Cell Biology | DNA Replication 🧬

What's up, ninja nerds? In this video today, we're going to be talking about DNA replication. But before we get started, please continue to support us by hitting that like button, commenting in the comment section, and please subscribe. Also, down in the description box, we have links to our Facebook, Instagram, and Patreon account. Go check that out. All right, ninja nerds, let's get into it.

All right, ninjas. When we talk about DNA replication, the first thing that we need to talk about is a couple of fundamental points, very important things that we're going to build on throughout the process of this lecture. And so, the first thing I need you guys to know is, what in the heck do we do DNA replication for? And the whole point is, is that in order for cells to be able to replicate and make more cells, we need the DNA within those cells to replicate because the DNA is pretty much the genetic portion of the cell. It's what makes a cell what it is. So, in order for us to really understand DNA replication, I really want you to understand that the whole purpose of it is to allow for cell replication. Okay? Or sometimes we refer to this as the cell cycle. Okay?

So, the cell cycle, I know you guys know the cell cycle. When in the cell cycle, what's kind of the really quick part of it? You start off, you go G1, then you go S phase, G2 phase, and then you go into the mitosis part, and then out of that, you get two cells. Where you take one cell, that cell enters into the G1, S, G2, goes through mitosis, and makes two cells. The big thing I want you to know is that DNA replication primarily occurs within a particular part of the cell cycle. When a cell's replicating, making more cells, it primarily occurs within the S phase. So, in the S phase, that is where DNA replication is occurring. So, the first fundamental that you need to know is, why do we perform DNA replication? In order for our cells to replicate, make more cells. So, whenever they go through their cell cycle, the particular point when the DNA is actually replicating is in the S phase of the cell cycle.

Now, real quick, what in the heck is cell replication? It's really simple. I'm taking this cell here, which has 23 maternal and 23 paternal chromosomes, and all I'm doing is I'm making two identical cells that look just like this. So, I have to replicate the DNA within this chromosome and the DNA within this chromosome, and I'll make two of this and two of this, and that's going to give me these two identical daughter cells. And that's the basic process of cell replication. So, that's the first thing I need you guys to know.

The second thing that I need you guys to know about DNA replication is that it occurs in what's called a semi-conservative model. What the heck does that mean, Zach? I got you. So, the next thing you need to know is that DNA replication is semi-conservative. Semi-conservative means, let's take that piece of DNA here, right? So, DNA has two strands, and we're going to call these, we're going to give them two names. We're going to call these blue DNA strands parental strands, or let's call them old strands. What I'm going to do is, when I want to replicate this DNA, I have to separate them. And we'll talk about how we do that. When you separate the DNA into separate strands here, I have two old parental strands separated. What I'm going to do is, I'm going to replicate the DNA in a complementary fashion. So, what does that mean, complementary? That means if, for example, this nucleotide was adenine, or A, this would be T. If this was T, this would be A. This was G, this would be C. If this was C, this would be G. You get the point. I'm going to make nucleotides that are complementary on that. But do you see how the color is different? And I synthesized a new strand, or a daughter strand, if you will. That's one aspect of it. I'm going to do the same thing to this other strand. So, I'm going to use this old parental strand and make a new strand with complementary nucleotides. On the other old parental strand, I'm going to again make DNA that is complementary to this old or parental strand. So, the whole concept here is that I start with old, if you will, let's use the term old, and I make new mixed with the old. That's really the easiest way of understanding DNA replication. I'm taking two old parental strands, separating them, and making two new DNA strands that are complementary to them. So, what I did is, I took this DNA and made two new double-stranded DNA molecules. Isn't that cool? And when I did it, I did it in this semi-conservative process.

The next really important thing that you guys need to know is that DNA replication occurs in a very, very specific direction. DNA replication has to occur, okay? The direction, okay, replication direction is very important. It's kind of annoying, we'll mention it a lot throughout the process of this lecture, but DNA direction always has to occur from the five prime end to the three prime end. I can't stress that enough, super important, it's going to come up a lot. What the heck does that mean? Really quickly, do you guys remember from the DNA structure video, what was on the five prime end of a nucleotide? The phosphate group. What was on the three prime end of the nucleotide? The OH group. So, when I'm adding nucleotides, I'm adding a phosphate group onto a three prime group of the preceding nucleotide. Let's show you an example of that.

So, let's say that we took this old DNA strand, we're going to do replication following the semi-conservative model here. I have two old parental DNA strands, separate them. I'm going to replicate it via the semi-conservative model, but I'm also going to follow this process where I have to replicate five to three. So, let's say on one end of this old DNA strand, I have a three prime end here. What does that mean? That means there's an OH group here. On the five prime end of this DNA strand, I have a phosphate group here. Okay? Same thing here, OH group, and then the phosphate group right here. So, that's basically what the five prime, three prime end is. And you guys remember that DNA is antiparallel. So, if it's three prime on one end, five prime on the other end, the other DNA strand has to be flipped. So, it's five prime on the same end, and then it's three prime, and three prime on the same end that it was five prime. That's important.

