The Semi-Conservative Replication of DNA
Transcript of Part 1: The Semi-Conservative Replication of DNA
00:00:05.21 Hello. I am going to talk to you about the semi-conservative 00:00:10.01 replication of DNA. Not so much about the technical details but about how Frank Stahl and I 00:00:16.19 ended up doing the experiment that showed that DNA 00:00:20.20 replicates by the two chains coming apart, 00:00:23.10 each making a new copy and then you get two, 00:00:27.10 each of which has one old chain and one new one. 00:00:30.24 It is hard to know where to start any particular story 00:00:34.06 but let me start with the publication in 1953 00:00:38.00 of the paper by Watson and Crick. There were actually two papers 00:00:41.29 in Nature. The first was about the structure 00:00:45.01 which was based on model building and a little bit of information from X-ray diffraction 00:00:50.15 The X-ray diffraction certainly did not tell you the structure. 00:00:54.09 It told you something about the repeat distance along the helix. 00:00:58.14 It told you that it was a helix and very little more. 00:01:02.05 The model building was model building, 00:01:04.27 so the structure certainly wasn't proven. 00:01:07.13 This was a proposal and many people didn't believe it 00:01:10.20 or maybe didn't even pay any attention to it. 00:01:13.10 The second paper proposed how the molecule might replicate. The two chains would separate, each would guide on its surface 00:01:22.26 the formation of a new chain, so you would 00:01:25.11 end up with two double helices, each one has 00:01:28.03 one of the old chains and one brand new chain. 00:01:30.23 That's called semi-conservative replication. 00:01:33.20 At Caltech, Max Delbruck who knew Jim Watson and was in correspondence with him 00:01:40.01 was pessimistic about this replication scheme. 00:01:44.05 Pessimistic about semi-conservative replication. 00:01:47.07 The chains were wound around each other, so to get them apart 00:01:50.19 unless you break them, you would have to unwind the basic double helix 00:01:55.18 to get the two arms separate. 00:01:56.27 Max thought this would be impossible, hydronamically impossible. 00:02:01.11 And so he proposed that maybe the two chains separated by a system of 00:02:05.21 breaking every several nucleotides along 00:02:09.12 the chain. And he and Gunther Stent published a paper 00:02:12.17 which proposed three different methods for DNA replication. 00:02:15.27 Semi-conservative, as Watson and Crick had predicted, 00:02:19.29 conservative, in which at least conceptually maybe there was 00:02:22.28 a way that a double helix could guide the formation of 00:02:26.09 another double helix just like it nearby 00:02:28.19 and so you would never have to separate the chains at all. 00:02:33.01 You'd have a brand new double helix and a completely old double helix, and that would 00:02:37.02 be the act of replication. 00:02:39.12 And then dispersive, the third way, break the single chains, separate the pieces, and then put everything back together again. 00:02:47.18 I visited Max in his office. I was in Chemistry. 00:02:52.24 I was a student of Linus Pauling. 00:02:55.01 Max was over in Biology, and he told me 00:02:57.04 about this problem, and it occurred to me that maybe one could 00:03:01.17 do an experiment to find out the mode of replication of DNA 00:03:06.04 based on the use of heavy isotopes. 00:03:08.15 I wish I had time to tell you about why I thought about heavy isotopes 00:03:12.08 but it did have to do with taking a course which was a piece of luck 00:03:16.11 Linus Pauling's course on the nature of the chemical bond. 00:03:19.10 in which deuterium and hydrogen bonds played an important role. 00:03:23.17 Nevertheless, the idea was to label DNA with something heavy, 00:03:29.00 I don't think I thought about, oh yes, it was deuterium I thought about at the time. 00:03:33.05 Then to label it by growing bacteria or phages 00:03:37.11 in heavy medium, deuterium medium, 00:03:40.00 and then switch the growth to light medium 00:03:42.15 and then put all this in the centrifuge and look so see where the 00:03:45.20 DNA went-up to the top if you adjusted the density right 00:03:49.12 if it was all light, down to the bottom if it was all heavy, 00:03:54.03 and in the middle if it was half heavy and half light. 00:03:57.05 That's an oversimplification, but that's the way I thought about the experiment 00:04:01.09 at that time. This was around 1954 sometime. 00:04:05.14 I then went to Woods Hole as a teaching assistant 00:04:08.25 for Jim Watson (he had lived at Caltech the previous term) 00:04:14.02 in the physiology course. 