Caught On Video: How DNA Replicates
You may recall from watching an animation in some science documentary the elegant way in which the double helix DNA molecule supposedly duplicates itself. An enzyme called helicase makes the helix unwind and unzip. Primers are added by a second enzyme. And then DNA polymerase moves in and rebuilds the matching half of each strand, leaving two molecules, each with one old backbone and one new.
Those animations may need some reworking. Writing in the journal Cell, researchers report that they’ve captured replication of a single DNA molecule on video for the first time. The process they observed doesn’t go quite as smoothly as those animations; some steps are quick, others slow. The process stops and restarts. Stephen Kowalczykowski, one of the authors of the paper, likened it to heavy traffic moving on the freeway, with lanes stopping, starting, and occasionally outpacing each other from time to time.
Stephen Kowalczykowski is Distinguished Professor of Microbiology and Molecular Genetics, and of Molecular and Cellular Biology at the University of California, Davis, California.
IRA FLATOW: You may recall from watching an animation in some science documentary– and there have been plenty of them– you know the elegant way in which the double helix DNA molecule duplicates itself. You know we always see it unzipping, and then duplicating, coming back together. Well, those animations may need some reworking. Because writing in the journal Cell, researchers report that they’ve captured the replication process for a single DNA molecule on video. They’re claiming it might be the first time it’s ever been done. The process they observed doesn’t go quite as smoothly as those animations we see on TV do.
Stephen Kowalczykowski is a professor at the University of California in Davis and one of the researchers on those findings. He joins me by phone from France this evening. Thank you for taking time out of your vacation, Dr. Kowalczykowski, to be with us.
STEPHEN KOWALCZYKOWSKI: Well, it’s my pleasure. It’s not a vacation– I’m out here for a conference– but I’m pleased to do this because I’m a longtime fan of the show, so, my pleasure.
IRA FLATOW: Well, thank you, you’re welcome. Thank you very much. How do you get a movie, make a movie, out of a DNA replication?
STEPHEN KOWALCZYKOWSKI: Well, it’s a lot of luck and a lot of work. So, what we do is initially need to trap or capture a single DNA molecule. And what we do alternatively is either attach it to the surface of a glass slide which we’re going to use in the fluorescent microscope or in some cases we attach it to a bead and capture that with an optical trap– a infrared laser. And then, of course, you can’t see DNA by eye, so what we do is we stain it with a fluorescent dye. That allows us to image it. And then the other thing you need to know is DNA normally is a coiled molecule so to see everything that’s going on DNA we need to extend it. And we typically do that by a solution flow. So flow will extend DNA that way flow extends things that are attached in the water like seaweed. We can also use a second optical trap but we didn’t use that in this particular study.
And then basically we need to introduce the proteins and we do that a number of different ways. So having done all that, and then there’s sort of a start button, and we start reactions different ways. In this particular case, we started them by adding all the nucleoside triphosphates and then if all goes well you can watch this in real time.
IRA FLATOW: Wow. And in the video you shared with us– which is on our website at sciencefriday.com/DNA– looks like tiny white streaks moving along a black field. What are we seeing there? Those streaks that you talked about, the fluorescent.
STEPHEN KOWALCZYKOWSKI: That’s correct. So at the very start zero time what you see are just bright white spots on a black background. And each one of those spots is a DNA molecule, is a template molecule. It’s a 8.6 kilobase pair of DNA molecule, which is visualized by virtue of this fluorescent dye that’s bound to it. And then when the video starts what you see is some good fraction of those template molecules elongate and they’re moving towards the right. And the reason they’re moving towards the right is because the flow is coming from the left. And at each one of those molecules, if you look carefully, you can notice at the right side it’s just a little bit brighter and that’s because there’s more DNA there. That’s the template DNA. It’s circular. And so what you’re watching is a replication of a DNA molecule in real time.
IRA FLATOW: And in your paper what you write about says is that what surprised you is that the replication is not like we see in the movies or the descriptions in the textbooks.
