Discovery of Telomeric DNA and Telomerase
Transcript of Part 1: Discovery Talk: Discovery of Telomeric DNA and Telomerase
00:00:05.19 Hello, my name is Elizabeth Blackburn and I'm going to tell you about 00:00:10.06 the discovery of telomeric DNA and of telomerase. 00:00:14.13 When I first started working on this question, 00:00:19.27 the ends of chromosomes, and these are chromosomes here, 00:00:23.29 were known to be very important for protecting the genetic material. 00:00:27.29 But nobody knew what was there. 00:00:32.11 Now, it was known that every chromosome had a long DNA molecule 00:00:37.22 that goes from one end of the chromosome to the other, 00:00:39.25 and that long DNA, of course, carries all the genes, 00:00:43.27 and every time that chromosome is replicated, the DNA has to be fully copied. 00:00:50.06 But it was also known around the 1970's that the DNA replication machinery of the cell 00:00:58.01 wasn't able to completely copy all the way out to the very ends of the chromosome. 00:01:03.02 And so the prediction of what might happen every time a DNA replicated, 00:01:09.04 in order for a cell to divide, 00:01:11.07 would look something like what's depicted in this slide here, 00:01:13.24 which would be that the chromosomal DNA end region 00:01:17.26 would get shorter and shorter and shorter until eventually the cells would have such short chromosomes 00:01:23.16 that the would be perhaps missing something from their ends, 00:01:26.26 and then not able to divide anymore. 00:01:29.08 And that was even predicted on these theoretical grounds 00:01:32.12 to be something that was called senescence, 00:01:35.26 which had been seen in cells growing in culture, 00:01:38.23 that they, in certain cases, could only go through a certain number of divisions, 00:01:42.26 and then they couldn't divide any further, but nobody knew why. 00:01:46.15 And so, how to approach this problem? 00:01:51.09 Well, I began it by taking advantage of a particular kind of organism, 00:01:57.00 this is called Tetrahymena thermophila, 00:01:59.03 and it's a single-celled, ciliated protozoan, 00:02:01.27 and you can see in this scanning electron micrograph here 00:02:04.28 all of the cilia all over the single cell. 00:02:08.24 And the particular reason for this choice of organism 00:02:12.01 was that these cells have within them some very short linear chromosomes, 00:02:17.17 in high numbers, and so what was known before 00:02:22.23 was that, well, nobody knew what was at the ends of the chromosomes in eukaryotes, 00:02:28.10 cells with nuclei, such as Tetrahymena, 00:02:31.04 certain very short, linear viruses that grow in bacteria, called bacteriophages, 00:02:38.24 had had the ends of their DNA analyzed, 00:02:43.22 and in this organism, Tetrahymena, the very short chromosomes were about the same kind of size, 00:02:48.01 and so I thought, well, I would try and look and see 00:02:50.26 what was at the ends of these chromosomes, 00:02:53.27 these mini-chromosomes. Now, at this point, DNA sequencing methods had not been developed. 00:03:01.07 And so, although this is hard for you to believe, probably, these days, 00:03:05.03 but that was the case. And so what I had to do was to rely on a different kind of approach 00:03:11.13 and that was to really try to stitch together pieces of information about the ends of the DNA, 00:03:18.04 that I could obtain by using labeling techniques, radio-labeling techniques, 00:03:23.24 which would incorporate radio labels into the DNA, 00:03:26.27 and analyzing by combinations of enzymes and chemicals 00:03:32.00 that would cleave the DNA into little pieces that were dependent upon the particular 00:03:39.27 building block bases of the DNA. 00:03:42.23 And patch together the sequences, sort of stitch it together like a jigsaw puzzle. 00:03:47.21 So, for example, I would cut the DNA up and see that there were, 00:03:52.09 for example, little motifs in it, like this one here, 00:03:55.01 where there was this motif of four cytosine residues in a row, CCCC, that's C4. 00:04:02.06 And in fact, furthermore, I could tell from some of these analyses 00:04:06.06 that there was a purine, that is an A or a G, 00:04:11.09 and then CCCC, and then an A or a G. 00:04:14.27 Because of the way I'd chemically cleaved this DNA up. 00:04:17.27 And furthermore, by quantifying what was there, I could find that there were actually 00:04:22.20 many, many repeats of this little motif, at the ends of these short mini-chromosomes, 00:04:30.22 and so, putting it all together, I was able to conclude 00:04:36.01 that these little tiny chromosomes ended in something that had really never been expected, 00:04:40.23 and that was a sequence, that I've drawn the strand which is the complement of the CCCC strand, 00:04:47.11 it's actually TTGGGG. And just repeated over and over and over and over again, 00:04:51.24 perhaps 20 to 50 different repeats. 