The SOS Response in Bacteria

00:00:14.09 Well, when I started my graduate work
00:00:18.14 in genetics the big question was
00:00:23.16 what on earth is this gene?
00:00:25.11 How can it do these amazing things
00:00:27.20 that it does? And it was a complete mystery.
00:00:31.04 That was the big question, that was the question that
00:00:34.11 attracted me to genetics and that made me want to spend my life
00:00:38.05 trying to get an answer or get part of the
00:00:40.13 answer. By that time, a lot was known about
00:00:43.09 how genes are transmitted in heredity
00:00:47.11 and some of the things that they control.
00:00:50.21 But nothing was known about the physical
00:00:54.12 chemical structure that was responsible for
00:00:57.14 what the gene could do. And it seemed very inaccessible
00:01:01.06 and very hard to get at. And that was the question
00:01:08.05 that was mostly on my mind. To find a way to work
00:01:10.17 that would help to decide what the gene
00:01:13.14 was like and how it could do what it was doing.
00:01:16.21 I was working with bacteria. Bacteria
00:01:20.17 had become a very good material to work with
00:01:23.04 in genetics, because in 1943 while I was a
00:01:28.11 graduate student, for the first time, it became clear
00:01:32.19 that bacteria had genes like other organisms.
00:01:35.16 Before that, the bacteriologists were telling us that
00:01:40.00 bacteria just change with the environment.
00:01:42.08 When it was established that they had
00:01:44.23 regular genes and they could be used for genetic
00:01:47.18 experiments, that's when I decided I wanted to work with
00:01:51.08 bacteria. I was just beginning the research for my PhD
00:01:54.22 and I had planned to do it with the fruit fly,
00:01:59.03 which was the standard organism in genetics
00:02:01.18 and a wonderful little bug.
00:02:03.09 And I was going to try to understand how
00:02:06.00 radiation induces mutation in fruit fly, Drosophila.
00:02:11.08 When I read that paper proving that bacteria
00:02:15.20 had genes, I really got excited and thought
00:02:19.16 that would be a wonderful material to work with.
00:02:21.21 I mean, they multiply every 20 minutes and you could
00:02:26.03 hold a billion of them in a little test tube.
00:02:28.05 And so just think how many generations
00:02:30.21 you could work with in very short time.
00:02:33.16 So, that's when my advisor at Columbia,
00:02:37.16 Professor Dobzhansky, saw how excited I was about
00:02:43.18 this new development using bacteria for genetics.
00:02:46.20 He suggested that I might want to do my PhD
00:02:49.20 research with E. coli instead of Drosophila.
00:02:52.18 And that's why he sent me to Cold Spring Harbor
00:02:56.16 for the summer. To learn how to work with bacteria.
00:03:00.10 Luria and Delbruck the ones who had discovered that
00:03:04.08 bacteria had genes and they were going to be
00:03:05.23 there in Cold Spring Harbor in that summer.
00:03:07.20 And so I did go and that was one of the
00:03:14.11 most fortunate events in my working life
00:03:18.19 that I've connected with Cold Spring Harbor.
00:03:21.16 Because it turns out, I actually stayed there for
00:03:25.00 11 years. I had a kind of beginner's luck,
00:03:29.00 I think. The very first experiment I did, I had
00:03:31.19 no idea what I was doing. The director at
00:03:36.15 the laboratory at Cold Spring Harbor handed me
00:03:38.20 a culture of E. coli in a test tube and he pointed
00:03:43.08 to an ultraviolet lamp, and said "Go. Induce mutations."
00:03:48.08 So I had to sort of feel my way as to how to work
00:03:53.07 with these bugs. I watched other people and saw
00:03:55.21 how they worked. And I started my very first experiment
00:03:59.05 and the first thing I needed to do was to decide
00:04:03.16 what does of ultraviolet light to use, because there were no
00:04:07.04 published information about that. So I was stabbing
00:04:12.11 in the dark at the first try. And in my first try
00:04:15.21 I overdid the dose, I used doses much too high
00:04:19.13 and the next day, when I looked at my
00:04:22.23 petri dishes, I found that I had put a million bacteria
00:04:26.22 on the surface of each of them. And the next day,
00:04:30.14 nobody grew. There was nothing on any of the plates.
00:04:33.16 Except one petri dish had four colonies
00:04:38.07 growing on it. So four bacteria out of a million
00:04:41.10 that I had put on that petri dish had somehow
00:04:44.00 survived this very high dose. And it occurred to me
00:04:48.23 that maybe those four were mutants resistant to radiation
00:04:54.11 because in Cold Spring Harbor at that point, there was
00:04:59.01 a lot of talk about resistance. Dr. Demerec was working on
00:05:04.17 resistance to penicillin, which had just been discovered.
00:05:07.13 And there was resistance to this, that, and the other that people
00:05:11.04 were working on. So I thought, why did they survive?
00:05:15.09 Just four bacteria out of millions?
00:05:17.11 And I cultured them and tested them
00:05:20.09 and sure enough, they could withstand at least 100x
00:05:23.17 more radiation, both ultraviolet light and later we found
00:05:28.08 x-rays, than the parent strain. It turned out
00:05:32.11 to be a clue to a whole new area that I was just starting
