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DNA Replication Basics
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0:00
Hi. In this video we will study DNA replication, which is the process of DNA a making a copy of itself to pass on to a new cell. The specific topics we'll talk about is that DNA is the template for DNA synthesis. We'll talk about four required elements for DNA replication. We won't be able to stress enough that DNA is always synthesized in the 5 ' to 3 ' direction. This will cause some problems for DNA synthesis, as you'll see.
0:36
As DNA is synthesized there are going to be leading and lagging strands because of the requirement that DNA is always synthesized 5 ' to 3 '. And in a second video we'll talk about the problem of DNA and telomeres. Remember that was one of the 3 essential elements of chromosomes. There's a problem replicating telomeres. And then we'll also talk a little bit about organisms which don't have DNA genome genomes. They have RNA genomes.
1:07
And what that means for our view of biology and what opportunities that gives us for biotechnology.
1:16
So in today's lecture, replication, we'll talk about DNA to DNA. In the next set of lectures on Wednesday, we'll talk about transcription and translation. These are words you have to memorize. Replication transcription, translation.
1:37
On this slide we see a double-stranded piece of DNA that's going to get replicated three times into eight copies of itself. The key point here being, again, that DNA replication to semi-conservative, meaning each of the daughter strands gets one of the original gold-colored strands and it synthesizes a new, approximately dark red strand. And as you can see, each of the daughter cells from any parent has one of the original strands and makes new ones.
2:13
Just as a little preview, we'll mention that DNA replication is not perfect, that errors get made, and errors are mutations and these can cause diseases such as cancer. And so certain cells have actually learned to hold on to the original strand. Stem cells have asymmetric DNA synthesis, but that is pretty rare in biology.
2:45
On this slide we see for requirements for DNA a replication. Replication requires an origin of replication, the site for it to begin. It will not begin randomly any place. DNA polymerase always requires a primer to get the strand going. That's a requirement of DNA polymerase. This is in contrast when enzyme we'll meet later today, in fact, RNA polymerase, which does not need a primer to start its synthesis. All polymerases need a template from which to copy, and all nucleic acid
3:26
synthesis goes in the 5-' to 3-' direction. And we'll again see some of the challenges that causes for the replication machinery.
3:37
On this slide we see a growing strand of DNA in red here. Here is the end of the existing piece of DNA. This is the 3 ' hydroxyl. Again, 1 ', 2 ', 3 ', 4 ', 5 '. Again, the "primes" are important because those are the positions of the sugar. The numbers without "primes" are for the bases. So you have the 3 ' hydroxyl group. A new nucleotide comes in and it will allow the hydroxylate group to attack this first phosphate group. You're going to cleave pyrophosphate out and you're going to have a
4:20
connection between the 3 ' carbon and the 5 ' carbon.
4:26
This connection will only occur if you are bringing the proper base to base pair with the existing base on the template strand. In this case G is on the template strand and C is the nucleotide that you're bringing in. Notice also that the template strand is anti-parallel to the growing strand. And that's depicted both explicitly with the labeling here and also in this particular instance with the sugars being drawn upside down.
5:00
If you were to bring in a base they didn't base pair with G, say an A, 999 times out of 1,000 the polymerase would not incorporate it here at the growing 3 ' hydroxyl group. Mistakes do happen very rarely, but most of the time that would get rejected.
5:23
On this slide, you see the second of the four requirements, and that is that DNA synthesis begins at replication of origins. This is a sequence, a piece of DNA a that is defined by a specific sequence. The sequences is recognized by certain proteins that bind to the replication of origin, and these proteins help unwind the DNA strands, creating a bubble. And now you're going to be exposing bases and allowing them to get replicated.
5:57
One important thing to remember here is that there are going to be four replication processes. There's going to be a replication process going in this direction on this strand, there's going to be a second one at that position going in that direction, there's going to be a third replication event going that way, and a fourth replication process going on at that end. So this is one replication bubble. There are two replication forks.
6:30
Here's one replication fork, here's a second replication fork, and at each replication fork both strands are being copied. So four simultaneous replication processes.
6:45
Here's a schematic of a growing replication bubble. Let's focus for the beginning just on the leading strand. We'll talk about the lagging strand in a second. The first thing I want to point out is that we can deduce the directionality of the DNA because of the fact that the leading strand is growing in this direction. We know that the leading strand has to be 5 '. DNA grows 5 ' to 3 '. That DNA a is anti-parallel, so if this is going 5 ' to 3 ' in this
7:21
direction, this top strand is 5 ' over here and 3 ' over here. So it's growing in the 5 ' to 3 ' direction. Since DNA is anti-parallel you know that this is the 5 ' end of this strand and this is the 3 ' end of this strand. And again, since this is anti-parallel 5 ' over here for this growing strand on the bottom. This is the 5 ' end and this is the 3 ' end. Now I'm going to erase some of these markings so that I don't get too confused. OK, so I have erased some of those markings.