00:04:56.26 Now, the question was how do these repeats get there? 00:05:00.05 Now, at this point, there were a lot of possibilities that you could draw on paper. 00:05:05.24 And often in science, what you do is you take an observation, 00:05:09.09 and you put it into the existing body of information, 00:05:12.07 and you say, well, how can I explain my new finding, given the context of all the information that is known. 00:05:18.21 So, we know about DNA, we knew about it's replication machinery, 00:05:22.18 we know how it recombined, and there were certain well-worked out rules by then 00:05:27.12 for this. But these kinds of DNA sequences were not obeying those rules. 00:05:35.10 And I'll tell you how they weren't obeying those rules in a moment, 00:05:38.16 but the first thing I think that's interesting is that 00:05:41.16 there comes a point when you're working in science, where you know a lot of things, 00:05:45.29 you're trying to fit your results into well-established principles, 00:05:49.02 and then it comes to a point where it just won't fit in that box anymore, 00:05:53.05 you just can't push it into that box anymore, 00:05:54.24 and you have to say, well, let's entertain some other possibilities. 00:05:58.18 Now the kinds of observations, and I won't go through them, 00:06:02.01 were that there were variable numbers of repeats on these small, linear chromosomes, 00:06:08.17 sometimes there were, you know, 30 or so, sometimes there were 50, 70, 00:06:11.17 you know, there was different numbers of repeats. 00:06:13.01 Well, that was already odd, didn't look like the bacterial viruses that people were familiar with, 00:06:19.02 and then, there were some other observations, 00:06:22.00 one of which came from the extraordinary biology of these particular organisms, 00:06:27.18 of which Tetrahymena thermophila is an example. 00:06:29.29 The way it's got very small chromosomes is because it has a stage in its development, 00:06:36.06 right after fertilization when it chops its chromosomes up into small pieces, 00:06:41.24 and then, these telomeric TTG4 repeats would appear at the ends. 00:06:48.09 How did they get there? 00:06:49.20 This was not obeying rules that were known for DNA, 00:06:54.05 DNA was supposed to be copied from DNA, or at least recombined with DNA that was very similar. 00:07:00.01 You weren't supposed to have two new sequences suddenly joined together. 00:07:04.01 And where did these repeats come from anyway? 00:07:06.10 So, I decided to look in extracts of the Tetrahymena cells 00:07:11.05 and see if one could detect if there was some enzymatic activity that might be able to 00:07:17.16 add this sequence, TTGGGG, over and over again, 00:07:21.05 to the ends of linear DNAs. I didn't know what kind of assay, really, to use at this point. 00:07:32.19 But I did know there was a stage in the life of these organisms 00:07:35.15 when the very short chromosomes got created, 00:07:38.00 and in fact, they were created by chopping up the DNA, for reasons that are a whole different story, 00:07:48.06 and then, the telomeric, as we called, repeat sequences, 00:07:53.05 were being somehow put onto the ends of these DNAs, 00:07:56.19 often in places where there were no such sequences there before. 00:08:02.11 So, how did such a different sequence get joined onto another piece of DNA? 00:08:06.18 That was what we were trying to think about, answering the question, 00:08:10.26 how did these repeats get put on. 00:08:16.19 And so, after a lot of trying different assay conditions, 00:08:20.13 I finally found a mixture that was able to give me the first hint 00:08:24.28 that something was happening, because I could see increasing amounts of this sequence 00:08:29.00 being synthesized, apparently from nothing, in the test tube. 00:08:33.03 And then at that point, I was joined by a new student in my lab, Carol Greider. 00:08:38.17 And so the challenge now was to simplify down this reaction 00:08:45.11 that we had going on in order to be able to see if there really was an enzymatic activity 00:08:52.04 that truly was doing what these experiments had been hinting at. 00:08:56.09 And so Carol was able to refine and strip down the assay 00:09:00.23 to its bare essentials to get to this following point, depicted here. 00:09:05.11 So, we would make a DNA oligonucleotide, which is this colored bar here, 00:09:10.05 made up of the building blocks, you can see, that looked like the end of a chromosomal DNA. 00:09:15.04 And then, by adding the extract of Tetrahymena cells, 00:09:19.05 right at a stage when they were known to be making new telomeres, 00:09:24.06 because the chromosomes at that point in their life cycle were being chopped up, 00:09:28.08 and telomeres were being added. 00:09:30.04 So, I reasoned that they might be enriched for any such enzymatic activity, 00:09:34.27 if such a thing existed. 