Skip to main content
Loading Explore guides…
DNA Replication Dynamics · Acedra
Home
Explore
Biology
DNA Replication Dynamics
Sign in to save
Chat
If YouTube asks you to sign in, open the video on YouTube.
Watch on YouTube
Reload player
Transcript
42 segments
Copy
Hide video
0:03
DNA replication. I will split the entire process into two videos - the dynamics of elongation, and the biochemistry of DNA polymerization. This video will focus on the dynamics. More specifically, we will cover the trombone model of elongation, and focus on the leading and lagging strand synthesis, and finally we will inspect the lagging strand and see how okazaki fragments mature into a continuous DNA strand. We will start where we left off in the initiation
0:35
step, which is the replisome… so let me quickly draw it out. It has the RNA primers as the starting points for the polymerization, which is performed by the DNA polymerase III. The primers are made by the moving primosome. And all these components are held together in a complex called the replisome by the clamp loader complex which also carries an open clamp with itself. We have already talked about this stuff in the videos on initiation
1:05
but as a reminder, keep note that the primosome and the polymerase on the leading strand are both going in the same direction if you look at their respective template strands. The polymerase on the other side however, is going in the opposite direction of the moving replisome, and that as we will discuss makes up the lagging strand dynamics. Again as a reminder, the DNA polymerase III is loaded onto the strands because of the Beta-2 clamp, and the clamp
1:34
moves along or slides along with the moving DNA polymerase III. One other thing to pay attention to, is that there are these short RNA primers, however, there are multiple of these as we will see on the lagging strand. Now to help build our understanding about the elongation dynamics, I will label some random positions as a b c d and e, and these will be the locations at which an RNA primer is made by the primosome. Again, these locations
2:04
are randomly selected to help you understand the process. Now, let's dive into the details of the dynamics of elongation. Our focus is on the primosome and replisome movement, as it approaches the position c. If we redraw the replisome at position c, we will start to see some interesting details. So again, in the replisome, the leading strand is synthesized without any interruption because the polymerase III moves in the direction of the replisome.
2:35
Now because the synthesis is in the direction of 3’ end on the newly synthesized strand, the polymerase which is polymerizing the primer at position B will eventually reach the end of the template, so essentially it has moved away from the replisome. And of course to complete the picture you have to show that it is all connected by the clamp loader complex. Since we are at position C now, the primosome will make a new RNA primer here. And now comes
3:04
the interesting bit: There are some weird things to notice here. First, is the tau protein, which seems like it is extended so it can reach the DNA polymerase that is moving away, but in reality, the tau protein cannot extend… it is not exactly elastic like that. Second thing to notice is that the new primer is synthesized while the DNA polymerase is already polymerizing from the previous primer. The third thing is this single stranded DNA that
3:34
gets exposed because it hasn’t been used as a template just yet. And all these three problems so to speak, occur at the same time during elongation. At this interesting juncture, we can start to add answers to these three problems. The first problem of fixed tau protein length can be easily solved if instead the DNA is looped.. Since DNA is pretty flexible, and it can be bent. So if we redraw the loop, it may look something like this. So in this
4:05
DNA loop, you have got the strand that is being synthesized from the primer at position B, and in addition, you have this new primer at position C, ready for polymerization or extension. And as expected, the clamp loader will load a beta clamp onto the RNA primer which is then followed by the loading of the DNA polymerase III. The other thing we noted is this single stranded DNA, which hasn;t been used as a template yet. To protect this
4:33
DNA from enzymatic digestion, single strand binding proteins will come and bind to it and stabilize the single stranded DNA, and as the DNA polymerase moves on the template, these proteins are kicked out. By the way, these single stranded binding proteins are everywhere, even this portion that is ahead of the strand B. If you notice the RNA primer at position c, you see that the leading strand didn’t need new RNA primers, it is still
5:02
continuing without interruption, and this is why it is leading the DNA replication and so it's called the leading strand. On the other hand, we have these portions of DNA that have not been converted into DNA yet, so this strand is lagging behind in DNA replication, hence the name lagging strand. This loop that we discussed is also sometimes called the priming loop, and textbooks will also sometimes call it the trombone loop - trombone is a
5:33
musical instrument. This is because the loop between the clamp loader will change as this polymerase and replisome move, and this resembles the slide of the trombone which changes in size when it is played. So we have talked about position a b and c, and now it is time to move to position d and e. The next position en route is position d, and replisome will move towards that. Before we get there, there is one important detail that you shouldn’t
6:03
miss - this DNA polymerase that started from position b, will eventually reach the end and hit the RNA primer at position a, and when it does the DNA polymerase will be released from that spot - we will come to this detail later in this video, when we discuss okazaki fragment maturation. For now, let's get back on this moving replisome and see what happens when it reaches position d. First thing first, as usual, the leading strand takes the win by polymerizing the template
6:34
uninterruptedly. Now, how about the lagging strand? Well, since we are at position D, we will get an RNA primer and now, the primer at position C will be caught in the loop, which has been used to copy the template DNA. And here is the clamp loader complex that keeps all things together. Now once again, since there is an available primer at position d, a new clamp can be loaded at that position followed by the loading of the DNA polymerase,
7:02
which will start copying the template DNA. Just to fill the details, the single stranded DNA is decorated by the single strand binding proteins. Towards the end of the loop you have the position b, which was used to replicate that portion of the DNA, followed by a primer and so on. As the DNA polymerase from the position C, hits the RNA primer at the position B, the polymerase will fall off. Now, that we have done this for a b c and d positions,
7:30
you have probably noticed a pattern, the leading strand synthesis keeps going without any pauses or breaks. But on the other side, you have these small breaks, which means that the lagging strand is synthesized in small segments. Another way to say this, is that the leading strand is replicated continuously, and the lagging strand is replicated discontinuously because you need new primers for these short segments. We have already said that this trombone loop
8:00
that is made to replicate the lagging strand, changes in size as the components of the replisome move forward. And all these dynamics of the movement is what is explained by the trombone model of elongation. Here is the problem with this model. Funny enough, it has only been observed in T4 and T7 bacteriophages, which are a type of a virus. But we are talking about E coli, or bacteria or prokaryotes in general. And NO one has any evidence that this model is true for prokaryotes.