So, when DNA replication occurs, it has to occur five to three. So, here's the three prime end. When I make a new DNA strand, it has to be five prime first, three prime end here. And then what will I do? I'll add another nucleotide, and then this connection here will be between what? I'll have a three prime end here and a five prime end of this next nucleotide. I'll have a three prime end here. I'm going to add another nucleotide. So, when I make nucleotides, I make them and synthesize them from five to three. Okay? Same thing if I'm going to do it off of this strand. Here's the five end, here's the three end of the parental strand. If I want to make the new strand, this is the five prime end. So, I'm going to be synthesizing DNA in which direction? In this direction here, right? And again, doing this according to the complementarity rule. Okay? So, that's the important thing I need you guys to remember is that DNA replication occurs in a five to three direction.

The last fundamental thing that is really important here is that DNA replication is bi-directional. And you're like, why the heck do I need to know that? I think oftentimes when we're looking in textbooks, we only focus on one end where DNA replication is occurring. But what's really important is, we're going to talk about this in a second, but what we do is we take the DNA and we already have an idea that we're going to separate the two older parental strands away from one another so that we can create new DNA from that. When we do that, we create these little ends here, these little Y-shaped regions called replication forks. Okay? So, it's called the replication fork, and you have two of them, one on this end, one on this end. There's going to be enzymes called helicases, which are going to come in and unwind the DNA on both sides, moving it in this direction and moving it in this direction. And then enzymes called DNA polymerases, which we'll talk about, they're also going to move into these areas and follow the helicase, synthesizing new DNA off of that parental strand in a bi-directional fashion.

So, the big fundamentals I need you guys to take away is that, why do we do it? DNA replication in order for cells to replicate and make more cells. It occurs in a semi-conservative fashion, taking old, making a mixed old and new double, two double-stranded DNA molecules. It occurs in a five to three direction, and it occurs bi-directionally from what's called the origin of replication, or where these replication forks are. Okay?

Now that we have the fundamentals, let's now talk about the steps of DNA replication. All right. So, the first thing that we have to talk about when we're talking about the stages of DNA replication, there's three stages of DNA replication: initiation, elongation, and termination. Okay? Initiation, elongation, and termination.

Initiation is really an easy process, it's not too hard to remember. So, what happens is, let's say that here I have my double-stranded DNA, okay? And let's say there's a particular region in that double-stranded DNA which is really, really a nice little area that I want to go and I want to separate the DNA so that I can create two separate DNA strands, parental strands, that I can use as templates to make new DNA. That area is going to become our origin of replication. All right? So, this area is really what's going to be our origin of replication. And why do we need to know this? Okay? So, whenever we need, we're picking this spot, how do we determine what we're picking that spot? Like, okay, I know that there's a bunch of regions on the DNA, why is this point here the particular origin? And there's a really cool reason why there's particular nucleotides in this region which are really highly concentrated with adenine and thymine. So, it's an adenine and thymine rich area.

Now, let's talk about this for a quick second. Why would I pick adenine and thymine as the area that I really want to target as compared to guanine and cytosine? Do you guys know why? So, adenine and thymine, how many hydrogen bonds are there between them? Two. How many hydrogen bonds are there between guanine and cytosine? Three. It's going to be easier to break two hydrogen bonds than it is to be able to break three hydrogen bonds. So, in this area, there's going to be particular areas which are really concentrated with adenine and thymine nitrogenous bases or nucleotides, and that's going to be better suited as the area we want to kind of separate. Why? Because there's only two hydrogen bonds, and that's going to require less energy to break those hydrogen bonds as compared to guanine and cytosine. Okay? So, that's the first thing. We have particular areas.

Now, the next thing I need you guys to understand is, in eukaryotic cells, is there only one origin? Okay? There's just one area here where there's a lot of adenine and thymine? I just have enzymes bind to that portion and separate it, and the enzymes just work from the center and go to the ends? No. What's really important that you guys need to know is that in eukaryotic cells, there are multiple origins. Sometimes represented, or E.C. origins of replication. So, that's really, really important. So, for example, I may just be representing this one portion, but there may be another origin of replication right here, and another origin of replication here, which is really rich in adenine-thymine nucleotides. Okay? So, that's one important thing.

The next thing is, what type of structure is going to bind onto these areas and help to break the bonds between the adenine and thymine? What do we have? There's a really interesting protein. It's got one heck of a name. They always do, don't they? And this protein here is called the pre-replication pro-pre-replication protein complex. One heck of a name. So, here we're going to draw a cute little enzyme here. Okay? This is a cute little enzyme, and this enzyme is going to come in and bind onto these areas. And when they bind onto the areas, they separate the adenine and thymine nucleotides in that area to separate the DNA. What is this protein here called that separates them? It's called a pre-replication protein complex. So, again, it binds to the origin of replication and separates the adenine-thymine nitrogenous bases.

Now, once it does that, what's that going to look like? Well, let's take the next step. We had this protein bind on to the origin of replication where there's a lot of adenine and thymine nucleotides. We separated the bonds between them, the two hydrogen bonds, and we create this little like bubble, if you will. You know what we call this? It's not hard, it's called the replication bubble. That's literally what it's called. I know it sounds crazy, but now we kind of form this little bubble due to this whole process, and it's called the replication bubble.

Now, once we form this replication bubble, there's a couple things that I need you guys to know. We have separated the nucleotides. So, if you imagine here, we're not showing them, but let's say that here I show a couple, you know, here's my nitrogenous bases that are coming off this sugar phosphate backbone, right? Which is made up of again, the deoxyribose sugars, the phosphate groups, all that. And these are just my nitrogenous bases that are popping out off of it, right? So, I'm going to have these exposed now. So, these were once really connected nicely together, like in these regions. I really separated them, so they're really kind of like vulnerable right now. And you know what's really important? These, when you separate them, they want so badly to re-anneal with one another. All right?

