mechanisms include rolling-circle replication, telomere?telomere recombination and t-loop-mediated extension (FIG. 1). End protection: no need for telomerase The ability of telomeres to dodge the DNA- damage response is a consequence of their association with a set of proteins that protect genomes. Poxvirus has a covalently-closed hairpin at each end of its double-stranded DNA (dsDNA) genome (reviewed in REF. 4). Controlled nicking of the hairpin provides the 3? OH group that is necessary for DNA replication. A similar strategy is used by linear plasmids in the Lyme disease spirochete Borrelia burgdorferi (reviewed in REF. 4). A complication of this replication strategy is that it generates a circular dimer, which requires a specialized reaction for conversion into monomers. Retroviruses prevent losing their chromosome ends by having their reverse transcriptase execute an elaborate ter- minal jump (reviewed in REF. 5) and aden- oviruses fix their end-replication problem with a proteinaceous primer, known as termi- nal protein, that is covalently attached to the 5? ends of its genome 6 . Alternative solutions to the end-replica- tion problem also occur in eukaryotic genomes (FIG. 1).An extreme example is seen in dipteran insects 7 ,an order that includes the model organism Drosophila melanogaster. Although most insects have telomerase-made TTAGG-repeat telomeres, the dipterans have lost this system. The widespread occurrence of flies and mosquitoes suggests that life Telomerase: who needs it? Most eukaryotes use telomerase to compen- sate for the loss of chromosome-terminal sequences 1 (reviewed in REFS 2,3). The reverse-transcriptase component of telom- erase is conserved and has been identified in vertebrates, invertebrates, plants, fungi and many unicellular organisms, including cili- ates, kinetoplastids and even Giardia. The success of the telomerase-based system is further illustrated by the striking conserva- tion of its product, the telomeric-repeat DNA. The majority of eukaryotes have a telomerase that synthesizes TTAGGG repeat arrays, making this hexamer one of the most pervasive nucleic-acid sequences in nature. Some of the telomeric repeats are slightly different; for example, plants have TTTAGGG repeats, but only a few eukary- otes (mostly budding yeasts, such as the well-studied Saccharomyces cerevisiae) have telomeric repeats that depart substantially from the TTAGGG motif. Despite its prevalence, telomerase is not the only way terminal sequences are main- tained. Many different ways to circumvent the loss of terminal sequences have surfaced from studies of viral, prokaryotic and eukaryotic Figure 2 | Proposed structure of the human telomeric complex. a | Human telomeres are comprised of a 2?30-kb array of duplex TTAGGG repeats, ending in a 100?200-nucleotide 3? protrusion of single-stranded TTAGGG repeats. This DNA can exist as a t-loop in which the 3? overhang invades the duplex-repeat array forming a displacement (D) loop of TTAGGG repeats. Other configurations cannot be excluded. POT1 binds to the single-stranded TTAGGG-repeat DNA. Two double-stranded TTAGGG- repeat-binding factors (TRFs), TRF1 and TRF2, are associated with the duplex repeats. b | TRF1 and TRF2 each recruit other proteins to the telomere (such as the tankyrases and RAP1). Some aspects of the complexes depicted have not been shown to exist in vivo. For example, it is possible that TRF1 and TRF2 form multiple complexes, and some of the protein interactions that are depicted have not been firmly established in vivo. The primary function of the TRF2-containing complex (left) is to protect chromosome ends. The TRF1-containing complex (right) has a role in the regulation of telomerase-mediated telomere maintenance. In addition to the proteins shown, Ku70/80, DNA-PK cs and the Bloom helicase might be bound to telomeres. The human telomeric complex is reviewed in REFS 15,43. ERCC1/XPF is a nucleotide- excision-repair endonuclease; the MRE11 complex is composed of MRE11, RAD50 and NBS1; PINX1, PIN2-interacting factor-1; POT1, protection of telomeres-1; TIN2, TRF1-interacting factor-2; WRN, Werner syndrome helicase. 5? 