00:05:37.21 to work on. And it led me to understanding that ultraviolet
00:05:43.00 light does a lot more than just induce mutations.
00:05:46.14 It activates what we now know to be 43 genes, at least
00:05:55.11 43 different genes in E. coli that are quiet and
00:05:59.08 silent when the cell is healthy, but are suddenly
00:06:03.19 activated and start turning out their product when the
00:06:09.10 DNA is damaged by radiation or some other agents.
00:06:14.01 My first mutation sort of led me into
00:06:18.10 perceiving that this was going on.
00:06:21.07 And it was a discovery of what we call the SOS response
00:06:25.05 in E. coli, which implies that it's a life saving mechanism
00:06:31.02 which it is indeed. Because of the activities that are turned on
00:06:35.19 by these genes that are activated by the damage
00:06:40.07 promote the repair of the damage and the survival
00:06:45.13 of the cell in a variety of different ways.
00:06:48.02 And the survival of the population, actually.
00:06:51.14 And I think that my experience in Cold Spring Harbor
00:06:54.20 was what sort of softened me up to think in terms of repair
00:07:00.11 because one of the people I met at Cold Spring Harbor was
00:07:04.01 a scientist named Barbara McClintock, who won the Nobel Prize
00:07:08.01 in her 80s, I think. For the amazing work that she did
00:07:12.18 in genetics. It was a pleasure to get to know her and
00:07:16.23 one of the things that I remember best about being
00:07:20.01 at Cold Spring Harbor was the many times that I spent
00:07:24.14 in Barbara McClintock's laboratory, looking over her shoulder
00:07:28.02 and watching her work. When she was making her major
00:07:31.10 discovery, when she found that genes actually can
00:07:35.22 move from one location to another, this was very
00:07:39.06 revolutionary. We always thought that a chromosome
00:07:43.03 was very stable and genes stay put, but
00:07:46.04 she showed that they can jump and they can have
00:07:49.15 important effects when they do. At any rate, one of the things
00:07:54.16 that Barbara was working on, and she talked about this
00:07:59.23 at our meetings at Cold Spring Harbor, she was studying
00:08:05.17 how chromosomes join together when they're broken.
00:08:08.22 Because chromosomes can break as a result of
00:08:13.07 various treatments, and when they do, she noticed
00:08:18.12 that they come together very rapidly.
00:08:21.12 The broken ends join and she was very puzzled about
00:08:28.00 how that could work, but she did make a point of saying
00:08:32.23 that they could -- the broken ends could find each
00:08:36.02 other over a long distance through the very different
00:08:39.02 parts of the nucleus, they would sort of aim at each other.
00:08:43.08 There seemed to be a force that was bringing them together.
00:08:45.12 And since genes are in chromosomes
00:08:49.17 and chromosomes are being repaired
00:08:53.22 when they're broken, it seemed to me that there
00:08:57.03 must be some kind of molecular repair going on
00:09:00.20 inside the chromosome that would involve carrying the gene.
00:09:04.02 So I think that exposure to the way broken chromosomes
00:09:08.07 are repaired made me think in terms of repair when I
00:09:13.02 was considering what was happening in irradiated bacteria.
00:09:17.07 Before the DNA is damaged, let's say by radiation,
00:09:21.18 the DNA is quietly replicating itself, copying itself.
00:09:26.15 And when DNA is damaged by ultraviolet light,
00:09:32.09 for instance, which changes one of the elements
00:09:34.21 of the DNA and stops the replication, the enzymes that are
00:09:40.15 copying the DNA cannot get by that damaged spot.
00:09:44.13 So the whole thing stops. And that's lethal if nothing happens.
00:09:49.07 Because if the replication is stopped and there's nothing
00:09:54.15 that can restart it, that's the end of that cell.
00:09:58.04 But we learned later that E. coli and all cells
00:10:04.15 including human cells, have developed repair mechanisms
00:10:08.07 that can in various ways, take care of that damage and
00:10:14.03 get rid of it, one way or another. There are a number
00:10:16.10 of different ways to do it. And usually, they work very efficiently
00:10:21.04 and the damage just goes away and the replication
00:10:23.18 continues. And that happens, as I say, in a number of
00:10:28.18 ways. For instance, if you shine visible light on the
00:10:32.21 E. coli after it's been damaged by ultraviolet
00:10:35.18 that will take care of getting rid of the damage. The visible
00:10:39.14 light splits that lump that's sitting on the DNA
00:10:43.16 and makes it go away. So that's an immediate repair.
00:10:47.08 And then there's another repair mechanism
00:10:50.03 that's called the "cut and patch" mechanism.
00:10:52.16 Actually, excision repair. Which detects the damaged
00:10:58.16 spot, cuts one strand of the DNA on one side of the
00:11:02.23 damage, cuts on the other side, and thereby removes
00:11:07.06 a small piece of the DNA containing the damage.
00:11:10.22 And that leaves a gap, see, DNA has two strands,
00:11:14.20 so there's one strand that's still in tact and the other one has
00:11:19.23 a piece taken out of it by this cut and patch mechanism.