So, what is this protein that really helps to protect these vulnerable separated parental DNA strands? You know, it's really ironic, you know, sometimes science is there's a big old protein that comes and binds to this end. It would do the same thing on this one. And this protein, you know what it's called? It's called the single-stranded binding protein. I'm not even joking, that's literally the name of it. And it's perfect, it's not hard to remember. Why? Because it's a protein binding to a single parental strand. So, you would have one over here, I'll just draw a portion of it, but you'd have the same thing over here, another single strand of binding protein binding onto this parental strand. And what is the purpose of these single-stranded binding proteins? That's what I really want you to remember. One of the functions is that it prevents the parental strands from re-annealing. What the heck does that mean? What's that term, re-annealing? From reconnecting to one another, right? So, that's important. So, it prevents the parental strands from re-annealing to one another because they honestly, they really want to click back to one another. The other thing that they do is that when you have these parental strands separated as kind of these single strands, if you will, they're very vulnerable to very nasty little enzymes. There's some nasty little Pac-Man-like enzymes that want to come to the area and break the phosphodiester bonds. These are called nucleases. So, what these single-stranded binding proteins do is they kind of act as a barrier and protect these single strands from exonucleases or endonucleases. So, again, it prevents, it protects from nucleases. Okay?

So, so far, pre-replication complex binds to the origin of replication, the AT-rich area, separates it, forms a replication bubble. Single-stranded binding proteins bind, protect them from exonucleases and endonucleases, prevent re-annealing. Whenever you do that, separating them, you create a replication bubble. At the ends of them, you have replication forks. Helicases, highly ATP-dependent, hop in there and start unwinding the DNA in front of them.

After they do that, something happens which is really important that we definitely need to know. Let's come down here, and this is a really important area that I really, really need you guys to understand. So, let's say that we come back to this point here again. What we have binding right here to these two ends, single-stranded binding proteins. We're not going to show it on the top, but the same thing here. And then again, what enzyme? Let's just focus on this area right here, just on this replication fork. What enzyme would be in this area here, really working and unwinding the DNA in front of it? Again, that's called helicase. What happens is, as helicase continues to unwind the DNA, the whole purpose of that, as it unwinds the DNA, separates the strands so that enzymes would be able to use those parental strands to make new DNA. Okay? But there's a problem that happens. As DNA helicases just, you know, going through those mofos and unwinding the DNA constantly in front of it, it bunches up the DNA in front of it, distal to it, okay, from that replication fork. So, distal to the replication fork, downstream, the DNA starts bunching up and it creates things, these things called supercoils. Okay? It creates these things called supercoils, and this is caused by the helicase really unwinding the DNA. Why are supercoils bad? If you really bunch up the DNA in front of it, it's going to really impede the helicase from continuing to unwind the DNA. It's going to face a lot of restriction because it's really bunched up here, so it'll just keep getting bunched up until you relieve those supercoils. So, we need enzymes that can come in there and fix these supercoils where the DNA is really, really tightly wound.

So, how do we do that? All right. So, what little enzymes do we have or special little things that come into play to really alleviate these supercoils? These enzymes are crazy interesting. So, these guys are really cool, and they are called topoisomerases. They're shaped like a T, right? So, they're called what? Topo-isomers. Now, topoisomerases, there's actually a couple different types. Okay? There's particularly type 1, type 2, and then there's type 4. For the most part, they all do the same kind of thing. And what is that? Okay, this enzyme has two little arms, if you will. Let's say here, it has one little arm and another little arm. On one arm, it can go to where this area of the supercoil is. Let's say that the supercoil's right here. It can use a little enzyme, a little domain of it, and cut this DNA strand. If it cuts the DNA strand, what does that allow for it to do? It kind of allows the DNA to kind of unwind a little bit and unravel. And so, there's a particular name to this domain on that topoisomerase enzyme. It's called a nuclease domain. And what does it do? It creates a cut or breaks the phosphodiester bond in the DNA strands, one, maybe two DNA strands. Allows it to unwind. There's a problem with that, though. If I just cut the DNA strand and allow it to continuously unwind, that may be problematic. The DNA could continue to fragment. I don't want that. So, what I do is I use this other arm. And after the DNA supercoils have been alleviated, so let's now kind of draw new DNA after the supercoils have been alleviated. So, we alleviated the supercoils, we got rid of all of those overwinding of the DNA. Look, now it's beautiful, it's not overwound. But we have a break in that DNA, and that's a problem. So, now we need to use the other arm of this topoisomerase, where we cut this portion here and allow it to unwind. We need to use this portion called the ligase domain. And once it's unwound, this portion here, what does it do? It re-stitches this area back together after it's unwound the supercoils. Okay?