STEPHEN KOWALCZYKOWSKI: Right, so basically, there are many ways of thinking about what you should see. The classic picture is that you have a leading strand polymerase that moves at a constant rate and then the second polymerase, the one that copies the strand in the opposite direction, has to move discontinuously. So it should move in shorter pieces. And what we expected was to see a continuous movement on the leading strand, and we didn’t see that. What we instead saw that the leading strand polymerase would stop sometimes, and in fact, it would change speed sometimes, and then it would do this in a random way, so it showed stochastic variations in speed punctuated by these pauses. And that was unexpected. And what we could then see subsequently is that the DNA helicase that you mentioned in the introduction would oftentimes proceed independently of the polymerase.
IRA FLATOW: I’m Ira Flatow. This is Science Friday from PRI, Public Radio International.
So if I could paraphrase what you said and tell me if I get this right. You know you have the two halves of the DNA ladder, the double helix, they’re sort of zipping down and then each side is going to get reproduce to bring the other half that it needs back. But as it was described to me it’s sort of like traffic on a clogged highway on a freeway. You have two cars next to one another and instead of them both coming together smoothly, one starts, one stops, you know, [INAUDIBLE] the car next to you, but you all wind up together.
STEPHEN KOWALCZYKOWSKI: Right so you’re describing the analogy I gave on the terms of the publication. And what that analogy refers to is the coordination of the two polymerases, the leading strand and the lagging strand. One of the other things we expected to see was that there’d be some way that the polymerase has to go through more steps would tell the one that doesn’t go through all the steps that it would need to control and communicate with it with regards to how far it’s gotten into synthesizing its strand. And we didn’t see any evidence of this coordination. But rather what we saw is the stochastic variation.
And the analogy on the freeway was that oftentimes when you’re driving down the freeway traffic and drivers are driving seemingly at random speeds. And oftentimes you think you’re gaining on this person next to you but then oftentimes you’re delayed or slowed and the other person has the opportunity to speed up. And if this is done completely stochastically, at the end, neither of you gain on the other yet you wind up at the same place. And this analogy applies to the way replication works, which is that they don’t coordinate with one another but rather change speeds and pause stochastically.
IRA FLATOW: Well what this says in your paper is that because they’re not zipping together at the same speed that unzipped they may remain vulnerable to damage for a longer period of time than suspected.
STEPHEN KOWALCZYKOWSKI: Well in some ways that is a potential problem. So what happens is, let’s say, in terms of coordination of leading and lagging strands synthesis, that the so-called fragments, these partial fragments, Okazaki fragments, can be different sizes. But the gaps are made up because at some opportunity the lagging strand polymerase can go fast. But the other problem is that if the polymerase stops the helicase that unwind DNA ahead of it creates single strand DNA which, as you pointed out, can be a problem. But the helicase just doesn’t continue unabated. In fact, it continues but it slows to only one fifth of its speed and this we referred to as a dead man’s switch. So that there’s a built in control mechanism.
IRA FLATOW: We’ve run out of time, Dr. Kowalczykowski. I have to say goodbye but we’ll pick this up later. Stephen Kowalczykowski is distinguished professor of microbiology and molecular genetics at UC Davis.
One last thing before I go, I hope you’ve been listening to and enjoying our new podcast Undiscovered. This week on the show it’s the 1940s. A team of scientists at General Electric team up with the military on a series of radical experiments in weather control.
SPEAKER 1: This is something that the military saw one way and the scientists saw another way. The scientist thought, how great if we could steer hurricanes so that they don’t hit the shore, and you know, if we could steer hurricanes away from big cities that would be wonderful. The military was thinking, wow, we could really mess with Cuba.
IRA FLATOW: But one of GE’s staff, a young press writer by the name of Kurt Vonnegut, was watching closely and soon the company’s science would inspire some of his most iconic stories. If you’re a Vonnegut fan, you can’t miss this one. Check out Undiscovered on Apple Podcasts, Pocket Cast, or wherever you’d like to listen, or visit undiscoveredpodcast.org.
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