3? POT1 MRE11 RAD50 NBS1 RAP1 WRN ERCC1/XPF TRF2 Tankyrase1/2 PINX1 TRF1 POT1 TIN2 T-loop a b (TTAGGG) n (AATCCC) n � 2004 Nature Publishing Group PERSPECTIVES The way in which t-loops might allow cells to distinguish chromosome ends from sites of DNA damage is less clear. As detailed below, t-loops resemble a strand-invasion intermedi- ate in homologous recombination (FIG. 1) and are also very similar to a presumed intermedi- ate in replication-restart events (FIG. 3).Perhaps these intermediates in DNA repair and replica- tion are ignored by the DNA-damage-surveil- lance machinery and t-loops escape detection that way. It is also possible that present-day t-loops are masked from DNA-damage sensors through coating with telomeric proteins. How are t-loops formed? In vitro, TRF2 has the remarkable ability to create t-loops when provided with a short stretch of duplex telomeric DNA that ends in a 3? overhang 21,22 . The frequency of t-loop formation by TRF2 is low, however. So, in vivo, TRF2 is likely to be assisted by other factors in the process of t-loop formation (FIG. 3).Human telomeres contain several recombination and repair pro- teins that could promote the strand invasion of the 3? telomeric overhang. One of these is the MRE11 complex, composed of MRE11, RAD50 and NBS1 (Xrs2 in S. cerevisiae). In S. cerevisiae, this trio has a role in homolo- gous recombination, a reaction that resem- bles t-loop formation (reviewed in REF. 23). The MRE11 complex is brought to the telomere by TRF2, which is consistent with a chromosome ends. Some of these protect- ing factors bind along the duplex array of telomeric repeats; others bind to the single- stranded part of the telomeric DNA. Loss of these factors leads to telomere uncapping, a dysfunctional state that induces a DNA- damage response and results in inappropri- ate processing of chromosome ends by DNA-repair enzymes such as nucleases and ligases. An example of a protecting factor is TRF2, a small dimeric protein that binds to the dou- ble-stranded TTAGGG repeat arrays of mam- malian telomeres (FIG. 2;reviewed in REF. 15). Loss of TRF2 results in activation of the ATM checkpoint kinase and induction of a p53-dependent cell-cycle arrest. The uncapped telomeres are also attacked by an endonuclease that cleaves off the telomeric overhang and they can become fused together by DNA ligase IV. TRF2 must somehow pre- vent these disastrous events and recruits a large complex of other factors to telomeres to accomplish this task. Another example of a telomere-protection factor is the S. cerevisiae single-stranded DNA (ssDNA)-binding pro- tein Cdc13,which protects telomeres against exonucleolytic attack and prevents the activa- tion of a DNA-damage checkpoint by chro- mosome ends (reviewed in REF. 16). Telomeres of the fission yeast Schizosaccharomyces pombe are protected by a TRF2-like factor, Taz1, and a Cdc13-related protein, Pot1 (reviewed in REF. 17). Since each of these factors is recruited to telomeres through direct binding to the telomeric-repeat array, telomere protection depends on the maintenance of their recognition sequences at chromosome ends. So, although telomerase is generally required for the synthesis of telomeric repeats, the enzyme is not needed for telomere protection per se as long as there is enough telomeric DNA 18,19 . End protection by t-loops? How do telomeric proteins protect chromo- some ends? Some early models simply envi- sioned the formation of a proteinaceous cap over the vulnerable telomere termini. This protection strategy probably operates in the macronucleus (the vegetative nucleus) of hypotrichous ciliates 20 . A new model proposes that telomeres are protected by forming a specific configuration ? the telomeric loop, or t-loop 21 .T-loops are created through the strand invasion of the 3? telomeric overhang into the duplex part of the telomere (FIG. 2).They were discovered by electron-microscopic analysis of purified, protein-free telomeric DNA from human and mouse cells but are now also known to occur in many other eukaryotes (BOX 1).The strand invasion of the 3? overhang displaces the G-rich strand of the duplex telomeric repeat array (FIG. 2).This displacement loop (D-loop) can be detected at the base of the t-loop through coating with the ssDNA-binding protein (SSB)from Escherichia coli.The amount of SSB that can be loaded onto isolated human t-loops is consistent with a D-loop of ~150 nucleotides, which suggests that most of the 3? overhang is tucked into the duplex part of the telomere. It is also possible that a short stretch (<100 nucleotides) of the 5? strand partakes in the strand invasion, creating a structure that is involved in homologous recombination and is known as a Holliday junction. The size of the t-loop is highly variable and might not be important for telomere function. More likely, the key func- tion of the t-loop is the sequestration of the 3? end. T-loops seem to be a good strategy to pro- tect chromosome ends from fusion. It is unlikely that DNA ligase IV could fuse telom- eres in the t-loop configuration and the strand invasion of the 3? overhang will stave off nucleolytic attack. So, some of the main threats to telomeres would be taken care of by simply changing the configuration of the telomeric DNA into the t-loop structure. