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T-Loops and the Origin of Telomeres E PERSPECTIVES 66. Steinman, R. M., Mellman, I. S., Muller, W. A. & Cohn, Z. A. Acknowledgments eukaryotes evolved. The presence of t-loops at Endocytosis and the recycling of plasma membrane. H.S. is supported by the Norwegian Cancer Society and the J. Cell Biol. 96, 1–27 (1983). Research Council of Norway, and J.G. by the Swiss National present-day telomeres and their association 67. Lafont, F., Lecat, S., Verkade, P. & Simons, K. Annexin Science Foundation and the Human Frontier Science Programme with proteins that have evolved from RDR XIIIb associates with lipid microdomains to function in Organization. apical delivery. J. Cell Biol. 142, 1413–1427 (1998). factors might be remnants of the original 68. Aniento, F., Gu, F., Parton, R. & Gruenberg, J. Competing interests statement telomere system. Furthermore, the relative An endosomal βcop is involved in the pH-dependent The authors declare that they have no competing financial interests. formation of transport vesicles destined for late ease with which many eukaryotes can main- endosomes. J. Cell Biol. 133, 29–41 (1996). tain telomeres without telomerase might 69. Gu, F., Aniento, F., Parton, R. & Gruenberg, J. Functional Online links dissection of COP-I subunits in the biogenesis of reflect this ancient system of chromosome-end multivesicular endosomes. J. Cell Biol. 139, 1183–1195 DATABASES replication. This proposal ends with a dis- (1997). The following terms in this article are linked online to: 70. Gu, F. & Gruenberg, J. ARF1 regulates pH-dependent Interpro: http://www.ebi.ac.uk/interpro/ cussion of the advantages of the telom- COP functions in the early endocytic pathway. FYVE domain | PX domain erase-based system that could explain the J. Biol. Chem. 275, 8154–8160 (2000). LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ 71. Daro, E., Sheff, D., Gomez, M., Kreis, T. & Mellman, I. ANXA2 | VPS34 emergence of this almost universal mecha- Inhibition of endosome function in CHO cells bearing a Saccharomyces genome database: nism for telomere maintenance. temperature-sensitive defect in the coatomer (COPI) http://www.yeastgenome.org/ ε component COP. J. Cell Biol. 139, 1747–1759 (1997). Fab1 | Vps4 | Vps10 | Vps23 | Vps36 | Vps37 RT 72. Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E. & Swiss-Prot: http://us.expasy.org/sprot/ a 5′ Mellman, I. Cytoplasmic coat proteins involved in EGFR | HRS | LAMP1 | LAMP2 | PIKFYVE | TSG101 ′ RNA template endosome function. Cell 83, 703–713 (1995). 5 3′ 3′ 73. Press, B., Feng, Y., Hoflack, B. & Wandinger-Ness, A. FURTHER INFORMATION Mutant Rab7 causes the accumulation of cathepsin D Jean Gruenberg’s laboratory: RT and cation-independent mannose 6-phosphate receptor http://www.unige.ch/sciences/biochimie/website/pages/gruen b Genomic RNA ′ AAAAA 5′ in an early endocytic compartment. J. Cell Biol. 140, berg.html 5 3′ 1075–1089 (1998). Harald Stenmark’s laboratory: 3′ 74. Zerial, M. & McBride, H. Rab proteins as membrane http://folk.uio.no/stenmark/ organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001). Access to this interactive links box is free online. c 5′ 3′ 3′ d OPINION 5′ 3′ 5′ 3′ 3′ T-loops and the origin of telomeres e ′ Titia de Lange 5′ 5′ 3 3′ Figure 1 | Solutions to the end-replication Most eukaryotes stabilize the ends of their The second problem is that cells must distin- problem. a | In eukaryotic chromosomes, the linear chromosomes with a telomerase- guish their natural chromosome ends from maintenance of terminal sequences is primarily based system. Telomerase maintains sites of DNA damage, so that checkpoint acti- facilitated by telomerase, which extends the 3′ specific repetitive sequences, which protect vation and inappropriate repair are avoided. terminus using reverse transcriptase (RT in the chromosome ends with the help of Present-day telomeres solve both problems. figure) and an RNA template. b | Dipteran insects telomere-binding proteins. How did this The protection and replication functions solve the end-replication problem by retrotransposition, a pathway that is analogous to elaborate system evolve? Here, I propose are currently provided by a telomere system the telomerase pathway in that a reverse that telomere function was originally that comprises the telomere-specific reverse transcriptase uses the 3′ end of the chromosome mediated by t-loops, which could have transcriptase known as telomerase, an array as a primer for DNA synthesis using an RNA been generated by prokaryotic DNA- of telomeric repeats that are created by template. c | Experiments in telomerase-deficient replication factors. These early telomeres telomerase, and telomere-specific proteins Kluyveromyces lactis have provided evidence for would have required only the presence of a that bind to the telomeric repeats. The rolling-circle replication in which the 3′ end is few repeats at chromosome ends. telomeric proteins protect chromosome extended on an extrachromosomal circular template as a means of extension