00:09:36.03 So, we made extract from cells right at this stage, 00:09:40.04 and found just by adding simple salts, and two simple building blocks of DNA, 00:09:44.29 dGTP and dTTP, that in fact, repeats were added to the ends of these DNA chromosome-end mimics. 00:09:54.19 So, this was very exciting for us, because this said, ah ha, 00:10:02.14 this really would be a potential way of solving this problem 00:10:05.29 of DNAs getting shorter and shorter because, now here's a way of making DNA get longer. 00:10:12.08 So, we had an enzyme activity, which was working in the test tube, 00:10:16.13 and adding nucleotides that corresponded to the telomeric DNA sequences, 00:10:21.13 to the ends of chromosomes, and we had to name this enzyme, 00:10:26.25 because we couldn't say Tetrahymena thermophila telomere terminal transferase too many times, 00:10:31.10 and so, there was a bit of a kind of a discussion through the lab, 00:10:36.00 and actually Claire Wyman in my lab came up with the name telomerase, 00:10:39.06 which we thought kind of made sense, because here was a telomere, 00:10:42.06 the "telomer", and then "ase", sounds like an enzyme, 00:10:46.11 and we were thinking of polymerase, where, you know, 00:10:49.28 a polymerase is something that makes a polymer, 00:10:51.27 so, we said, well, here's telomerase, that makes a telomere. 00:10:55.02 So, we were happy to add this new word, which eventually made its way into the dictionary. 00:11:01.11 Now, just to give you an example of the kind of way these reactions looked, 00:11:07.14 what this is depicting here is an autoradiogram, 00:11:12.15 which is a fractionated mixture of the reaction products, 00:11:20.24 which have been labeled with trace amounts of radioactivity, 00:11:23.24 and then they've been fractionated in electrophoresis, 00:11:27.08 in what's called a DNA sequencing gel, 00:11:29.02 and they start with just the very short DNA oligonucleotide, 00:11:35.19 we call it a primer, and then the DNAs get longer and longer and longer, 00:11:39.26 and you can see that there's this lovely repeating pattern, 00:11:43.04 and that pattern is the 6-base repeats 00:11:45.05 of the TTGGGG motif being repeated over and over. 00:11:49.06 And you can see that with time, more and more of this gets added to the ends of chromosomes. 00:11:55.03 So, this was a very visual kind of demonstration of this activity 00:11:59.21 and we could quantify it and do many experiments to try and understand its nature. 00:12:04.00 One of the things we found out, not too long after first finding this activity, 00:12:09.28 was that the enzyme actually had within it an essential ribonucleic acid component. 00:12:16.12 And what this does is depicted here. So, it had a ribonucleic acid component in it 00:12:23.24 which made the enzyme very sensitive to the enzyme ribonuclease. 00:12:27.26 Not what you'd expect if it were just a protein enzyme. 00:12:30.24 And within this RNA, there was a sequence, shown in blue here, 00:12:35.10 which was the exact complement of the TTGGGG sequence that we found 00:12:42.28 was being synthesized in the test tube. 00:12:44.28 So, with such a very strong hint, we decided this really was the enzyme 00:12:51.09 and now the challenge was to find out if this really was behaving in the cells 00:13:01.21 as it was behaving in the test tube, because what we were finding in the test tube 00:13:06.23 was that a DNA, such as a mimic of a chromosomal end, shown in black here, 00:13:12.02 could be elongated by addition of nucleotides and copying the template, 00:13:17.14 this was our model, and that would now make the chromosomal end longer. 00:13:21.29 Was that really happening in cells? 00:13:25.05 So, the answer to this gave us a bonus answer as well. 00:13:32.26 We were answering this question by making small changes in that blue sequence here, 00:13:38.20 in the RNA, and then putting RNAs with the changed sequence into a cell, 00:13:45.02 and asking if the changed sequence in the blue sequence caused a change in the DNA that was added. 00:13:52.18 For example, if this templating mechanism, as it's called, 00:13:56.19 were really true, then if we changed a particular C into a G, 00:14:01.12 then instead of being copied by templated synthesis into a G, 00:14:05.28 it would now be copied into a C, or for example, if we changed one of those A's into a G, 00:14:10.19 it would not be copied into a C, instead of into its normal T. 00:14:15.24 And in fact, that was what we were able to show in experiments that were done in cells 00:14:23.07 by Guo-Liang Yu and John Bradley and Laura Attardi. 00:14:26.19 Now that also gave us, very unexpectedly, another answer to this question. 00:14:34.23 How do Tetrahymena cells respond when telomerase is not working? 