8:00
And now let's imagine what's going to happen as this strand keeps growing. This strand will have to burrow in to the center here to keep this unwinding, to keep copying the strand. And it will just keep on going for as long as there is a template to be copied.
8:21
This motion is depicted sort of in these three snapshots. The smallest bubble, a little larger bubble, and a larger bubble, just because as you keep unwinding at this location and this location to keep copying, the bubble will get larger and larger as you're copying it. And as you can see for the leading strand, as the bubble gets larger, it just keeps extending and it keeps getting larger and keeps getting larger. Same thing is true on this end.
8:53
The leading strand on the bottom is just going, going, going, and it will just continue to make a very long strand as long as it is needed.
9:05
The problem for the lagging strand is that it synthesizes in the wrong direction. As this bubble grows larger going in this direction, on the bottom in the lagging strand, you are still required to synthesize in the 5 ' to 3 ' direction. So what happens is, the polymerase jumps ahead, sequences back, jumps ahead, sequences back, jumps ahead, sequences back. We'll see that better on another slide. But the point here is just no that's a lagging strand has to put down many
9:40
primers and it can synthesize many small little pieces of DNA, which then later on will have to get stitched up.
9:50
In this picture, we see half of a replication bubble. We see one replication fork. The rest of the replication bubble would be here, on this side.
10:02
And as this replication fork moves out wider and wider, farther and farther away from the center of the origin of replication, we're going to look at the consequences. Now, I have to apologize. These figures are not very related. Notice that down here we have 5 ' at the top, and here it's a 3 '. So you'd have to flip this over for these two to relate. That's not important. This is the orientation that we showed in the previous slide.
10:31
Here we have a leading strand synthesizing 5 ' to 3 ' and it's going to just continue to synthesize as long as you can go. The first amount of material that's synthesized is in dark red. Then we look at it a little later and you can see that in lighter red or pink there is additional synthesis, so the most recently synthesized base of the leading strand is the one that's furthest most near the replication fork. For the lagging strand, again, it's a little more complicated.
11:05
Remember, here is the center of the replication of origin, so this piece was synthesized in the 5 ' to 3 ' direction. And then the polymerase jumps ahead to this one and it's going to synthesize in the 5 ' to 3 ' direction until it bumps up against this fragment. And then, as you see, these pieces are in dark red. There was a little more synthesis in pink, right here, until it stops. And then it jumps ahead again and is synthesized the 5 ' to 3 ' direction.
11:45
One more slide of the growing replication bubble to emphasize what gets synthesized most recently-- again, you can always think of the oldest pieces of DNA in general are those that are at the center of the origin of replication. This is a symmetrical structure here, so these are the oldest pieces that are here. This one is the first part of the leading strand. Once again, the book has flipped the orientation, but that shouldn't bother you because you're getting good at looking at the 5 ' to 3 '
12:14
direction of things. So this is a very old piece of DNA. This is an old piece of DNA. This is the first lagging strand. This is the first lagging strand on the side. At some time later, the forks have moved out and you can see in red what's been synthesized.
12:34
So if I were to ask you what is the last piece of DNA that has been synthesized in the lagging strand, you would go to the replication fork, find the first piece that you come to, and then you would go to the end. Here's the end. So here is, by symmetry over here, those are the last pieces of DNA that have been added to the lagging strand.
13:05
So in this slide we're going to look at some of the details of the lagging strand synthesis. We're not looking at the leading strand here, so this is just half of one replication fork. The replication fork is moving in this direction, and approximately here is the origin of replication. So as we've seen before in the lagging strand, you jump ahead, you synthesize back, you jump ahead, you synthesize back. Here we're synthesizing back. And then there'd be another step.
13:36
We would have a primer here and we would synthesize back because you always have to synthesize in the 5 ' to 3 ' direction.
13:46
But now we can see some of molecular details and some of the problems. First of all, why is some of this drawn in blue and some of this draw in red? On this slide, DNA is in red and RNA is in blue. Now, we're just replicating DNA so we don't need any RNA to be there. We don't want any RNA in the final product, so what is it doing? It turns out that RNA can be synthesized by an enzyme called DNA primase, which is an enzyme that adds RNA to a DNA template.
