Telomeres and Telomerase: the Path from Maize, Tetrahymena and Yeast to Human Cancer and Aging
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COMMENTARY Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging Elizabeth H Blackburn, Carol W Greider & Jack W Szostak The telomere problem research report that described the frequent join- the 1970s, Joe Gall had been delving into the Scientific discoveries are each individual and ing of broken chromosome ends, “no case was processes by which some organisms produce http://www.nature.com/naturemedicine occur by their own unique path. However, there found of the attachment of a piece of one chro- extra copies of the genes for ribosomal RNA are key ingredients that set the stage for them. mosome to the end of another [intact chromo- (rRNA). This occurs, for example, early in Many of these ingredients were important in some]”1. In 1938, Muller named the natural ends development, when large amounts of protein the discovery of telomerase: talking with sci- of chromosomes ‘telomeres’2. But neither Muller synthesis must occur rapidly. Joe had discovered entists from different fields, paying attention to nor McClintock had the tools to understand the that the genes encoding rDNA are amplified unusual findings and taking the risks of doing molecular nature of these chromosome ends. on circular DNA molecules that are present in crazy experiments. We will describe how a com- The question of the molecular nature of the very high numbers in the developing oocyte in bination of these ingredients and productive chromosome end only became meaningful in frogs. He then found that the same thing hap- collaborations led us to postulate and discover 1953, when the structure of DNA was described3. pened in a very different organism—the cili- telomerase. By the 1960s, Arthur Kornberg had discovered ated protozoan Tetrahymena thermophila—but The earliest functional description of telo- DNA polymerase and its mechanism had been this time, the rDNA was amplified into linear Nature Publishing Group Group Nature Publishing 4 6 meres was by geneticist Hermann Muller when determined . This understanding posed yet DNA molecules. Tetrahymena contained large he used X-rays to fragment chromosomes. another question about DNA ends—how was numbers of nearly identical, relatively short 200 Muller, working with fruit flies, and Barbara their complete replication ensured? Because minichromosomes. This was the material that © McClintock, working with maize, converged on DNA polymerase could only extend a preformed Liz decided to use to analyze natural ends of the same conclusion around the same time: that primer, it could not copy the very end of a linear chromosomes. the natural ends of chromosomes are different DNA; this became known as the DNA end-rep- There was no road map for how to do this. from those created at the site of a chromosomal lication problem5. By the early 1970s, studies of But Joe Gall had already shown that a fraction of break. The natural ends were somehow protected DNA bacteriophage genomes had shown that the molecules were circular when extracted from from the frequent rearrangements that occur at the answers to the DNA end-replication problem cells, a property reminiscent of phage lambda, broken ends. As McClintock wrote in a 1931 differed between one virus and another6. How, the linear DNA of which circularizes to replicate. then, was the DNA at the very end of eukaryotic Because Liz had grown to believe that nature uses Elizabeth H. Blackburn is at the Department chromosomes arranged? In 1975, Liz Blackburn elegant and universal solutions, she thought that of Biochemistry and Biophysics, University of arrived at Yale in Joe Gall’s lab to do postdoctoral the lambda ends might be a possible model for California, San Francisco, 600 16th Street, research, having recently completed her gradu- the molecular nature of the Tetrahymena termini. MC 2200, San Francisco, California 94158-2517, ate work in Fred Sanger’s group in Cambridge, Thus, she decided to use in vitro the DNA ‘repair’ USA. Carol W. Greider is Daniel Nathans Professor England, where DNA sequencing was being reaction of DNA polymerase, which had been and Director, Department of Molecular Biology invented. Liz wanted to apply her knowledge successfully used by Ray Wu and colleagues to and Genetics, Johns Hopkins University School from the Sanger lab to understanding the molec- sequence the cohesive ends of the lambda phage of Medicine, 603 PCTB, 725 North Wolfe Street, ular nature of chromosome termini. family genomic DNAs7. Baltimore, Maryland 21205, USA. Jack W. Szostak This was a fortunate choice, because the is Professor of Genetics at Harvard Medical School, Telomeres go molecular: mysterious DNA molecular ends of the rDNA turned out to Department of Molecular Biology and Center for termini have discontinuities (the significance of which Computational and Integrative Biology, Simches One of the most daunting aspects of addressing remains mysterious to this day) within the Research Center, 85 Cambridge Street, Boston, the question of the DNA at chromosomal