So, again, the topoisomerases, what were their function again? To unwind the supercoils. Now, why in the heck did I take all this time to mention the topoisomerases and there's different types? Let me explain why. Topoisomerases can be in both cells. So, we primarily haven't really discussed that DNA replication can occur in bacterial cells, and it can occur in eukaryotic cells, or we call it bacterial cells, like prokaryotic cells, and eukaryotic cells. Human cells primarily have type 1 and 2 topoisomerases. And in prokaryotic cells, they primarily have 2 and 4. So, again, type 1 and 2 are primarily for eukaryotic cells, and then type 2 and 4 are primarily for prokaryotic cells. Big thing I need you guys to know is that type 1 topoisomerase does not require any ATP to unwind the supercoils. So, let's put that next to it. That just for this one, just for this one, it, no ATP is required in order for it to perform this unwinding of the supercoils. But for type 2 and 4, let's pick a different color so that we don't confuse it here. For type 2 and 4, these do require ATP in order for them to unwind the supercoils. There's also one more thing, I don't want to get too far in depth in it, that type 2 and 4 can do that are a little bit different from type 1. And that's that if you really look in the textbooks, they can actually take and cut that little supercoil area, allow for unwind, and then insert in what's called negative supercoils, which also helps to kind of relax the DNA and prevent that kind of bunching up region. Let's not get too far in depth in that. What I really want you guys to know is why in the heck did I spend so much time talking about these topoisomerases? In your USMLEs, you have to know particular drugs that we can target on these. In eukaryotic cells, I want you to think about a reason, just try to think for a reason, why would I want to target this enzyme in eukaryotic cells when this is important for DNA replication? If these enzymes aren't really working, DNA replication won't occur. When a eukaryotic cell, what if I have a cancer cell? So, if I have cancer, the cells will continue to replicate. So, they'll replicate and replicate and replicate. Well, I could maybe use some drugs that could target these topoisomerases in my cancer cells and prevent them from replicating. How do I do that? Well, there is a particular name of, there's a couple drugs that you guys definitely need to know for topoisomerase 1 in eukaryotic cells. The cancer drugs that we can use for topoisomerase 1 is called irinotecan and what's called topotecan. And again, these are anti, like they're chemotherapeutic drugs that are going to inhibit the topoisomerase 1 in the cancer cells. The type 2 in eukaryotic cells, we can use drugs called etoposide and teniposide. And I'm going to explain how they do that because that's really important. I really want you to understand how they do this. But that's for the eukaryotic cells. Think about prokaryotic cells. If you were infected by a bacteria, and a bacteria infected your lungs, and it just kept replicating and replicating within your lungs, could I maybe target the topoisomerase 2 and 4 and prevent that replication process of the bacteria in my lungs? Yes. So, in bacterial infections, let's say because it's a prokaryotic cell, and bacterial infections, these bacteria will continue to keep replicating. So, if I use particular drugs that can maybe prevent the DNA from replicating in these bacterial cells, that's important. And we can do that via inhibiting the topoisomerase, primarily type 2. And we use the drug called fluoroquinolones. Some of you may have heard of these, ciprofloxacin, levofloxacin, ofloxacin, all of those guys. They're inhibiting the topoisomerase 2. But I really want to take a second because I never understood this completely until I really dug into the mechanism of how they actually do this. I really want to quickly say how they do it. What these drugs are really doing is these drugs, so let's kind of just put here, drugs, they're exciting in increasing the activity of the nuclease domain. What the heck is that going to do? That's going to just chop all those like portions of the DNA, and it's going to continue to fragment them. Right? Remember we had another, another domain of this enzyme that re-annealed them and kind of stitched it back together to prevent that fragmentation? That was the ligase binding domain. What if I use drugs to enhance the nuclease domain but inhibit the ligase domain? So, now I'm going to have this enzyme go in and kind of cut where the supercoil is, but I'm never going to re-stitch it together. The DNA will just fragment over time. And that's important because then you can't replicate the DNA within what kind of cells? Eukaryotic cells, like cancer cells, or bacterial cells. And that's why those drugs are important. Doesn't it make sense? All right, cool. It's important to take a clinical application and tie it to the basic foundational science. Okay?

So, we kind of went through, talked about the topoisomerases. That was the big thing, and how they unwind the supercoils. Let's get back to the foundational science. And now that we've talked about that, the next thing that we have to go into is elongating the DNA.

All right, ninja nerds. So, we already know we have a replication bubble. We got replication forks. We have our protein here called the single-stranded binding protein, which is stabilizing these single strands. We have the helicase enzymes that are in these replication forks, working like a son of a gun to unwind the DNA. We've got those topoisomerases over here that are kind of unwinding those coils. Okay? Now we've really separated this, we've stabilized it, and we're ready to begin elongating the DNA. Okay? Here's what's really interesting. There's an enzyme that comes into play here, and it does something really cool. It's called primase. So, an enzyme called primase will come into play here. So, what primase does is, it's an enzyme that lays down, okay, that gets, makes what's called RNA primers. So, this takes a really quick turn where we've got to understand, Zach, you just said that we're making DNA. Why the heck would I make RNA? There's a reason. There's an enzyme that we'll talk about a little bit later called DNA polymerase 3 that will make DNA, but the only way it can do that is if it has some type of primer or three prime OH to build off of. So, what's the purpose of this primase? This enzyme, it lays down RNA primers, which enable DNA polymerase, particularly type 3, to make DNA. And I'll kind of show you that in a little bit, in a second, okay?

