Okay. So now let's talk about the steps of replication. The first step of replication involves unwinding the double helix. So remember, the double helix is all double helical and attached to each other. But in order for replication to occur, each one of those strands has to act as a template, and it can't act as a template if it's bound to the other strand. So something has to come in and, like, break those hydrogen bonds between the nucleotides so that the DNA double helix will be unwound into two single strands. So the enzyme or the protein that's responsible for this is called DNA helicase, and it attaches to DNA and unwinds the double helix by breaking those hydrogen bonds.
Now, once those hydrogen bonds are broken, stranded pieces of DNA. Right? They are single-stranded in some areas and still double helical, where the helicase hasn't reached yet. So it's not an entirely, like, single-stranded piece of DNA, but it's pretty much single stranded in that area. And so there are these special proteins called single-strand binding proteins that come in and bind to those single-strand regions. So the regions that have been unwound already, and that prevents them from reforming. Right? Because these hydrogen bonds are going to just spontaneously form. Right? You don't need any catalysis of energy or anything like that. So if they're close to each other, they're just going to bind back together unless something prevents them. So this is kind of just like a hat almost that gets put on that DNA to protect it from binding to the other strand, which is still close to it.
And then as the DNA is unwound, what happens is a process called supercoiling, which you may have heard me talk about before. If not, I'm going to talk about it later in this same topic. But supercoiling is essentially where the DNA helix just gets so wound up on each other. You can think of it as a rope. You have two ropes wrapped around each other, and you just keep spinning them. Right? They're eventually going to super coil on themselves. They are going to be wound up really tight.
Now, as the DNA is being unwound in one area, that actually results in that rope spinning that I'm talking about at other sorts of above, or I don't want to use the word downstream, but it's essentially what it is. But it's a different area ahead of that unwinding. So that portion of the DNA is going to be unwound, but it's not unwound yet. And because it's not unwound yet and at this portion being unwound, it's creating this tension in this DNA region and that will result in this kind of rope coiling or what's called super coiling, where it coils in on itself. And there are special enzymes that actually get rid of that, or else the DNA wouldn't be able to be replicated because it would just be so tightly wound up. And those enzymes are called topoisomerases. You may also see it as DNA gyrase, depending on the book you use, but it’s the same thing.
Here you have this double helix. Right? And you have to unwind. You have to break these double hydrogen bonds in order to be able to unwind the double helix to leave them for replication. So here you have your helicase, it’s coming in and it’s breaking these hydrogen bonds. As it’s breaking them, the single-stranded binding proteins are coming in, binding to these single strands and preventing them from reforming these hydrogen bonds that helicase just broke. And as it’s doing this, this region is becoming supercoiled because you can’t just unwind it here without it winding up here. If you don’t believe me really, get some rope or some string or something and try it out for yourself.
And so these topoisomerases come in and they prevent the DNA from supercoiling on itself. So that's the first step, is the unwinding. Then once it's unwound, replication can begin, and replication involves the use of a lot of important enzymes, which we are going to have to learn the names of. So the first thing is the Pol III enzyme. Some of you may see it as the Pol III holoenzyme, depending on your book, but essentially the Pol III holoenzyme consists of DNA polymerase III and some other proteins, accessory proteins that help it replicate.
Now DNA polymerase III is what replicates the DNA. Any of these accessory proteins that are found in this holoenzyme are just accessory proteins. Right? Now, DNA polymerase III has to have these accessory enzymes because by itself, it doesn’t really like to attach to DNA for too long. It’s like I got really other things to do. And so, DNA polymerase III would add about 10, 12 nucleotides, and then it would fall off and be like, oh, I’m ready to go do something else. But these accessory proteins help attach the polymerase to the DNA for much longer so that it can replicate thousands upon thousands of nucleotides without falling off.
So DNA polymerase does the replicating, but without accessory proteins, it would be really inefficient at it. Now to start replication, you have to have DNA polymerase III, but DNA polymerase III actually cannot be the initiator. It can’t be the first thing. It replicates it, but unless you tell it where to start replicating, it’s just not going to do its job. It has to be, you know, it’s kind of like a four-year-old, giving a four-year-old a task. You have to make sure that it’s doing it and starting in the right place. You have to make sure the four-year-old is going to stay at that topic, you know, like you have to help them along. DNA polymerase is very much like a four-year-old).