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | APRIL 2004 | 325 Box 1 | T-loops across evolution: who?s out of the loop? T-loops are a conserved feature of telomere structure. They have been found, not only in vertebrates, but also in the germline DNA (found in the micronucleus) of the hypotrichous ciliate Oxytricha fallax 44 .They also occur in the flagellate protozoan Tr ypanosoma brucei. Although trypanosome telomeres are as long as human telomeres, their t-loops are tiny, often less than 1 kb in length (REF. 45).Extremely large t-loops, up to 50 kb in size, are seen in Pisum sativum (peas) 46 .Key pieces of information on t-loops are still missing, including what percentage of telomeres carry this structure, how t-loops vary through the cell cycle, and their functional significance. We also have no information yet on t-loops in S. cerevisiae and S. pombe. This is mainly because of technical reasons. First, there is the crosslinking problem ? in order for t-loops to be detected, one must introduce inter-strand crosslinks, so that branch migration will not resolve the structure during the isolation of the telomeric DNA. The crosslinking step is performed on isolated nuclei (when the telomeres are still in their native configuration) and the crosslinking agents used are psoralen and UV light. Unfortunately, the T?A steps (as in TTAGGG) that are the substrate for psoralen are missing in the telomeric DNA of S. cerevisiae. The second problem has to do with the purification of telomeric DNA. For mammalian, plant and trypanosome telomeres, isolation of the telomeric fragments is based on a simple trick. The genomic DNA is reduced to very small fragments (~1 kb) by digestion with several frequently cutting restriction enzymes 21 .Under these conditions, the telomeres, which lack most restriction-enzyme recognition sites, remain large (often ~10 kb) and can be separated from the bulk of the genomic DNA on a sizing column. This trick does not work for organisms like S. pombe and S. cerevisiae,which have telomeres in the 1-kb size range. The only setting where t-loops are known to be absent is found in the macronucleus of hypotrichous ciliates. Although the micronucleus of these ciliates might have t-loops, their macronuclear telomeres do not. The macronucleus contains highly amplified gene-sized DNA molecules at a remarkable copy number of 2 x 10 7 per nucleus and an average size of just a few kilobases. Rather than containing long telomeres with t-loops, these DNA molecules are capped by ultrashort telomeres with termini that are hidden inside a tenaciously bound protein complex 47 . � 2004 Nature Publishing Group 326 | APRIL 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES A link between t-loops and RDR Several factors that are known to be associated with present-day telomeres, including compo- nents of the MRE11 complex and the single- stranded telomeric proteins POT1 and Cdc13, are distantly related to proteins that play a role in RDR in prokaryotes (TABLE 1). RDR was first discovered in bacteriophages 28 ,but more recently it has become clear that RDR is also crucial for replication of the bacterial-host genome (reviewed in REFS 29,30). RDR is nec- essary for the reinitiation of a broken replica- tion fork after replication has encountered a lesion. Such lesions are so frequent that repli- cation of the E. coli genome often requires an RDR event to be completed. So, the process- ing of broken forks by RDR represents an ancient pathway for the repair of DNA ends that was in existence before the evolution of eukaryotes. The early steps in RDR resemble the reac- tion that is required for t-loop formation (FIG. 3).In T4-phage replication, a 3? overhang is generated with the aid of gp46/47 and is coated with the ssDNA-binding protein gp32 (which is related to SSB). The strand- exchange factor UvsX (related to bacterial RecA) mediates strand invasion in homolo- gous sequences, thereby creating a D-loop. If a replication fork is established at this site (requiring several specific loading steps of the replication machinery), semi-conservative replication can take place. An alternative pathway is bubble-migration synthesis in which the 3? end is extended while the D-loop migrates forward. The latter pathway would require further lagging-strand synthesis to render the DNA double stranded. At least some of the proteins that are proposed to promote t-loop formation in mammals are evolutionarily related to RDR factors. The telomeric protein POT1 shares an OB-fold (a structural motif often used for binding to ssDNA) with the T4 protein gp32 and E. coli SSB. MRE11 and RAD50, which