So, primase comes in. So, imagine here, I just have like this cute little enzyme here called primase. Okay? And this cute enzyme comes in here. And let's say here, what's this strand up here? This top part. Remember, we're going to say that this is the three prime end where the OH would be. This is the five prime end where the phosphate would be. And again, the opposite strand here would have to be antiparallel. So, five prime end here, three prime end here, right? We already know that the primase is going to come in, and it's going to read the nucleotides, and it has to go in a particular fashion. It reads it from the three all the way to the five end. So, what does it do? The first thing it does is it reads the DNA strand from three to five. After it reads it from three to five, what did I tell you that's super important? What does DNA replication occur? Even though this isn't DNA, it's the same concept. It synthesizes RNA primers or nucleotides in a five to three fashion. So, it's going to take and make a couple nucleotides, generally it's about 10 nucleotides. We're only going to draw a couple here, but it'll have, has to be again, five prime starting here. And I'm just going to make a couple, I'll make like four nucleotides here, okay? So, here it's going to have five all the way to the three prime end here, okay? That's my five prime end here that I just started with, and I'm synthesizing it in the three direction. Now, the reason why this is important is on that three end, what do we have here? The OH. That's what's on the three prime end. I need that OH. The reason why is another enzyme called DNA polymerase type 3 comes in.

So, there's an enzyme called DNA polymerase type 3, and he comes in, and he needs that three prime OH from the RNA primers in order for it to continue to build nucleotides. So, again, a big thing I need you to remember is it needs the three prime OH of RNA primer in order to carry out its activity. If it doesn't have it, it can't do it. So, now that it has that, this DNA polymerase comes in and it says, "Okay, I have my three prime OH region. Perfect. Now, what I'm going to do is I'm going to read my DNA, and I'm going to do it the same way that the primase did. I'm going to read the DNA from the three direction to the five direction." So, I'll read it, boom, boom, boom. Once I read and figure out what kind of nucleotide is, then I'm just going to synthesize those nucleotides in the five to three direction. And it's the same process here. So, now let's make a different color since it's a different enzyme. We kind of picked red over there. So, we're going to start off and we're going to say, "Okay, I'm going to take that OH and I'm going to add a phosphate group onto it of a of another nucleotide." So, when I do that, I'm going to continue to keep synthesizing in a five to three direction, moving towards the replication fork. Again, when I do that, I read it, I say, "Okay, let's say that this is adenine, I'll put a thymine. This is guanine, I'll put a cytosine. This is cytosine, I'll put a guanine," and so on and so forth. And I'll just keep reading the nucleotides three to five and making a DNA strand in what direction? Five to three. Okay? And again, important to remember, it needed this three prime end of that OH of the RNA primer to build off of it.

Now, here's what's really interesting. The primase will give it kind of a little leading point, and the DNA polymerase will just go all the way towards the replication fork. This strand is very continuous, where there's just one RNA primer and then DNA the rest of the way. This strand is very important. We give it a particular name. The strand that's very continuous, where the DNA polymerase moves towards the replication fork, is called the leading strand. Okay? It's called the leading strand. On this other strand, which we're going to talk about, is called the lagging strand. Something different happens where you're still going to have the are the primase. It'll come to this area, okay, on this other strand. And again, this is the three end on this part, five end on this part. And what it'll do is it'll read it from three to five and then synthesize a couple nucleotides from five to three. So, this is the three end. It's going to synthesize from five to three. And again, the same thing will happen. We created a primer of a couple nucleotides with a three prime OH end that the DNA polymerase type 3 can build off of. So, now the DNA polymerase type 3 will just pop on and say, "Oh, perfect. I have my three prime end to use. I'm going to go ahead and just read the DNA from three to five and synthesize it from five to three." Okay? So, I'm going to do all that perfectly.

Now, something interesting happens where it's going to look, it looks perfectly the same. You're like, "Zach, I don't get the difference here." Let's say that the helicase continues to unwind the DNA. So, it continues to unwind the DNA. Something interesting happens that we have to talk about, okay? So, now let's come down here. So, let's say, let's pretend right, that for a second here, we have that primer. Let's kind of continue off this. Let's say that the helicase unwound the DNA a little bit more, and we along, we kind of opened up the DNA and created a more longer length of nucleotides. So, again, let's say that here we had that primase came in here, read this from one end. Again, this would be your three prime end, this would be your five prime end. So, it'll read from three to five and synthesize from five to three, creates a little primer with a three prime OH end. The DNA polymerase 3 says, "Okay, perfect. I have everything I need. I can continue to grow." And let's say that it just came up to like this point here. But then you kind of unwind the DNA again. That DNA polymerase 3 doesn't stop. It just keeps on going and keeps on moving, reading the DNA from three to five and continuously synthesizing nucleotides from five to three. So, again, this would be your five prime end, this would be a three prime end on this other chain. This is on the leading strand. It continues. On the lagging strand, here's where it's a little bit different. Let's say that we continue to move on here, and let's say that like at this point here, this was where the previous RNA primer was from above, where it had, again, reading this portion of the DNA. This is the five prime end of this part, three prime end of this part. It read this sequence of DNA from three to five, and let's say that it synthesized a couple nucleotides to give your RNA primer from five to three. And then what did we say happen from that part above? We have the DNA polymerase 3 use that three prime OH end, read the DNA from three to five, and synthesize the nucleotides from five to three. Here's what happens. The primase laid down a primer here, but the DNA polymerase 3 has to use that primer to continue to keep building off. If you unwind the DNA a little bit more now, now you have a couple of the nucleotides. So, now let me kind of just, so we have enough room here, let's say I draw a couple more nucleotides. So, now here I have a couple more nucleotides. Now, that primase, after it just made down this primer for the DNA polymerase 3 to use, it comes down to the next part of the replication fork and it says, "Okay, here I got another three prime end here. Let me again read from three to five and synthesize a couple nucleotides from five to three." So, I laid down my RNA primer. DNA polymerase says, "Okay, cool. I got my three prime OH end here. Let me go ahead and use that to make my DNA, and I'm going to read the DNA from three to five and synthesize it from five to three." Do you notice something really interesting here? On this strand, which we called again, what did we call this strand? Well, we had one primer and then DNA for the continuous way towards the replication fork. We called this the leading strand. So, the big thing I want you to know is that you have one RNA primer and then a continuous DNA strand from that point on. On this strand, called the lagging strand, something different happens where you have a couple RNA primers, okay, and then stretches of DNA between those RNA primers. This kind of like broken up portion where there's RNA, DNA, RNA, DNA. And if we continue to keep elongating it, we'd have more RNA, DNA, RNA, DNA. This gives a particular name, which is called Okazaki fragments. Okay? Okazaki fragments. And again, it's basically where you have multiple RNA primers and multiple stretches of DNA stretches. Okay? So, multiple DNA stretches and then multiple RNA primers. It's a mix of them, and that's a problem, okay? Because you're going to see now, there has to be another thing that we have to do. We want everything, when we replicate DNA, it has to be all DNA. We can't have it be DNA with a little bit of RNA. So, we're only using these primers as just kind of a point to build off of. After we've built some stuff, we're just going to go in and cut those things out because we don't really need them anymore.