00:14:39.26 We weren't actually initially setting out to do this experiment, 00:14:44.29 but serendipitously, one of those changes in the template sequence, 00:14:49.11 or in fact a couple of different ones, 00:14:51.18 gave the sorts of effects that are shown here. 00:14:54.21 So, certain changes, for reasons to this day we really don't understand, 00:14:59.26 would cause the enzyme, instead of simply synthesizing 00:15:04.03 the corresponding repeated sequence with the corresponding mutation in the DNA 00:15:10.20 that was copied from the template, instead these changes caused the enzymes to just choke up, 00:15:16.07 and it wouldn't work, it just basically stopped the enzyme working, 00:15:19.17 as though it just sort of got choked up in its active site and can't work properly. 00:15:25.02 Now, that was very lucky for us because it allowed us to say, 00:15:27.28 what happens when an enzyme that doesn't work is present in the cell? 00:15:32.11 Now, what happened was that the telomeres started getting shorter and shorter, 00:15:39.04 and then over the course of about 20-25 cell divisions, 00:15:42.05 they progressively got shorter and shorter and then the cells ceased to divide, 00:15:46.14 altogether. And so that told us something very important, which we can sort of 00:15:53.05 summarize here. Tetrahymena cells are normally immortal; 00:15:57.23 that is to say, they keep multiplying, pretty much forever. 00:16:02.09 However, all we had to was to inactivate telomerase 00:16:07.10 by this very small, surgical strike, in the central RNA component of the enzyme, 00:16:13.11 which inactivated the enzyme, and now the cells became, if you will, mortal. 00:16:19.21 They could have a certain number of cell divisions 00:16:21.29 during which their telomeres progressively shortened, 00:16:25.01 and got too short, and then the cells ceased to divide. 00:16:27.26 So, loss of functional telomerase was leading to progressive loss of telomeric DNA 00:16:35.29 from the chromosome end, just as had been predicted 00:16:38.18 from the original predictions of DNA replication 00:16:43.03 if there weren't some compensatory mechanism. 00:16:47.23 And so, many cells have sufficient telomerase, and so they can now maintain telomeres at various lengths, 00:16:56.20 but maintain them sufficiently well so cells can keep multiplying. 00:17:00.21 We discovered telomerase really by trying to answer a very basic question 00:17:05.13 which was how do chromosomal DNAs solve their problem of incomplete replication. 00:17:12.19 And it was very much driven by first of all the idea that if there's some interesting problem in biology, 00:17:20.07 you want to go for it and try and understand how nature is working, 00:17:24.07 I think in the back of my head, I was always aware that nature tends to be very conserved 00:17:30.04 in many of its most fundamental molecular mechanisms, 00:17:33.19 as have been amply learned from the central dogma, 00:17:37.07 of DNA information going to RNA going to protein, 00:17:42.02 although interestingly, this was a case in which a perfectly normal cellular enzyme 00:17:46.15 was breaking the rule, and RNA was being copied into DNA, 00:17:50.24 something that people before had been thinking was only the kind of things that certain viruses, 00:17:57.15 and retro-elements did, but they didn't realize until we found telomerase 00:18:02.08 that in fact, copying RNA into DNA can be a normal part of a normal cell's life. 00:18:08.09 So, we did think that this was probably fundamental and likely to be conserved 00:18:14.29 throughout at least eukaryotes. I think what was unknown 00:18:18.19 was really what the implications of this might be. 00:18:21.04 And that has taken many years to work out and is still very much in the process 00:18:26.19 of trying to be worked out, as it affects, for example, what happens to humans, 00:18:32.00 because we seem to live our lives with a sort of transition, if you will, 00:18:37.06 between situations in which telomeres are shortening, 00:18:39.26 and situations in which telomeres are lengthening. 00:18:42.12 And how that balance, and dynamics, are all played out over human life is something that's very interesting, 00:18:48.22 and may well have implications for long term progression, 00:18:53.23 for example, towards certain disease states. 00:18:57.17 And so I think what I take home as a message from this 00:19:01.00 is that one really wants to understand how biology works by working at it 00:19:07.19 in the most sort of curiosity-driven, question driven ways. 00:19:13.04 And not necessarily trying to ask, you know, 00:19:18.05 the question of, you know, some application, but just simply trying to understand how things work, 00:19:23.18 because I think we won't predict, necessarily, what the ramifications 00:19:27.20 of that would be. That's certainly been the case 00:19:30.13 in our adventure in working with telomeres and telomerase.