ends telomeric repeat tract DNA that allowed DNA Massachusetts 02114, USA. was the enormous length of the chromosomal polymerase to label them readily in vitro using e-mails: [email protected], DNAs of eukaryotes. DNA cloning methods radiolabeled triphosphate substrates. Liz was [email protected] or [email protected]. had not yet been invented, so to be able to study then able to piece together the DNA sequence harvard.edu. the ends, short chromosomes were needed. In of the telomeres by combining a variety of NATURE MEDICINE VOLUME 12 | NUMBER 10 | OCTOBER 2006 1133 COMMENTARY in vitro labeling and other analytical techniques, ciliates16,17. The question then became: how mid, resulting in a linear replicating plasmid with and she came up with a very un-lambda-like was the telomere repeat added? stable DNA ends. The experiment was technically surprise. The rDNA minichromosome end In considering telomere formation in simple to perform, and the prediction—that lin- sequence and the structure at the termini of Tetrahymena, Liz wrote in 1982: “...the sequences ear plasmid molecules would be seen instead of these molecules were complex and unlike common to the macronuclear DNA termini the usual circles—would be trivial to confirm. any previously described. At each end of the must be acquired by these subchromosomal Armed with a purified Tetrahymena telomeric Tetrahymena rDNA molecules there were segments during their formation. Two types of DNA fragment supplied by Liz, Jack generated around 50 tandem repeats of the hexanucleo- routes can be envisaged: Telomeric sequences a few nanograms of a linearized yeast plasmid tide unit CCCCAA—TTGGGG on the comple- are transposed or recombined onto the devel- capped with Tetrahymena telomeres, and intro- mentary strand—with the latter (G-rich) strand oping macronuclear DNA termini, or the simple, duced the DNA into yeast cells. He obtained a bearing the 3ʹ OH end at each end of the linear repeating telomeric sequences are synthesized dozen or so transformants that were analyzed DNA. The C-rich CCCCAA-repeat strand had de novo onto these termini by specific synthetic by Southern blotting. The result was immedi- single-stranded discontinuities in at least a por- machinery”13. This idea for de novo telomere ately clear: over half of the colonies maintained tion of the repeat array. Oddly, too, the number addition was further supported by ongoing the introduced DNA in linear form. This result of tandem repeats per end was heterogeneous experiments in yeast. was confirmed by more detailed DNA analysis19. among the population of purified molecules, This result showed that telomeres could function ranging from an estimated minimum of 20 to Telomere function transcends kingdom across phylogenetic kingdoms, implying remark- up to about 70 (ref. 8). Late in 1977, Liz gave boundaries able functional conservation. a talk describing the unusual molecular fea- By 1980 we understood the molecular struc- tures of the rDNA ends at the University of ture of Tetrahymena telomeres, but the con- Yeast telomeres reveal conservation of California, San Francisco to Herbert Boyer’s nection between this structure and the special telomere structure group in the Biochemistry Department. In the properties of telomeres remained obscure. These linear plasmids then provided the http://www.nature.com/naturemedicine discussion after her presentation, a member of Whether this surprising structure was unique ideal vector for cloning yeast telomeres. Jack the Boyer lab asked whether the heterogeneity to Tetrahymena and its ciliate relatives or was removed one Tetrahymena telomere, generat- in the number of CCCCAA repeats in the DNA more broadly conserved was also unknown. ing a linear DNA fragment that could not be population might arise by addition of repeats The answers to these questions began to emerge maintained in yeast. He then went fishing for to the chromosome ends. Liz was intrigued but from an unlikely collaboration between Jack functional yeast telomeres by joining random at the time could see no known way this could Szostak and Liz. Jack had recently completed pieces of yeast genomic DNA onto this linear occur. Looking back, this conversation seems his graduate and postdoctoral work with Ray DNA fragment; only when the missing telo- prescient, as later Liz would find that, in fact, Wu at Cornell, where he had begun to study mere was replaced with a yeast telomere could this addition does occur. recombination in yeast. In 1979, he had set up the DNA survive in yeast as a linear plasmid. his own lab at what was then the Sidney Farber Three of the expected linear plasmids were New telomeres are added to fragmented Cancer Institute in Boston, and was investigat- recovered, and Southern blots of genomic Nature Publishing Group Group Nature Publishing 6 Tetrahymena chromosomes ing the highly recombinogenic nature of DNA yeast DNA showed that the linear plasmids In collaboration with Meng-Chao Yao in Joe ends in the budding yeast Saccharomyces cere- indeed carried a functional yeast telomere 200 Gall’s lab, Liz showed that the same termi- visiae.