So, now let's talk about the next part, which is we've started to kind of create these primers that we needed to build the DNA off of. Now we don't need the primers and we got to get rid of it. How do we do that? The next thing that you guys need to understand is, okay, we've used our RNA primers for the DNA polymerase 3 to kind of build off of and make DNA from. We don't need those primers anymore. We got to get rid of them. So, let's draw the diagram that we had previously, which again, we only had what? A little stretch of RNA primer here, and then the rest of the length down going towards the replication fork, which again, what is this strand here called? The leading strand. Is going to be all DNA. Okay? So, now this is really important. We're going to talk about one more thing in just a second. And again, if you guys remember, on this strand, the lagging strand, we had a couple RNA primers that were in between the stretches of DNA, creating what's called Okazaki fragments on the lagging strand, right? We talked about that. So, now the next goal here is that we have to remove those RNA primers. But before we do that, I got to mention one more thing.

So, before we talk about how we remove these RNA primers, I really want to take a quick second here to explain something else that DNA polymerase type 3 can do. So, we know that it reads the DNA, okay, from three to five, and then synthesizes nucleotides off of the RNA primer from five to three. But it also has one other function. It's called a proofreading function, which is very important before we talk about the RNA primers. And this proofreading function is helpful to prevent mistakes. And what it does is, let's say, okay, it reads three to five, reads all the nucleotides from a three to five direction, and then synthesizes nucleotides in a five to three. After it does that, it says, "Okay, let me check my work." It goes back and it finds the connection between this point. Says, "Okay, is this a good connection? Yeah, that's a good one. A and T are connected together. Oh, G and C are connected together. A and G. Oh, this isn't a correct, uh, complementary kind of base pair. I need to cut that out." So, it reads from three to five, and if it finds any mistakes, it uses what's called a three prime to five prime exonuclease activity, where it says, "Okay, let me read here. I'm going to read it and it says, okay, three to five, I'm reading A T, and again, it's complementary base. It should be T. Should be A. G A. Oh, not a correct one. I'm going to cut that out. Read it again. Make sure I have it. Okay, it was G that has to be C." And synthesize the correct nucleotide in the five to three direction. So, big thing I need you to remember for DNA polymerase type 3: reads DNA three to five, synthesizes nucleotides five to three, proofreads back in three to five, and if that's incorrect, uses a three to five prime exonuclease to cut it out and put in the correct complementary nucleotide. Very important.

Okay, now we got to get rid of these RNA primers. How do we get rid of the RNA primers? Well, the DNA polymerase 3 is not the answer. The next enzyme, as if there isn't enough enzymes, is called DNA polymerase type 1. So, DNA polymerase type 1 comes to the rescue. And what it does is, it starts here and it finds this. Okay? So, let's say here we had our three prime end here, five prime end here. The new strand would be synthesized from five to three. What this enzyme will do is it'll come in and it'll cut out these primers going from the five to three direction. So, it removes primers, or it plucks those little primers out in a five to three exonuclease activity, right? Particularly for what? To pluck out the RNA primers. So, this guy will come in and it'll say, "Okay, pluck, remove that one. Pluck, remove that one." And then what it'll do is, once it plucks those out, it then says, "Okay, I'm going to read this strand from three to five." So, it reads the one that it plucked out and says, "Okay, that's an adenine. What do I need to add here? I need to add in thymine. Oh, this is thymine. I need to add in here adenine." So, it plucks off the primers, then what does it do? It reads the DNA from three to five, and then it synthesizes from five to three. You know what else it can do? One more function. Let's say, okay, it plucked off the RNA primer, reads it as adenine, puts a thymine. Reads it as guanine, accidentally puts an adenine. Has to go back and proofread it, though, because that's always the thing that they have to do. Proofreads it and says, "Not a good connection. I don't want that. What do I need to do? I need to pluck that thing out of there and put the correct nucleotide." So, the last thing DNA polymerase type 1 can do is again, it has that proofreading type of activity where it can do what? It can read from three to five. It finds an incorrect base pair connection. It cuts it out. And when it cuts it out, it cuts that at a three to five exonuclease type of fashion. Okay?

