Basic Medical Research Award
Elizabeth Blackburn, Carol Greider, and Jack Szostak
For the prediction and discovery of telomerase, a remarkable RNA-containing enzyme that synthesizes the ends of chromosomes, protecting them and maintaining the integrity of the genome.The 2006 Albert Lasker Award for Basic Medical Research honors three scientists who predicted and discovered telomerase, an enzyme that replenishes the ends of chromosomes. In so doing, they unearthed a biochemical reaction that guards cells against chromosome loss and identified the molecular machinery that performs this feat. The work resolved perplexing observations about chromosome termini and explained how cells copy their DNA extremities.
In the 1930s, scientists surmised that protective capstelomeresensure the propagation of chromosomes during cell division and prevent them from inappropriately melding with one another. The physical nature of these structuresand how they are constructedeluded researchers until Elizabeth Blackburn, Carol Greider, and Jack Szostak performed their groundbreaking investigations in the late 1970s and 1980s. Blackburn showed that simple repeated DNA sequences comprise chromosome ends and, with Szostak, established that these repeats stabilize chromosomes inside cells. Szostak and Blackburn predicted the existence of an enzyme that would add the sequences to chromosome termini.
Greider and Blackburn then tracked down this enzymetelomeraseand determined that each organism's telomerase contains an RNA component that serves as a template for the creature's particular telomere DNA repeat sequence. Szostak found that budding yeast unable to perform the telomerase reaction lose their telomeresand chromosomesover multiple generations. Eventually, the organisms stop dividing. In addition to providing insight into how chromosome ends are maintained, Blackburn's, Greider's, and Szostak's work laid the foundation for studies that have linked telomerase and telomeres to human cancer and age-related conditions.
The Beginning of the Ends
In the 1930s, Barbara McClintock (Lasker 1981) and Hermann Muller independently inferred that the natural termini of chromosomes display special characteristics. Unlike ends generated by DNA breakage, they don't fuse with each other. Furthermore, only chromosomal fragments containing intact ends persist when a cell duplicates. A distinct structure must seal chromosomes and confer these properties, Muller reasoned. He dubbed chromosome termini "telomeres" from the Greek "telos" for end and "meros" for part or segment. However, no one knew what made telomeres different from randomly generated ends.
A second telomere-related conundrum arose after researchers deciphered how eukaryotic cellsthose with nucleicopy DNA. The enzyme that performs this reaction should be unable to fully replenish linear DNA due to a peculiarity of its mechanism; each round of replication should generate a molecule missing a few building blocks, called nucleotides, on the DNA's end. As a result, linear chromosomeswhich house genes in eukaryoteswould shorten every time a cell divides. In 1972, James Watson (Lasker 1960) speculated that organisms with linear chromosomes need a strategy to maintain chromosome tips, a theory that became known as the "end-replication problem." In parallel, Alexey Olovnikov suggested that the gradual loss of chromosome ends would lead to cellular senescence, a dormant state in which cells remain alive but can no longer divide or perform their normal functions. Although scientists discussed possible solutions to these problems, they did not have ways to test their ideas.The Ends in Sight
In the late 1970s, Blackburn wanted to determine the sequence of DNA at the ends of a eukaryotic chromosome. Joseph Gall, then at Yale University, had found that the ciliated protozoan Tetrahymena thermophila contains many DNA minichromosomes. Because the molecules are small but abundant, the number of ends relative to the rest of the DNA is large. This feature of Tetrahymena allowed Blackburn, working in Gall's lab as a postdoctoral fellow, to gather enough ends to sequence. Each was composed of a six-nucleotide sequence (CCCCAA) that was repeated 2070 times. Similar sequences turned out to reside in other ciliates, but no one knew whether this odd feature appeared in distantly related organisms.
In 1980, Blackburn, by then running her own lab at the University of California, Berkeley, presented her work at the Nucleic Acids Gordon Research Conference. After her talk, she spoke with Jack Szostak, a yeast geneticist from Harvard Medical School. They decided to add the Tetrahymena repeats to the ends of linear DNAs and test whether the resulting DNA would persist in budding yeast. Szostak knew that non-chromosomal linear DNAs in yeast normally insert themselves into chromosomes or are destroyed by cellular enzymes, presumably because they behave as if they result from random fractures. The Tetrahymena sequences provided the first hope for yeast to retain such linear DNAs. The experiment worked, despite the vast evolutionary distance between budding yeast and Tetrahymena. The Tetrahymena telomeres protected the linear yeast DNA, allowing it to pass reliably from one generation to the next.
