Discovery of Telomeric DNA and Telomerase

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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.

This Talk

Speaker: Elizabeth Blackburn

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: February 2012

Educator Resources for this Talk

Talk Overview

In this talk about the discovery of telomeric DNA and telomerase, Elizabeth Blackburn explains that with each round of replication, the protective repeats, or telomeres, on the end of chromosomes shorten, eventually leading to cellular senescence. Blackburn and her colleagues reasoned that there must be an enzyme that rebuilds the lost telomere so cell division can continue. She explains how this enzyme, called telomerase, was found and discusses its key role in cellular aging.

Speaker Bio

Elizabeth Blackburn

Dr. Blackburn is a leader in the area of telomere and telomerase research. She discovered the molecular nature of telomeres-the ends of eukaryotic chromosomes that serve as protective caps essential for preserving the genetic information – and discovered the enzyme telomerase, which replenishes telomeres. Blackburn is currently a Professor in the Department of Biochemistry and… Continue Reading