8:35
Trombone model is therefore simply a model, it is nonetheless the best explanation we can have. Just to make things super clear, when I say that the trombone model has no evidence of existence in prokaryotes, all I am referring to is the existence of the trombone or priming loops. Everything else like discontinuous and continuous replication and the replisome movement is true and has been directly studied. So, all this loop changing
9:03
its size and trombone the musical instrument analogy, is this even true? Theoretically it makes sense, but that's only until a better more direct mechanism is discovered. Since we are on the topic of T4 and T7 replisome and the trombone model, people have also seen multiple priming loops in viruses instead of just one loop, and some researchers think that it may apply to prokaryotes as well. For multiple loops to exist the primosome
9:33
has to move pretty fast from the position D as soon as the primer is made, so that there is enough DNA to be looped out between position d and e. And that should give you another DNA loop. Again, these multiple priming loops are seen in bacteriophages, but no one has actually visualized their existence in prokaryotes. And this completes our discussion on the trombone model and leading and lagging strand synthesis. The major takeaway from this portion is that
10:02
the leading strand is synthesized continuously, whereas the lagging strand is made in small segments. The leading strand synthesis is pretty boring and simple. All the interesting bits occur in the lagging strand because there are these gaps that appear periodically. And these gaps will continue to appear in the lagging strand as replisome moves in the forwards direction. So if we simplify our visualization and only look at the DNA that is replicated,
10:30
we see that a single primer is used on the leading strand to make DNA, and it is continuous but the lagging strand has multiple primers that are used to initiate replication. So you get these small DNA segments going in 5’ to 3’ direction that have RNA in the 5’ end. Not only that, the junction between the primer and the preceding 3’ end of the DNA has a small gap because the DNA polymerase III sometimes will not fully complete the
11:00
synthesize, so you have a few nucleotides that are not replicated. And these are the places where the DNA polymerase was released from, it happens every single time the polymerase III hits the RNA primer in front of it. These segments of the DNA are usually 1000-2000 bases in length, and these segments of DNA are called okazaki fragments, named after Tsuneko Okazaki who discovered them. Now we are ready to understand the okazaki fragment
11:31
maturation stage. The idea of this process is simple. You want to convert the discontinuous lagging strand into a continuous strand by joining the adjacent okazaki fragments. Not only that, you also have to convert the RNA primers into DNA. We are making DNA from DNA, so all nucleobases should be DNA bases, not RNA bases. So the process of okazaki fragment maturation can be understood if you can solve 3 problems. First is something we have already
12:02
hinted at in the first half of this portion, which is that every single time the polymerase hits the RNA primer, it needs to fall off, or rather it will fall off.. So the question is how exactly does the DNA polymerase III fall off, because if it does not fall, the okazaki fragment maturation process cannot start. The second problem is how do we remove the RNA bits and replace it with DNA? Because remember this is DNA replication, and you
12:29
don’t want ribo-bases in your DNA. Then the final question is how to join the adjacent okazaki fragments because DNA polymerases cannot seal these 5’ and 3’ links. Let's solve these problems and start with the process of releasing DNA polymerase III. There are two models to explain this: The first is the collision model, which has undergone multiple revisions. Note that this is a model with little direct strong evidence. The problem
12:59
is simple, there is an okazaki fragment in front of the moving DNA polymerase III, which is connected to a tau protein of the clamp loader complex. The first iteration of the model suggested that when the DNA polymerase III reaches the 5’ end of the okazaki fragment in front, there is some sort of competition between the beta 2 clamp and the tau protein for interaction at the junction. Sounds weird? That is because it totally is! In the second
13:29
iteration the collision model was modified to suggest that there is some internal destabilization going on in the alpha core of the polymerase that causes it to dissociate from the junction - this iteration was very vague. And now the current collision model revises the second iteration and suggests that there is a weakening interaction of the alpha core at the junction because it cannot move ahead. This specifies the destabilizing interaction of the second
13:58
iteration, which goes further and points to the OB fold domain in the alpha core that is involved in weakening and therefore destabilizing the alpha core which causes the polymerase to release. The second model is the signaling model, which invokes that primase involved in the DNA polymerase release from the lagging strand. So if we look at the trombone loop, the primase makes an RNA primer. And this RNA primer synthesis destabilizes the DNA
14:30
