Discovery Talk: The Semi-Conservative Replication of DNA

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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.

This Talk

Speaker: Matthew Meselson

Audience:

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

Recorded: September 2011

Educator Resources for this Talk

Talk Overview

In 1953, Watson and Crick proposed a double-helical structure for DNA and suggested that it replicated in a semi-conservative manner. This method of replication was not universally accepted as correct, however. In this talk, Meselson recalls the events that led him to meet Frank Stahl and to plan and execute the now famous experiment proving that DNA does indeed undergo semi-conservative replication.

Speaker Bio

Matthew Meselson

Dr. Meselson has made important contributions to the areas of DNA replication, repair and recombination as well as isolating the first restriction enzyme. Currently, he is Professor of Molecular and Cellular Biology at Harvard University, where his lab studies aging in the model organism bdelloid rotifers. Meselson is also a long-time advocate for the abolition… Continue Reading