of chromosome Telomerase could have been a later ends from being recognized as sites of DNA ends. d | In Saccharomyces cerevisiae strains that innovation with specific advantages for damage and also regulate telomere-length lack telomerase, telomeric sequences can be telomere function and regulation. maintenance by telomerase. maintained with a break-induced replication The complexity of the telomerase-based (BIR)/recombination-dependent replication (RDR)- At the time of their switch to linear genomes, telomere system has raised the question of like pathway in which one telomere uses another eukaryotes must have evolved a mechanism how it evolved. Here, I discuss the possibility telomere as a template for extension. e | T-loop formation using terminal repeats and extension of to manage their chromosome ends. that eukaryotes originally existed without the the invaded 3′ end might be another mechanism Chromosome ends create two major prob- benefits of telomerase or telomeric-DNA- of telomere maintenance. The figure only shows lems. The DNA-replication machineries of all binding proteins. I propose that these first the elongation of the 3′ ends. For each pathway, cells, regardless of their genome organization, eukaryotes could have stabilized their chro- elongation of the 5′ end will require further use short RNA primers to initiate DNA syn- mosome ends using the t-loop structure. If (lagging-strand) DNA synthesis, which could take ′ thesis. Removal of the terminal primer at the the ends contained a few terminal repeats, place concomitantly with 3 -end extension or in the next round of DNA replication. The blue boxes end of lagging-strand synthesis leaves a small t-loops could have been generated by factors in parts a and b signify the sequence in the gap that cannot be filled in. If left unfixed, this that were involved in recombination-depen- telomerase RNA and in the retrotransposon RNA, gap leads to the loss of terminal sequences. dent replication (RDR), a form of DNA repli- respectively, that will be added to the This is known as the end-replication problem. cation that was already in existence before chromosome end by reverse transcription. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | APRIL 2004 | 323 © 2004 Nature Publishing Group PERSPECTIVES T-loop without telomerase has been possible, if not a 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 5′ POT1 (TTAGGG)n 3′ retrotransposons, HetA and TART. Occasionally, a new HetA or TART element (AATCCC)n will jump onto a chromosome end, using the ′ b 3 chromosome terminus as a primer to Tankyrase1/2 reverse transcribe its RNA genome. While MRE11 RAD50 WRN haphazard, this system of balancing the loss of NBS1 TRF2 PINX1 terminal sequences clearly works well. TRF1 Interestingly, telomerase loss has also ERCC1/XPF TIN2 POT1 occurred in several species of Coleoptera (beetles)8, an order of insects that is closely RAP1 related to the diptera. Figure 2 | Proposed structure of the human telomeric complex. a | Human telomeres are comprised Genetic disruption of the telomerase path- of a 2–30-kb array of duplex TTAGGG repeats, ending in a 100–200-nucleotide 3′ protrusion of way has revealed the ease with which eukary- single-stranded TTAGGG repeats. This DNA can exist as a t-loop in which the 3′ overhang invades the otes get by without this enzyme. S. cerevisiae duplex-repeat array forming a displacement (D) loop of TTAGGG repeats. Other configurations cannot be strains lacking telomerase lose their telomeres excluded. POT1 binds to the single-stranded TTAGGG-repeat DNA. Two double-stranded TTAGGG- gradually and eventually perish due to chro- repeat-binding factors (TRFs), TRF1 and TRF2, are associated with the duplex repeats. b | TRF1 and mosome instability9.But survivors arise read- TRF2 each recruit other proteins to the telomere (such as the tankyrases and RAP1). Some aspects of the ily and are so frequent that spontaneous complexes depicted have not been shown to exist in vivo. For example, it is possible that TRF1 and TRF2 10 form multiple complexes, and some of the protein interactions that are depicted have not been firmly mutations cannot explain their occurrence . established in vivo. The primary function of the TRF2-containing complex (left) is to protect chromosome They can use two homologous-recombina- ends. The TRF1-containing complex (right) has a role in the regulation of telomerase-mediated telomere tion pathways to elongate their telomeres. maintenance. In addition to the proteins shown, Ku70/80, DNA-PKcs and the Bloom helicase might be One pathway is dependent on Rad50 (a com- bound to telomeres. The human telomeric complex is reviewed in REFS 15,43. ERCC1/XPF is a nucleotide- ponent of the Mre11 complex, see below) and excision-repair endonuclease; the MRE11 complex is composed of MRE11, RAD50 and NBS1; PINX1, involves recombination between tracts of PIN2-interacting factor-1; POT1, protection of telomeres-1; TIN2, TRF1-interacting factor-2; WRN, Werner syndrome helicase.
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