are recruited to telomeres by TRF2, have evolved from the T4 RDR proteins gp46 and gp47. Furthermore, vertebrate RAD51 and its related paralogues are the eukaryotic versions of the strand-exchange factors UvsX and RecA. Interestingly, a RAD51 paralogue was recently implicated in telomere biology in chicken cells 31 . Finally, UvsX loading seems to be facilitated by UvsY, a protein that is func- tionally related to the RAD52 family of recombination proteins. A mammalian mem- ber of the RAD52 family, RAD54, has been implicated in telomere function 32 . According to the t-loop?RDR compari- son, one could speculate that t-loop forma- tion involves the generation of a 3? overhang, t-loops, there is a risk that the reaction would go too far. If a Holliday junction is formed, its resolution could lead to deletion of the t-loop, resulting in a much shorter telomere. Interestingly, sudden large dele- tions occasionally happen at over-elongated yeast telomeres (known as telomere rapid deletions (TRDs)) and a t-loop-like struc- ture has been proposed as an intermediate (reviewed in REF. 27). Clearly then, the homologous-recombination factors at telomeres must be highly controlled and it is expected that telomere-specific compo- nents in the telomeric complex govern this regulation. role in establishing the protective t-loop state. Additional components of the TRF2 com- plex might be the Werner syndrome (WRN) and Bloom syndrome (BLM) helicases 24 , which could facilitate the unwinding step that is involved in t-loop formation. Furthermore, this family of helicases has the ability to disrupt G?G (Hoogsteen) base pairs 25,26 ,a feature that might be necessary to remove G?G base-paired structures from the 3? TTAGGG repeat overhang, so that it can anneal to the CCCTAA repeat strand of the duplex telomeric tract. If telomeres indeed employ homologous- recombination factors for the formation of Figure 3 | T-loop formation resembles initiation of RDR. a | Recombination-dependent replication (RDR). After the demise of a replication fork, RDR can reinitiate replication. In RDR of phage T4, a 3? overhang is formed by end-resection involving gp46/47, and coated with the single-stranded-DNA- binding protein gp32. Strand invasion and exchange takes place after loading of the strand-exchange factor UvsX. A D-loop is formed and is coated with gp32. gp59 mediates the assembly of the replication fork and replication is continued. Only a subset of RDR factors from T4 are shown (see TABLE 1 for further details and Escherichia coli orthologue). Similar steps might be required for t-loop formation. b | Capped- telomere formation. A 3? overhang has to be generated and a strand invasion/exchange step will take place to form a D-loop. The relationship between a subset of telomeric proteins and some of the RDR factors is depicted (see TABLE 1 for further comparison). In RDR, D-loop formation is followed by assembly of a replication fork and semi-conservative replication (or bubble-migration synthesis). Presumably, this latter step is blocked at telomeres resulting in a stable t-loop state (capped telomere). Telomerase-independent telomere elongation could result from the loss of factors that block RDR at telomeres. 5? 3? 3? POT1 5? 5? 5? 3? 5? 5? 3? 5? 3? 3? 5? 5? 3? 3? 3? 5? 3? 5? 3? 5? 5? 5? 5? 3? 5? 3? 3? 3? 3? Leading strand Lagging strand 5? 5? 5? 3? 5? 3? 3? 3? Leading strand Lagging strand D-loop D-loop gp32 Leading strand Lagging strand Unreplicated Replication fork demise gp59-mediated replication-fork assembly Normally blocked? Lagging strand Leading strand ? End resection ? Strand invasion and exchange ? D-loop formation ? gp32 coating of D-loop gp32 (SSB) UvsX (RecA) gp46/47 POT1 RAD51D MRE11 complex TRF2 RECQ helicases (BLM/WRN) ? Strand invasion and exchange Capped telomere Recombination-dependent replication a b (TTAGGG) n (AATCCC) n (TTAGGG) n (AATCCC) n (TTAGGG) n (AATCCC) n � 2004 Nature Publishing Group PERSPECTIVES requirement for the generation of a linear chromosome is the (accidental) formation of a few terminal repeats. Neither the size nor the sequence of these early terminal repeats would have been important. Indeed, as each end is replicated by a cis-acting process, the two ends of a linear chromosome could have contained entirely different sequences. One amusing possibility, given the close ties between telomerase and which is then coated by POT1. The next step would involve strand exchange, and one of the RAD51 paralogues could be a player in this reaction. These early steps would also require the MRE11 complex and RAD54. TRF2 could orchestrate these events through its inherent ability to promote the t-loop configuration and would also