00:04:17.13 And one day as a teaching assistant in that course 00:04:20.17 Jim Watson and Sydney Brenner, who were teaching the course, 00:04:24.11 were in an upstairs room in a building called the Lillie building, the Marine Biological Lab, 00:04:30.24 and Jim looked through the window and pointed down at a tree 00:04:33.18 under which was sitting a man who was, you couldn't tell from a distance, 00:04:38.05 but he was selling gin and tonic. 00:04:40.18 He had a big jug of gin and a big thing of tonic, and some ice and some glasses and some limes, 00:04:45.22 and he would sell gin and tonic to passersby, and with the profits he could buy some gin and tonic for himself. 00:04:51.06 It was called the gin and tonic tree. 00:04:53.29 But Jim was saying that this guy thought pretty much of himself, 00:04:57.15 so let's give him a really tough experiment to do in the Physiology class, 00:05:02.14 the famous Hershey-Chase experiment done by two people over a period of time, 00:05:06.02 Hershey and Chase, and see if Stahl could do it all in one afternoon 00:05:08.28 by himself. Well I thought that was pretty rotten to gang up on this poor guy 00:05:13.13 So I went down and introduced myself to him and told him what lay in store for him. 00:05:18.07 And he told me what he was doing in phage genetics 00:05:21.07 and that he would be at Caltech the next year. 00:05:23.28 So we agreed to try to do this experiment together, ourselves, on how DNA replicates 00:05:29.03 when he got to Caltech. I had to finish my X-ray crystallography first 00:05:33.25 before Frank would let us start because he said it would be bad for my character to go ahead and start some new project 00:05:40.14 when I hadn't finished my thesis work, 00:05:43.03 which was X-ray crystallography. 00:05:44.19 Finally I got the X-ray crystallography done 00:05:47.21 and we could start the experiment. 00:05:50.05 Frank and I decided that we should develop the method 00:05:52.26 for doing this density gradient centrifugation 00:05:55.24 and so we spent quite a long time, more than a year, 00:06:00.00 developing a method that could separate macromolecules in a density gradient. 00:06:04.09 And to do that we put in pure DNA 00:06:07.03 into a centrifuge and turned it on to see what was happening 00:06:10.16 and to our amazement a density gradient was forming 00:06:14.20 before our very eyes. 00:06:16.14 This was because the cesium ion is quite heavy, quite dense, and it 00:06:22.10 tends to settle to the bottom in a powerful centrifugal field 00:06:26.09 and the diffusion wants it to go back up to redistribute it 00:06:29.25 and at equilibrium you have a density gradient in which there is more cesium chloride 00:06:34.25 near the bottom than near the top. 00:06:36.27 And so the density gradient forms automatically. 00:06:40.08 We didn't expect this. 00:06:41.13 We saw it happen, we thought we would have to make a preformed density gradient 00:06:45.00 in the centrifuge cell 00:06:49.00 but the centrifuge itself makes the density gradient. 00:06:51.14 So to make a long story short, we grew bacteria in heavy nitrogen medium 00:06:57.14 and then after many generation of growth, so that everything would 00:07:01.26 be labeled with heavy nitrogen, we switched the bacteria, centrifuging them and resuspending them 00:07:07.25 in a light medium and took samples at various times. 00:07:11.26 The first experiment Frank had warned me I would mix it up if I did both directions at once, 00:07:18.14 if I did heavy to light and light to heavy. 00:07:20.16 And I said, "no, I'll color code the tubes," and I mixed it up completely. 00:07:25.25 So it was unclear exactly which tubes were which. The second experiment 00:07:30.28 we labeled experiment number one and that is what we published, 00:07:35.14 along with experiment number 2, which was a repeat. 00:07:38.23 And what we found of course was that 00:07:42.09 the bacteria at one generation had only one kind of DNA. 00:07:46.25 It had a density half way between heavy and light. 00:07:49.26 And at the second bacterial generation, there were two kinds of DNA, 00:07:53.19 half heavy, and fully light. 00:07:56.27 Now we had to write this paper up. Actually we were rather sluggish in writing it up 00:08:01.06 and so Max Delbruck took us to the marine lab 00:08:04.26 at Corona del Mar, and locked us up in a tower room. 00:08:08.00 He literally did. Mary Delbruck, Max's wife, would bring us meals, 00:08:11.25 but then locked the door again, 00:08:15.05 until we- and there was a typewriter- 00:08:16.13 until we produced a draft manuscript, which we did. 00:08:20.06 But there was a question how to write this up. 00:08:22.01 There were two ways that we discussed. 00:08:24.20 One way is to start with a hypothesis, the Watson-Crick hypothesis, 00:08:28.25 and say here's a test, we do this experiment. 00:08:31.26 and see if it works out the way they said it should. 