polymerase on the other end of the loop. The interaction or signaling is thought to be mediated through the changes in tau proteins of the clamp loader complex. So the synthesis of the RNA primer is somehow the trigger for the polymerase release, and this also suggests some sort of timing mechanism that makes sure that the polymerase isn’t released just before it reaches the end. If you thought that the previous model was weird, well this
14:55
one is even weirder. As bizarre as they appear, there is evidence for both in the bacteriophage replisomes. In prokaryotes it is still unclear how the process actually occurs, but there appears to be a consensus that the OB-fold collision model may be more plausible. And this solves the first problem, and now let's discuss the last two problems and find a solution. Now before things get confusing, let me make it clear that the DNA polymerase III is the
15:25
one that makes okazaki fragments, and following the collision or signaling DNA polymerase III is released, and once it is released from the junction, it is not coming back. And after the release the junction will look something like this, without some unprocessed single stranded DNA template left to copy. These unprocessed nucleotides or the gap in between two unlinked nucleotides is called a nick. In this case, the one side of the nick is
15:57
RNA and the other side is DNA, so we have to remove the RNA primer and fill the gap. So these Nicks in the DNA are recognized by DNA polymerase I. Again, a very important point, it is the DNA polymerase I not III, which recognizes the nicks. This enzyme is similar but slightly different from polymerase III. The C terminal domain of Pol I has two major activities. It has 5’ to 3’ polymerase activity, which is used to make name DNA from
16:30
a DNA template. The second activity is the 3’ to 5’ proofreading activity or exonuclease activity, and this removes any mismatched bases, or single stranded 3’ DNA overhang as well, and that is why it is called exonuclease. The C terminal domain is also referred to as the klenow fragment. The N terminal domain of the pol I, has a 5’ to 3’ exonuclease activity, which is used to remove the RNA primer. Now lets see how to fill the gap, remove the RNA primer and replace it with DNA.
17:06
There are two mechanisms for this process. These are not speculative models, these are factual mechanisms that exist and have strong evidence to prove them. The first is the Nick translation, where the Nick in the lagging strand is recognized by the DNA polymerase I. So Pol I binds and it removes the ribonucleotides one by one by using its 5’ to 3’ exonuclease activity in the N terminal domain. The polymerase will move in the 3’ direction, and as it
17:40
chews the RNA primer, at the same time, it will also add dNTPs to the 3’ end of the previous fragment. So there is RNA removal and DNA synthesis occurring at the same time. Oh, one special thing to note is that DNA polymerase I loading is not dependent on a clamp. And because of the lack of the beta clamp, the processivity of the DNA polymerase I is low, so it only works in the range of 20 to 100 nucleotides. Which means that it
18:13
will chew from the 5’ end upto 100 bases, and at the same time make a copy of about 100 bases of temple DNA. This is perfect because remember that the RNA primer length optimally is around 10-12 bases but they can sometimes be as long as 100 bases, so this makes sure that all RNA primer is removed even if it means that you have to eat a bit into the okazaki fragment. And after processing about 100 bases, the Polymerase I falls off, and
18:43
you are left with a DNA DNA junction that has an open 5’ and 3’ end. The Polymerase cannot join these ends, so another enzyme called DNA ligase comes in and joins these 5’ and 3’ ends. To summarize… in nick translation, the rNMPs are removed one by one and dNTPs are used to replace them one by one at the same time. Oftentimes RNA degrading enzymes like RNases H can also join to help remove the RNA primers, but that still requires
19:18
that DNA polymerase I actually does the final completion. The second mechanism to do this exact thing is called strand displacement, which appears to be more widely accepted than nick translation. This is similar to the nick translation but instead the DNA polymerase I moves too fast and the exonuclease activity doesn’t have much time. Essentially, the polymerization rate is so fast that there is not 5’ end available for degradation.
19:47
Well, let me show you what that means. Essentially you make new DNA using the template, but the 5’ end of the strand in front gets displaced. This length of the displaced strand is equal to the processivity of the enzyme which is about 20-100 nucleotides. The displaced strand is also referred to as the single stranded overhang or more commonly known as a flap. Once this Flap is available, the Polymerase I FEN activity comes into action. FEN stands
20:20
for Flap endonuclease, which is essentially another activity of the 5’ to 3’ exonuclease domain, and it can target both single stranded DNA and RNA. And as soon as it finds the flap, it will cut it out and release it away. And you end up with the same type of 5’ and ‘3 end structure, which can be joined by the help of a DNA ligase. And you can imagine these two mechanisms happening at all junctions in the lagging strand, and as a result the
20:52
discontinuous lagging strand is converted into a continuous strand. And this completes our discussion on dynamics of elongation. In the next video we will discuss the biochemistry
Here to help you learn
Sign in to keep a private conversation with your course tutor.
Ask me anything about the material…
Sign in to chat