serve to bring (some of) the other factors to the telomere. The t-loop structure that is now established resembles RDR before capture of the replication machinery or bubble- migration synthesis (FIG. 3). The t-loop-evolution model The similarity between present-day telomeres in their t-loop configuration and the initia- tion of RDR indicates that there might be a simple scenario for telomere evolution. In this model, the first linear chromosomes had two or more repeats at their ends that are remod- elled by RDR enzymes into a t-loop (FIG. 4).It is easy to see how these t-loops could have solved the end-replication problem using the enzymes involved in RDR. Capture of the lagging- and leading-strand replication machinery at the base of the t-loop could have elongated chromosome ends by semi- conservative DNA replication. Alternatively, bubble-migration synthesis could have extended just the 3? end. The product of the latter reaction is identical to that of telom- erase and would require further DNA synthe- sis to create the complementary strand. Although haphazard and unregulated, any mechanism of extension of the 3? end of the t-loop could have solved the end-replication problem. The t-loops would also have the ability to protect the ends from nucleases and ligases. Furthermore, chromosome ends bearing t-loops might not have been detected as sites of DNA damage. The prokaryotic DNA-dam- age response (the SOS response) is triggered by RecA bound to ssDNA. If the first chromo- some ends had very short 3? overhangs, the amount of bound RecA might have been too little for the activation of the SOS response. In this way, the ends of the first linear chromo- somes would have achieved a protected state. The t-loop-evolution model is attractive because there is no need to postulate the emergence of new protein functions when the first linear chromosomes were created. The newly formed eukaryote would have dealt with its chromosome ends using a pre-existing major pathway for the repair and processing of DNA ends. The only NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | APRIL 2004 | 327 Figure 4 | The t-loop-evolution model. With a few notable exceptions, prokaryotes lack chromosome ends (a), and the first linear chromosomes (b) are proposed to have been capped and maintained by t-loops. The only requirement for this early form of telomere function is the presence of a few terminal repeats at each chromosome end. The length and sequence of the repeats is not important and can be different at each terminus. Using the prokaryotic recombination-dependent-replication (RDR) pathway, these repeats form a t-loop at each end, providing a mechanism for extension of the terminus. The t-loop structure is also proposed to protect the ends from nucleases and ligases. The SOS DNA-damage response is presumed not to be activated by t-loops. In present-day eukaryotes (c), telomere maintenance is primarily executed through the telomerase pathway. The telomerase reverse transcriptase (RT in the figure) could have evolved from pre-existing retrotransposon reverse transcriptases. T-loops might still be formed in some (but not all; see BOX 1) eukaryotes, providing telomere protection, and potentially regulating telomerase access to the telomere terminus. These t-loops are not used for telomere replication (except in unusual circumstances). The advent of telomerase has the advantage that all telomeres contain the same repeat sequence allowing emergence of specific trans-acting factors that can facilitate telomere protection and regulation of telomere length. 5? 5? 3? 3? RT RT AAAAA Genomic RNA Linear genome with terminal repeats Circular genome Retrotransposon T-loop phase Telomerase phase Telomerase Telomere capping by T-loops and telomere-specific proteins Template RNA a b c Telomerase-mediated telomere maintenance Table 1 | Relationship between T4 RDR factors and mammalian telomeric proteins Function in T4 RDR T4 Escherichia coli Eukaryotic orthologues Role at mammalian orthologue or functional homologues telomeres? ssDNA binding gp32 SSB RPA (DNA replication) Unknown POT1, Cdc13 (telomeres) Yes (POT1) Strand exchange UvsX RecA RAD51 family Yes (RAD51D) Loading of UvsX UvsY RecO/RecR RAD52 family Yes (RAD54) End resection gp46/47 SbcCD MRE11/RAD50/NBS1(Xsr2) Yes POT1, protection of telomeres-1; RDR, recombination-dependent replication; RPA, replication protein A; ssDNA, single-stranded DNA, SSB, single-stranded-DNA-binding protein � 2004 Nature Publishing Group 328 | APRIL 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES they were instrumental for the identification of the telomerase reverse-transcriptase gene 42 . In a twist of fate, the t-loop-evolution model would suggest that these ultra-short, protein- capped structures might be among the most highly evolved of present-day telomeres. Titia de Lange is at the Laboratory for Cell Biology and Genetics, Box 159, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. e-mail: delange@rockefeller.edu doi:10.1038/nrm1359 1. Greider, C. W. & Blackburn, E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405?413 (1985). 