So, the big difference that if you ever get asked between what in the heck is the difference between DNA polymerase type 1 and DNA polymerase type 3, really the big difference is that this guy can do everything type 3 can do, it's just it has that five to three prime exonuclease activity where it plucks out the RNA primers. Everything else is the same, though.

Now, the next thing is, DNA polymerase type 1, we talked about on the on this leading strand. On the lagging strand, it's a little interesting. It'll come in and again, it'll use its five to three prime exonuclease activity. So, again, let's use our combination here of what we know. This was three, this was five. So, antiparallel, this has to be five to three for the old strand. The new strand would then be what? Read three to five, synthesize five to three. So, this DNA polymerase will come in, and it'll start moving down. And at this point, it'll pluck off an RNA primer. And then what will it do? Read three to five and synthesize five to three. Then proofread it. Is it correct? Oh, it is. Okay. If it's not, use my three to five prime exonuclease to cut it out and put in a new one. It goes to the next one, plucks off the RNA primer, and does the same thing. Reads three to five, synthesizes five to three, proofreads three to five. Then it just keeps doing that and plucking these things off. Here's the difference, though. In the lagging strand, it creates like a couple gaps. These actually don't completely kind of fuse together. So, let's kind of draw where we had this here, and we'll create a little space between these points here where the RNA primers were. So, there's kind of a little space here, let's draw it here in orange. So, on the lagging strand, it creates like a little space where it can't like really.

fuse these ends where the primers were to the original DNA. Okay, so it plucked the RNA primers off and put nucleotides, but it wasn't able to perfectly fuse these ends on the lagging strand. One more enzyme. You're like, "Dude, I can't do no more." I promise, one more. This enzyme is called ligase. So, it's called ligase. Now, ligase will come in on that lagging strand and fuse the DNA ends together. Okay, those basically where those Okazaki fragments were, it'll come and it'll say, "Okay, here's these ends here. I'm going to fuse these points together so that it's perfectly connected and continuous." And now we have a parental DNA with a new daughter DNA strand. Again, a parental DNA strand with a whole new daughter DNA strand that is all continuous and all in sequence. No RNA primers, no nothing, no breaks. It's perfectly set.

Now, the last thing that I want to talk about here before we go on to termination is we've elongated our DNA. We now took the old parental DNA and made new DNA. The reason why I want you to remember this is that there's, it's very important for us, Emily's, to connect foundational sciences with clinical significance. And so, in people who have HIV, okay, their T cells, okay, their T cells have, are infected with the particular virus called a retrovirus, and it's causing this virus to get incorporated into the DNA. And then from every point that on that these T cells replicate, they continue to replicate more of the HIV genome. So, there's drugs that we use to target this HIV virus and particularly the T cell replication process. And these drugs are called nucleoside reverse transcriptase inhibitors. You're like, "Holy crap, what the heck does that mean?" I just want you to remember that they're drugs that are used for HIV and they inhibit the replication process in T cells that have been affected with HIV.

Let me explain how this works. This is really cool. Let's say here, I just, I quickly put down an RNA primer, okay? And I have my DNA polymerase 3. It comes in here and it starts making some DNA, right? Uses that three-prime end of the RNA primer and starts making DNA. I give them a drug. Let's kind of put here an NRTI. And again, there's many different names of these, like didanosine, zidovudine. There's a whole bunch of these. But what I want you to remember is imagine these as what's called nucleosides. Okay? And what they do is, imagine here's my, my basically my, my ribose sugar, and then here I'm going to have my phosphate group, and then here I have like adenine. Okay? What they do, which is really interesting, is usually on your, your deoxyribose sugar, you should have an OH, right? And you need that OH on that three-prime end in order for the DNA polymerase to continue to keep adding. What they do is, is they remove the three-prime OH region. So now the DNA polymerase 3 will get another nucleotide. It'll read this and say, "Okay, this is adenine. I'm going to put a thymine or something like that." And I'm going to add in this drug. This drug kind of floats around kind of interestingly. And the DNA polymerase 3 will then say, "Okay, here, this is another nucleotide, just like the ones I've been adding. Let me add this one on." The only problem is, is it doesn't have a three-prime OH region. So, you know what happens here? Since there's no three-prime OH, you, DNA polymerase can't build off of that. Remember what I told you? DNA polymerase type 3 needs a three-prime OH region to build. If you give a drug that doesn't have that, can you continue to build off of it? No. So all the DNA replication from this point is inhibited. Are you able to replicate all the DNA within these T cells that have been affected with HIV? No. So that's how it does it. They're what's called kind of like analogs, nucleoside analogs, where you're kind of like, DNA polymerase 3 doesn't know the difference. It's just taking nucleotides and adding on. And all of a sudden, just by kind of chance, you have this drug that it attaches on. It doesn't know the difference. It goes to add another nucleotide on. It's like, "Hey, that didn't add on. What the heck? I can't add this nucleotide on." And the DNA never gets completely replicated. So it's a good, it's a good clinical point to understand that covers our elongation.