14:26
And this can be done without a template, meaning the first base that gets put down here does not have a 3 ' hydroxyl on which to attach to. It starts to novo. After a few bases of RNA, somewhere between 5 and 10, not a great deal, now you have an established primer. You have a 3 ' hydroxyl ready to be added and so DNA polymerase comes in and it's going to add a DNA nucleotide to the existing 3 ' RNA hydroxyl group. And then it's going to continue to synthesize DNA.
15:07
And it's going to synthesize until it hits the previous RNA primer and then it's going to chew way the fragments of RNA and lay down DNA. And it's going to stop when it hits this DNA until all that's missing is a phosphate group between the two sugars, or a bond between a phosphate group and a sugar. An enzyme called DNA ligase will add that phosphate, make that connection between the phosphate group and the sugar, making a complete strand of DNA.
15:46
So, for the lagging strand, we're going to use lots of RNA primers, we're going to synthesize a lot of short DNA a fragments in reverse, we're going to go until we hit RNA, chew it away, stop when we hit DNA again, and then we're going to seal the nicks. And then the last thing that I point out-- this is a term you have to remember-- that these short fragments of DNA on the leading strand are called Okazaki fragments, after the scientist who first discovered them.
16:24
This slide provides little detail about the enzyme DNA ligase. It's a two-step process, and it takes a molecule of ATP, which is used for RNA a synthesis, but it is also, as we mentioned, a high-energy intermediate molecule, which can be used to power different reactions. And the goal of the ligase enzyme is to seal a nick in the backbone. The first step, this adenosine triphosphate is added to the existing phosphate group to activate the 5 ' end.
17:01
And then, in a second step, that is going to be released as you get bond formation. And now you have an intact backbone of this DNA molecule. So, by hydrolyzing in this high-energy intermediate ATP, we've paid the energetic price to do this otherwise challenging reaction of sealing the DNA backbone.
17:29
So here's a more detailed picture of the replication fork and here is an annotated version. So, in this slide we see a lot of the proteins and a lot of the processes needed for the replication. Let's first talk about the unwinding step done by an enzyme called DNA helicase. This is going to unwind, since this is twisted around each other. You might remember from old telephone land-line cords, when they would get all twirled up that it would take some energy to pull that cord apart.
18:08
And that is something that is required here at this replication fork. If you would just pull these apart without unwinding, this part of the DNA a would get wound up even tighter and eventually you would have a situation which could not continue. We'll see on the next slide how DNA helicase case goes about unwinding the strand. If we look at the leading strand, DNA polymerase is just chugging along, adding new nucleotides in the 5 ' to 3 ' direction.
18:42
DNA polymerase will bind to DNA when it's getting started, and then this protein called the sliding clamp, or PCNA, will attach to the polymerase. And this helps lock the polymerase on so that it won't come off the strand prematurely. On the lagging strand, the action trails a little bit. Here is the polymerase with the sliding clamp and it's synthesizing this way. On the back end of this RNA primer, here we see the enzyme DNA primase,
19:19
which is actually adding an RNA primer because it doesn't need a template to do that. Excuse me, it doesn't need a previous primer to do that. You just put the bases down automatically. Eventually, this DNA polymerase will hit the DNA section, it will jump off, it'll come back here, and it will start synthesizing down until it hits this RNA primer, chews up this RNA, and stops when it hits the DNA. Now, single-stranded DNA is subject to various chemical and enzymatic
19:51
processes, so single-stranded DNA is protected. This is a protein called single-stranded binding protein, and it's going to cover the DNA until the polymerase gets there and starts doing its synthesis. Now, the last little wrinkle in this picture, which I won't go into but I'll let you know about, is that, in reality this polymerase is physically located pretty close to here. This is going to loop around, even though this is sort of the unfolded
20:23
geometry, and I think it's the only thing that's clear to see. If you follow this YouTube link, you can see a picture of how this is done. But, I warn you, it's a little complicated. Back to the helicase, here's the polymerase it's adding. This is the newly synthesized strand. This is the template that is being read, and we're going to unwind the replication fork that's moving in that way, so we have to unwind this the way in which the helicase relieves the torsional strain that's caused by the
21:00
unwinding is that it works with an enzyme also called DNA gyrase. It does this by cleaving one of the DNA strands, and then passing, essentially unwinding it one time and then re-sealing it to relieve the torsion created by the unwinding. Gyrases and helicases are both part of a larger class of enzymes called DNA topoisomerases, in which case it's the same DNA molecule but the topology is slightly changed. In this case, the number of times DNA is wound around itself.
21:47
And then here again is the video of the replication fork. We'll take a break now and then we'll go on to discuss how DNA is replicated at the ends of chromosomes. Thanks.
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