2. Nugent, C. I. & Lundblad, V. The telomerase reverse transcriptase: components and regulation. Genes Dev. 12, 1073?1085 (1998). 3. Lundblad, V. Telomerase catalysis: a phylogenetically conserved reverse transcriptase. Proc. Natl Acad. Sci. USA 95, 8415?8416 (1998). 4. Kobryn, K. & Chaconas, G. The circle is broken: telomere resolution in linear replicons. Curr. Opin. Microbiol. 4, 558?564 (2001). 5. Varmus, H. E. Form and function of retroviral proviruses. Science 216, 812?820 (1982). 6. de Jong, R. N., van der Vliet, P. C. & Brenkman, A. B. Adenovirus DNA replication: protein priming, jumping back and the role of the DNA binding protein DBP. Curr. Top. Microbiol. Immunol. 272, 187?211 (2003). 7. Biessmann, H. & Mason, J. M. Telomere maintenance without telomerase. Chromosoma 106, 63?69 (1997). 8. Frydrychova, R. & Marec, F. Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera). Genetica 115, 179?187 (2002). 9. Lundblad, V. & Szostak, J. W. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57, 633?643 (1989). 10. Lundblad, V. & Blackburn, E. H. An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell 73, 347?360 (1993). 11. Kass-Eisler, A. & Greider, C. W. Recombination in telomere-length maintenance. Trends Biochem. Sci. 25, 200?204 (2000). 12. Natarajan, S. & McEachern, M. J. Recombinational telomere elongation promoted by DNA circles. Mol. Cell. Biol. 22, 4512?4521 (2002). 13. Bryan, T. M., Englezou, A., Dalla-Pozza, L., Dunham, M. A. & Reddel, R. R. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines Nature Med. 3, 1271?1274 (1997). 14. Henson, J. D., Neumann, A. A., Yeager, T. R. & Reddel, R. R. Alternative lengthening of telomeres in mammalian cells. Oncogene 21, 598?610 (2002). 15. de Lange, T. Protection of mammalian telomeres. Oncogene 21, 532?540 (2002). 16. Lustig, A. J. Cdc13 subcomplexes regulate multiple telomere functions. Nature Struct. Biol. 8, 297?299 (2001). 17. Ferreira, M. G., Miller, K. M. & Cooper, J. P. Indecent exposure. When telomeres become uncapped. Mol. Cell 13, 7?18 (2004). 18. Singer, M. S. & Gottschling, D. E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase Science 266, 404?409 (1994). 19. Blasco, M. A. et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25?34 (1997). 20. Gottschling, D. E. & Zakian, V. A. Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell 47, 195?205 (1986). 21. Griffith, J. D. et al. Mammalian telomeres end in a large duplex loop. Cell 97, 503?514 (1999). 22. Stansel, R. M., de Lange, T. & Griffith, J. D. T-loop assembly in vitro involves binding of TRF2 near the 3? telomeric overhang. EMBO J. 20, E5532?5540 (2001). 23. de Lange, T. & Petrini, J. A new connection at human telomeres: association of the Mre11 complex with TRF2. Cold Spring Harb. Symp. Quant. Biol. LXV, 265?273 (2000). 24. Opresko, P. L. et al. Telomere binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases. J. Biol. Chem. 277, 41110?41119 (2002). a primer. On the basis of this relationship as well as other arguments, Eickbush has pro- posed a scenario in which telomerase evolved from a non-LTR retro-element 38 .The t-loop- evolution model explains how eukaryotes could have survived before telomerase emerged. In this way, the telomerase-based telomere system could have evolved at a later stage (although still very early) from the non- LTR retrotransposons that were already invading the eukaryotic genomes (FIG. 4).The view that telomerase evolved from a retro- transposon (rather than the other way around) is consistent with the presence of reverse transcriptases in prokaryotes. The telomerase-based telomere system brought enormous advantages. First, unlike the t-loop-replication strategy, telomerase can create new telomeres where none existed before. In doing so, telomerase can heal bro- ken chromosomes and speed up genome evolution by permitting chromosome frag- mentation and other rearrangements. Furthermore, telomerase has the advantage of homogenizing telomeric sequences. Whereas t-loop replication can lead to rapid divergence of the telomeric sequences, telomerase ensures that all telomeres are created equally. This is a crucial change in the system because it allows the use of sequence-specific trans- acting factors. With the emergence of telom- ere-binding