00:11:22.15 And another enzyme comes along and fills in
00:11:26.20 that patch, using the information on the other strand
00:11:31.04 to do it accurately. So that's another repair mechanism
00:11:35.04 that acts very quickly and that takes care of most of the
00:11:38.02 damage that happens. But sometimes, there's a kind of damage
00:11:42.03 that cannot be easily repaired by these mechanisms.
00:11:46.01 And that's when the SOS response happens.
00:11:49.22 So here you have damage sitting on the DNA
00:11:52.18 which the visible light couldn't help and the cut and patch
00:11:56.04 couldn't help for some reason, and then what happens is
00:12:00.07 the stalled replication, the place where the replication
00:12:04.21 has stopped, generates a signal that says "SOS!"
00:12:10.22 Basically. And then a number of things happen,
00:12:19.03 and I can't really describe them in great detail,
00:12:21.03 but the two strands of DNA come apart into a sort of
00:12:26.08 bubble and that's the signal for the SOS system
00:12:30.20 is single stranded DNA, which normally doesn't
00:12:33.11 occur in the happily replicating DNA. But the single strands
00:12:37.03 bubbling out cause another molecule to come and bind
00:12:42.05 those single strands. And that sets off a sequence
00:12:47.03 which leads to turning on 43 genes. You know,
00:12:52.15 there were a lot of people involved in that, and I haven't
00:12:54.11 mentioned all the people, but the SOS response was
00:12:57.03 a joint work. And this was a young man,
00:13:02.03 whose name was Miroslav Radman, he was a
00:13:05.09 postdoctoral fellow at Harvard and I had written a paper
00:13:10.18 in which I suggested that ultraviolet light was inducing
00:13:17.07 two different consequences in a similar way that seemed
00:13:24.18 as if they were being induced together. And Radman
00:13:31.06 a few years later, had linked to other ultraviolet effects
00:13:37.18 and suggested that they were both induced by the same
00:13:41.01 signal. And one of them was the same one that I had
00:13:46.13 linked, so we both linked A, I had linked it to B,
00:13:51.13 and he had linked it to C. And when we met, I guess it was
00:13:57.11 in 1972, he had just described his end of it. And
00:14:04.13 we met and we put together this observation that
00:14:08.12 I had linked two events that were caused by UV
00:14:12.14 and said that they were induced together, and he had
00:14:15.08 linked two others, and one of them was the common one.
00:14:18.22 So there we had three different ultraviolet
00:14:22.16 effects that we thought were induced in a common
00:14:25.23 way by a single signal. And that was really the beginning,
00:14:31.18 and by the way, Radman was the one who introduced
00:14:33.20 the term "SOS." And it was used in the sense of
00:14:38.13 the international distress signal. So in a way, our
00:14:42.21 putting together our two separate pieces of this
00:14:45.08 began the SOS response to be appreciated.
00:14:49.11 It was a great joy to be a part of the group that
00:14:53.21 was working on DNA repair. I mean, it was a small
00:14:56.15 international group, there must have been probably
00:14:59.13 I'd say 30-35 individual scientists and the group of
00:15:05.01 students around them and so on, who were trying
00:15:08.01 to understand how the DNA changes and how it's repaired
00:15:12.01 and all of that. And being part of that group was
00:15:17.00 one of the most joyful experiences I ever had
00:15:19.20 in science, because it was a group of people with a
00:15:23.06 very common set of goals and I sometimes hear
00:15:27.07 these days that there's a lot of competitiveness
00:15:29.07 in science. It doesn't ring a bell with the way it was
00:15:32.14 in my experience, at all. We shared our information,
00:15:36.23 we shared our strains, we cheered for each other
00:15:40.07 when somebody made an important discovery, and
00:15:44.00 it was a human experience that was extremely
00:15:47.01 satisfying. And when I look back at my work, that's a big part
00:15:53.00 of why I'm glad I did science.

This Talk

Speaker: Evelyn Witkin

Audience:

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

Recorded: September 2015

Talk Overview

Witkin provides a historical perspective on how working at Cold Spring Harbor (CSH) shaped her scientific career and led to the discovery of the SOS DNA damage response in bacteria. At CSH she worked with the pioneers of bacterial genetics, at a time when there was a major discovery every other week. As a PhD student Witkin observed that some bacteria were able to survive high doses of UV radiation. She speculated that these bacteria may have a mechanism to repair DNA making them UV resistant. These revolutionary ideas gave birth to the discovery that cells have a mechanism to repair DNA damage, the SOS response.

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Speaker Bio

Evelyn Witkin

Evelyn Witkin earned her BA in zoology from NYU in 1941 and started her graduate studies at Columbia University where she joined Dr. Theodosius Dobzhansky’s laboratory. She was interested in studying Drosophila genetics, but her interests changed when she realized that bacteria also have genes. She went to CSH in the summer of 1944 to start her… Continue Reading