The researchers then identified short, distinctive repeats on the ends of normal yeast chromosomes and showed that this yeast telomeric sequence was tacked on to Tetrahymena ends that were present on linear DNA in yeast. Because yeast added its characteristic sequence to Tetrahymena telomeres, telomeres must not serve as templates for additional telomeric sequences. This finding and the varied number of repeats led Blackburn and Szostak to speculate that an enzyme adds telomeric sequences to chromosome ends. Such an act would replenish the genetic material predicted to be whittled away by DNA replication. This idea differed radically from other suggestions scientists had proposed to solve the end-replication problem.The Means to the Ends
Blackburn, soon joined by Greider, who was then a graduate student, started seeking the hypothetical enzyme by looking for a substance that could affix telomeric repeats to chromosome ends in the test tube. Because Tetrahymena contained so many telomeres, the organism should provide a rich source of enzymes that act on them, the researchers reasoned. They added the contents of Tetrahymena cells to a mixture of radioactive nucleotides and small DNA pieces composed of the Tetrahymena telomere repeats, hoping to see the tagged nucleotides attach to the repeats. On Christmas Day 1984, Greider discovered that the Tetrahymena contents generated precisely the DNA pattern predicted for an enzyme that added the six-nucleotide repeats one building block at a time. Blackburn and Greider named the enzyme telomere terminal transferase.
Next, they wanted to figure out exactly how the telomere sequence was determined. They postulated that each organism's enzyme contained an RNA or DNA component that could serve as a template. An RNA-destroying enzyme obliterated the telomere terminal transferase activity, so Blackburn and Greider concluded that an RNA must play a crucial role. In a tour de force of biochemistry, Greider purified the enzyme, which they now called telomerase, and showed that it contained both an RNA and a protein subunit. In her own lab at Cold Spring Harbor Laboratory, she completed this work by isolating the RNA-encoding gene. It indeed carried a sequence that could specify the Tetrahymena telomere repeats. Furthermore, cleaving that sequence demolished telomerase activity. To prove that the internal template determines the sequence of the telomeric repeats, Blackburn altered the crucial sequence in the RNA component of telomerase; this perturbation resulted in production of telomeres that correspond to the new sequence. Later, Tom Cech (Lasker 1988) purified the protein portion of telomerase, which adds nucleotides one by one to the chromosome ends, according to instructions from the RNA component.
In the meantime, Szostak and his postdoctoral fellow Victoria Lundblad established that the inability to restore telomeres imperils the cell. They had been seeking yeast mutants that could not properly elongate telomeres. This scheme should identify genes that are crucial for telomerase function, they reasoned. Telomeres in such mutants would shrink, Szostak and Lundblad predicted, and strains harboring such defects would lose their chromosomes over many generations. At first, the yeast would grow normally, but as the genetic material disappeared, the microbes would stop dividing. The researchers found such a strainand named the gene responsible for the defect EST1 for "ever shorter telomeres." The approach subsequently led to the discovery of other proteins required for telomere stability, including telomerase's core protein. Furthermore, the finding provided the first experimental support for the end-replication problem: As predicted, the inability to replenish telomeres caused the structures to dwindle as cells reproduced. Moreover, the work implied that cells unable to solve the end-replication problem eventually senesce.
All's Well that Ends Well
The work by Blackburn, Greider, and Szostak set the stage for discoveries about the role of telomerase in human cancer and aging. Like their Tetrahymena and yeast counterparts, human telomeres are composed of a particular simple DNA sequence, repeated various numbers of times. Sperm and eggs manufacture telomerase, but most adult cells don'tand telomeres in most adult cells are shorter than those in sperm and eggs. Telomere attrition, at least in cells grown outside the body, leads to senescence.
Approximately 85 to 90 percent of human cancers reactivate telomerase (the rest maintain their telomeres through an alternative mechanism) and strong evidence suggests that the enzyme renders these cells able to proliferate uncontrollably by continually refreshing their telomeres. For example, adding the enzyme to certain human cells grown in culture dishes renders the cells immortal. Conversely, blocking the enzyme's action in lab-grown cancer cells can inhibit their growth or kill them. Scientists are pursuing compounds that thwart telomerase as a potential strategy for fighting cancer. Several clinical trials of such drugs are now under way.
Along similar lines, telomere erosion during a person's lifetime could curtail cell survival, thereby promoting age-related ailments. Evidence supporting this notion comes from studies of the rare human disease dyskeratosis congenita. One form of this illness arises from genetic defects in the RNA component of telomerase. Short telomeres limit the ability of certain tissues to replace themselves. As a result, the disease generates age-like conditions: It wipes out affected individuals' bone marrow, predisposes them to a variety of human cancers, and gives them splotchy skin, ratty fingernails, and prematurely gray hair. The work on dyskeratosis congenita demonstrates that withered telomeres can accelerate physical deterioration.
Blackburn, Szostak, and Greider pursued basic questions of cell biology and enzymology to unveil mysteries that have huge implications for human health. The impact of their work is certain to extend long into the future.
By Evelyn Strauss, Ph.D.