factors, cells can connect events at telomeres to other pathways, resulting in the precise regulation of telomere transactions that is seen in present-day eukaryotes. In other words, telomerase allows a more bureaucratic approach to telomeres in which large ?committees? of protein networks decide on events at chromosome ends. Once telomerase is firmly in control at telomeres, there might be a need to suppress the original t-loop-replication mechanism, as it could lead to unregulated telomere exten- sion. Again, a regulatory machinery that allows t-loop formation but keeps RDR as well as homologous recombination under control seems a likely solution. Here too, the logic of the present-day telomere might be that its telomerase-made repeats recruit spe- cific regulatory factors to keep the t-loop from reverting to its old RDR mode. With the advent of telomerase, some eukaryotes could even have avoided the hazards of t-loops by switching to an entirely different system. One example might be the telomeres in the macronucleus (vegetative nucleus) of hypotrichous ciliates, which are kept at such a short length (<40 bp) that t-loop formation is impossible (BOX 1).Decades ago, these short telomeres contributed to the birth of telomere molecular biology 39?41 ,and, more recently, retrotransposons, is that the first terminal repeats were created by retrotransposition. The relative ease by which eukaryotes can come up with telomerase-independent systems of telomere maintenance might be testimony to their past use of the t-loop- replication strategy. Some of the yeast strains that survive telomerase deletion have been proposed to use break-induced repli- cation (BIR) 33 ,a eukaryotic replication mechanism akin to RDR. Indeed, the reliance of these survivors on Rad52/Rad50 or Rad52/Rad51 is in keeping with the idea that BIR or RDR is at work (TABLE 1).In addition, some of the human ALT cell lines might simply have found a way to return to the t-loop-replication strategy. So, why is t-loop extension not happening all the time? In eukaryotes that use telomerase for telomere maintenance and have t-loops at their chromosome ends, regulatory steps might be in place to avoid inappropriate t-loop replication. Perhaps ALT cell lines lack factors that normally keep t-loops from entering an RDR (or BIR)-like pathway. There is one linear genome, discovered in the mitochondria of a ciliate, that might rep- resent a ?living fossil? of the original t-loop- replication and -capping strategy. Unlike most mitochondrial DNAs, the genome of Tetrahymena sp mitochondria is linear. Early electron-microscopy studies and later sequencing of the termini showed that the ends of this chromosome contain tandem direct repeats of about 50 base pairs (REFS 34,35). Remarkably, the terminal repeats diverge extremely fast and show no sequence simi- larity in related Tetrahymena sp 36 .In extreme cases, the left and right ends of the mito- chondrial DNA have different repeats. These findings are consistent with a t-loop-replica- tion mechanism, which allows repeats to diverge rapidly. Indeed, electron-microscopy spreads indicate that the Tetrahymena sp mitochondrial DNA can form t-loop-like structures in vitro 34 .It would be of interest to examine crosslinked Tetrahymena sp mito- chondrial DNA by electron microscopy to establish whether the ends occur as t-loops in vivo. The age of telomerase The telomerase reverse transcriptase TERT is related to the enzyme that is used by many retrotransposons, including retroviruses 37 . The telomerases are most closely related to the reverse transcriptases of non-long-termi- nal-repeat (non-LTR) elements, which date back to the origin of eukaryotes. They also have functional similarity to non-LTR reverse transcriptases, since both use a DNA 3? end as � 2004 Nature Publishing Group PERSPECTIVES Acknowledgements I dedicate this article to the memory of Anat Krauskopf. K. Kreuzer, J. Haber, V. Lundblad, R. Reddel, K. Hoke, and S. Buonomo provided extremely helpful criticism of these ideas. I thank J. Griffith for comments on this manuscript and for long- term collaborative efforts on t-loops. B. Bowerman is acknowl- edged for sage advice (?Evolution happened. Get over it.?). Space limitations prevented me from providing a thorough review of all relevant publications and I refer to the cited reviews for many important primary references. Research on telomeres in my labo- ratory is funded by the National Institutes of Health, the National Cancer Institute and the Ellison Foundation. Competing interests statement The author declares that she has no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/Entrez/ RecA | SSB Flybase: http://flybase.bio.indiana.edu/ HetA | TART OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM&cm d=Limits Bloom syndrome | Werner syndrome Saccharomyces genome database: http://www.yeastgenome.org/ Cdc13 | Mre11 | Rad50 | Rad51 | Xrs2 Schizosaccharomyces pombe gene database: http://www.genedb.org/genedb/pombe/index.jsp Taz1 | Pot1 Swiss-Prot: http://us.expasy.org/sprot/ BLM | NBS1 | RAD54 | TRF2 | WRN Access to this interactive links box is free online. 