So, it has to have these accessory proteins that are helping it stay on task, but it also has to have a special protein telling it where to start, and that protein is called a primase. It’s part of a primosome. Some of you may see it as a primosome, but essentially, primosome is just a big complex that contains the primase. Primase is the enzyme, and what primase does is it synthesizes RNA primers. Now the RNA primers bind to the region of the DNA that's going to be replicated. So the primers bind there, and it says, hey, DNA polymerase, you’re a four-year-old, you start here. And so DNA polymerase is, like, yay, there’s where I start. And it comes over and it, like, starts doing its thing, and the accessory proteins make sure it stays on task.
So DNA polymerase III recognizes the primers. It says, This is where I’m supposed to start. It starts DNA replication. Now eventually, notice that these are RNA primers. These aren’t DNA primers, right? But we’re trying to replicate DNA, not this mixture of DNA and RNA. So there’s a different polymerase, DNA polymerase I, that comes in after, you know, polymerase III is doing its thing, sort of comes in behind it and says, okay. This is RNA. It shouldn’t be RNA. So it takes out those RNA primers and makes them DNA, and that creates one long DNA strand, not some DNA with an RNA primer at the beginning.
Now the primers are important for both the leading and lagging strands, but you’ll notice that the leading strand only needs one, because it’s continuous, and so it just continuously replicates, whereas the lagging strand needs a lot more than one. One plus, one-plus, plus, plus, plus, because it needs one for every discontinuous fragment it makes. So let’s look at this. So here we have DNA, it's unwinding. Right? Here’s the helicase, and it’s breaking these DNA fragments as it goes.
Now the leading strand, so it was started back here. Right? The DNA was a double helix, and so it’s, the helicase probably started here. Right? So it started unwinding, DNA polymerase III came in, and it started to replicate. And it’s replicating in this direction, and as long as the helicase is still unwinding things, it’s going to go all the way to the end. The lagging strand, on the other hand, has to actually move in the opposite direction, so it’s moving this way and replicating. So as the helicase is unwinding, it is starting to synthesize fragments.
So here you have DNA polymerase III. So it’s already copied these regions. So it probably bound here and went this way, and then it bound here and went this way. And you can see that it’ll eventually bind somewhere here and go this way too. But that is how the DNA polymerase III is replicating. But like I said, it’s like a three-year-old, it needs accessory proteins to keep it there, and it also needs an RNA primer. Now these RNA primers aren’t very clear in this picture, but they’re here, essentially. So here’s the primer. Here’s this primer. So DNA polymerase probably started here, and it’s working on making this Okazaki fragment is what this is called. I’ll introduce that word in a second.
And then once it’s replicated this region, it’s going to start here where this primer is and replicate to here, to here where the other fragment met. Then the primates will come in and make sure that this is replaced and made into DNA, or the DNA polymerase I will make sure that these RNA primers are turned into DNA. So this is an overview of how DNA replication works. So like I said before, I wanted to introduce some more terms for you. So the lagging strand is replicated discontinuously, and this creates many DNA fragments. And I use the word Okazaki fragment because that’s what that fragment is called, and I’ll introduce that in a second.
So the helix is unwound. Primase adds RNA primers, and then replication starts with those primers, and it continues until it reaches the beginning of the strand or the previous fragment. Now this is occurring on the lagging strand, which is what we’re talking about here. These fragments are called Okazaki fragments. And once you have replication occurring on the lagging strand, you have a bunch of different Okazaki fragments after a while, right because you have just these then discontinuous fragments that are being formed. But eventually, you don’t want a bunch of DNA fragments. You need an entire DNA strand being replicated. And so the enzyme responsible for connecting those fragments is called DNA ligase, and it joins the Okazaki fragments together, which creates a single new replicated strand of DNA.
So here we go. So this is the image that we use at the very, very beginning, and here were two Okazaki fragments. And you can see DNA ligase has come in here, and it’s going to seal them together. So this, I will add DNA strands and will link them here, or just like usually a DNA nucleotide, it will link the two strands together, and then it will jump here and do the same thing for this strand that will be made and so on and so forth until all the Okazaki fragments have been connected together and become one DNA strand. So that is the overview, DNA replication or really just the detailed version of DNA replication, so hopefully, that’s clear. With that, let’s now move on.