00:08:36.13 And that is certainly one way to do an experiment, and Richard Feynman, who was very close to students in those days 00:08:41.26 He would come over to our parties and so on. He thought that we should write it up that way. 00:08:47.08 The other way would be to write up exactly what your experiment said, no more, no less 00:08:53.19 without reference to any hypotheses at all, 00:08:56.00 and then at the very end say whether it may agree with some hypothesis. 00:09:00.15 We chose the last way, partly because we thought, well, if you are trying to test a hypothesis, 00:09:07.01 the only way to really be sure that you are going to be right 00:09:10.03 is to know all possible hypotheses. 00:09:13.26 And since nobody can know all possible hypotheses 00:09:16.06 just because your experiment might agree with one of them 00:09:18.29 doesn't prove that that is the right hypothesis. 00:09:21.02 There could be another one that your experiment agrees with 00:09:24.20 So it seemed to us more elegant to write our paper 00:09:27.21 up in terms of subunits, and only at the end say, 00:09:30.19 "ah, these subunits could be the single chains of the Watson-Crick model," 00:09:35.13 which of course they were. So that is what we did. 00:09:37.22 And then this diagram here shows the result in terms of subunits 00:09:42.10 not DNA chains. What we did was blessed by a lot of accidents. 00:09:48.11 The accident of being at Caltech, 00:09:50.02 the accident of meeting each other at Woods Hole, 00:09:52.10 the accident of having Max Delbruck impress you 00:09:55.27 with his deep pessimism that DNA couldn't possibly replicate 00:09:59.07 the Watson and Crick work the way it does. 00:10:02.26 And finally the accident of finding out that the centrifuge itself 00:10:06.16 will make a density gradient, you don't have to 00:10:09.06 make a pre-existing one. 00:10:11.01 The effect of this experiment is worth saying something about. 00:10:14.13 When the DNA structure was proposed, a lot 00:10:16.14 of people didn't believe it. 00:10:18.01 And there were not a lot of references to it in the literature. 00:10:20.18 for the first several years after 1953. 00:10:24.04 After all, it was based on model building, which doesn't prove anything. 00:10:27.23 It looked so beautiful that some people were convinced 00:10:31.04 that it had to be right because it looked so right. 00:10:34.19 Other people I think were convinced that it couldn't be right because it looked too good to be true. 00:10:40.04 In any case, it was just a model. 00:10:44.09 The evidence from the X-ray diffraction was really not very supportive. 00:10:48.22 It showed that the repeat distance was a certain 00:10:52.04 distance and that it was helical, but you couldn't 00:10:54.14 deduce any of the details from those early X-ray pictures. 00:10:58.02 I think the effect of our experiment wasn't really a discovery, 00:11:02.22 It was a psychological effect. It made the DNA seem real. Suddenly you could 00:11:07.22 see bands in a centrifuge. 00:11:09.27 that were behaving just in the way Watson and Crick said they should. 00:11:14.02 And I think that the main value of the experiment was 00:11:18.04 that it had the psychological value of convincing a lot of people 00:11:21.18 that the DNA structure had to be right. 00:11:24.29 Let me say something about that molecule. 00:11:27.20 This molecule essentially gave orders to a whole period in the development of molecular biology. 00:11:34.12 Here is this double helix, standing there, 00:11:36.16 and it's saying, "Here I am. I have two chains. Go find out how I replicate." 00:11:41.13 "Here I am. I have four kinds of bases." 00:11:44.05 "Go figure out how that is converted into making proteins." 00:11:47.26 "I sit in the nucleus. Proteins are made out in the cytoplasm in eukaryotes." 00:11:52.21 "Go figure out what it is that takes this information from me out to the cytoplasm." 00:11:56.21 "My information being made up of these bases changes once in a while." 00:12:01.09 "That's called mutation. Go figure out how that happens." 00:12:04.08 "Every once in a while there is something called genetic recombination." 00:12:08.10 "Go figure out how I am..." What I am trying to say here 00:12:11.16 is that if I showed you a model of say a polysaccharide, 00:12:14.18 would it tell you what experiment you should do next? 00:12:17.11 This molecule, DNA, is like the wizard of Oz 00:12:20.11 standing there (except unlike the wizard, this is a true molecule) 00:12:24.04 standing there and telling you essentially the whole agenda 00:12:29.25 for the future of science for the next 20 years. 00:12:32.07 Now that's a completely different mood and attitude, I think, 00:12:37.03 from the science that went before it 00:12:39.15 and the biological science that came after it. 00:12:42.25 So that was currency of this time. 00:12:44.06 It was the DNA molecule telling you what the problems were, and what you had to go out and solve.