25. Sun, H., Karow, J. K., Hickson, I. D. & Maizels, N. P. The Bloom?s syndrome helicase unwinds G4 DNA. J. Biol. Chem. 273, 27587?27592 (1998). 26. Sun, H., Bennett, R. J. & Maizels, N. The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G?G paired DNAs. Nucleic Acids Res. 27, 1978?1984 (1999). 27. Lustig, A. J. Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion. Nature Rev. Genet. 4, 916?923 (2003). 28. Mosig, G. The essential role of recombination in phage T4 growth. Annu. Rev. Genet. 21, 347?371 (1987). 29. Kreuzer, K. N. Recombination-dependent DNA replication in phage T4. Trends Biochem. Sci. 25, 165?173 (2000). 30. Cox, M. M. et al. The importance of repairing stalled replication forks. Nature 404, 37?41 (2000). 31. Wei, C., Skopp, R., Takata, M., Takeda, S. & Price, C. M. Effects of double-strand break repair proteins on vertebrate telomere structure. Nucleic Acids Res. 30, 2862?2870 (2002). 32. Jaco, I. et al. Role of mammalian Rad54 in telomere length maintenance. Mol. Cell. Biol. 23, 5572?5580 (2003). 33. Le, S., Moore, J. K., Haber, J. E. & Greider, C. W. RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics 152, 143?152 (1999). 34. Goldbach, R. W., Bollen-de Boer, J. E., van Bruggen, E. F. & Borst, P. Replication of the linear mitochondrial DNA of Tetrahymena pyriformis. Biochim. Biophys. Acta 562, 400?417 (1979). 35. Morin, G. B. & Cech, T. R. The telomeres of the linear mitochondrial DNA of Tetrahymena thermophila consist of 53 bp tandem repeats. Cell 46, 873?883 (1986). 36. Morin, G. B. & Cech, T. R. Mitochondrial telomeres: surprising diversity of repeated telomeric DNA sequences among six species of Tetrahymena. Cell 52, 367?374 (1988). 37. Cech, T. R., Nakamura, T. M. & Lingner, J. Telomerase is a true reverse transcriptase. A review. Biochemistry (Mosc.) 62, 1202?1205 (1997). 38. Eickbush, T. H. Telomerase and retrotransposons: which came first? Science 277, 911?912 (1997). 39. Klobutcher, L. A., Swanton, M. T., Donini, P. & Prescott, D. M. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3? terminus. Proc. Natl Acad. Sci. USA 78, 3015?3019 (1981). 40. Lipps, H. J., Gruissem, W. & Prescott, D. M. Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova. Proc. Natl Acad. Sci. USA 79, 2495?2499 (1982). 41. Gottschling, D. E. & Cech, T. R. Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex. Cell 38, 501?510 (1984). 42. Lingner, J. et al. Reverse transcriptase motifs in the catalytic subunit of telomerase Science 276, 561?567 (1997). 43. Smogorzewska, A. & de Lange, T. Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. (in the press). 44. Murti, K. G. & Prescott, D. M. Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops. Proc. Natl Acad. Sci. USA 96, 14436?14439 (1999). 45. Munoz-Jordan, J. L., Cross, G. A., de Lange, T. & Griffith, J. D. T-loops at trypanosome telomeres. EMBO J. 20, 579?588 (2001). 46. Cesare, A. J., Quinney, N., Willcox, S., Subramanian, D. & Griffith, J. D. Telomere looping in P. sativum (common garden pea). Plant J. 36, 271?279 (2003). 47. Horvath, M. P., Schweiker, V. L., Bevilacqua, J. M., Ruggles, J. A. & Schultz, S. C. Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963?974 (1998). NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | APRIL 2004 | 329 ADDENDUM T-LOOPS AND THE ORIGIN OF TELOMERES Titia de Lange Nature Rev. Mol. Cell Biol. 5, 323?329 (2004) The author wishes to make a short addition to the section ?The t-loop-evolution model?. In the last paragraph of this section, the author raises the possibility that the linear mitochondrial DNA of Tetrahymena sp. represents a ?living fossil? of the original t-loop- replication and -capping strategy. Unfortunately, the author omitted to highlight studies on the linear mitochondrial DNAs of several budding yeast species (for example, Candida parapsilosis), which, like Tetrahymena sp. mitochondrial DNA, contain tandem repeats at their ends. Importantly, electon-microsopy analysis has shown that the ends of the budding yeast mitochondrial DNAs are in a t-loop configuration 1 . 1. Tomaska, L., Makhov, A. M., Griffith, J. D. & Nosek. J. t-loops in yeast mitochondria. Mitochondrion 1, 455?459 (2002). � 2004 Nature Publishing Group "
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