Let's hit it home with termination and take a quick second to talk about telomeres. All right, this is actually the easiest one of all. You're probably like, "Oh, please, Zach, I can't do any more." I know, this is a lot. But let's say that we have another enzyme, okay? That helicase enzyme. We're getting to the point where DNA replication has been completed. We got that enzyme. What was that enzyme that was working at these replication forks and just continuing to unwind the DNA, you know, in front of it? What are they called? The helicases. And then you had those enzymes, the DNA polymerases type 3 and type 1 and all those guys that were coming and basically reading the DNA three to five and synthesizing it five to three, proofreading in the three to five, all that good stuff. And you've synthesized the DNA. And the helicases are meeting each other at kind of replication forks that are about to abut one another. When this happens, when the helicases meet and you kind of unwind this portion of the DNA, what's going to happen? The helicases are just going to kind of say, "Oh, well, hey buddy, I guess I don't need to keep unwinding anymore." And what will happen is you're just going to kind of have this point here where the DNA polymerases will just hop off of the DNA because at this point, there's nothing else for it to read. And so usually, once it gets to that point, they'll just say, "Hey, I guess the helicases are done. There's no more unwinding for me." And then after that, the DNA polymerases will say, "Hey, I've already kind of hit all the nucleotide regions here. I'm done." And I've replicated all of my DNA. So that's important. So it's basically again, where there's multiple origins of replication and they're constantly moving towards one another. Whenever they moved and hit one another, the DNA replication at that point stops.

Now, there's something else that you have to remember, though. DNA replication will, you know, start at a point and then work bi-directionally. It's eventually going to go to the ends of the DNA or the chromosomes, which we call the telomeres. There's a particular nucleotide sequence at that end where the DNA polymerases have a really hard time being able to replicate. And that's very important. We'll talk about that next. Okay? But again, termination of DNA replication, it's really simple. It's when the DNA polymerases are moving towards one another at a replication fork and they've just stopped at that point. They hop off and they no longer perform their function. There's one other part of it, which is with the telomeres, which we're going to finish off with.

All right, so the next thing that we're going to talk about is telomeres. Right? So DNA replication, there's a little interesting issue that happens at the telomeres. So one thing I need you guys to know is that telomeres, they really shorten over time. So let's say that here we look at some chromosomes, which again, made up of DNA and proteins. Well, let's primarily think of it as DNA. And let's say that this goes through, goes through a replication cycle a couple times. I want you to notice what happens to the ends. Right? So when you look at a chromosome, there's two primary kind of like structural points. The point in the center, right, which is called your centromere, and then the ends. Okay? These points here, and these are called your telomeres. Now, what happens is, over time, as your cells continue to keep replicating, the DNA replicates. Watch what happens to the telomeres. They get shorter and shorter and shorter. As that continues to happen, there's a worry with this. And let me explain what that worry is. Obviously, your DNA has particular areas which code for RNA. RNA can then get translated to proteins. What are those called? They're called genes. So let's say that I have a gene right here. The telomeres will be there. And their primary function is that they will, it's common for them, this to happen where the telomeres were short and short and shortened. But the whole purpose of them is that telomeres don't code for anything. That's very important. Let me write that down. Telomeres do not code for any RNA. So do not code for RNA. In other words, you can't take the DNA from a telomere, make RNA, make protein. That's important. But let's say here, there is a gene there that can make RNA. The telomeres will kind of sacrifice themselves because DNA replication doesn't occur at this point for a particular reason. We'll explain why. And so because of that, they prevent gene loss. So they kind of like take the hit for us, if you think about it. They're like, "Don't worry, I don't code for any RNA, so you don't have to continue to replicate me." So with each replication cycle, the telomeres will shorten and shorten and shorten. But that's okay. And for a particular reason, because they do not code for RNA and they help to prevent gene loss. But here's the problem. Eventually, you're going to get to a point where the telomeres will shorten so much that it can interfere with the genes. Once that happens, where the cell has reached the replication limit, where it can't replicate anymore, it's reached its maximum number, there's a particular term that you guys need to know, and it's called the Hayflick limit. It's called the Hayflick limit. And that is basically the maximum amount of times that this, this kind of DNA can replicate before it starts to involve genes.

Now, let's talk about why these telomeres are shortened. Really quickly. Let's say here we take and let's say that we use this as our example. This is our leading strand and this is our lag strand, right? Again, let's just say here we have our three-prime end. We're going to use our RNA primer here. And then from here, the RNA primer was made by the what enzyme? Primase. DNA polymerase type 3 will then add on to that three-prime end and start making DNA continuously all the way down the leading stream. Remember, it was continuous. So this will happen all the way down, okay? On the leading strand. The lagging strand is where it becomes a problem. Remember, you have that three-prime end, right? And the five-prime end. The primase will have to add on to that three-prime end. So let's say it adds on right here at this three-prime end. When it does that, it gives a little primer. And then again, DNA will build, built off of that. Here's another primer. And then DNA would be built off of that, right? So we kind of know that process. This is pretty much, you know, a review of what we just talked about. But here's what's different here. Watch what happens, you guys. Your minds are about to get blown. Remember DNA polymerase one? What does he come in and do? Comes and plucks off the RNA primer and then makes DNA, right? On the leading strand. On the lagging strand, it'll come off and pluck this portion here, okay? That RNA primer. And when it plucks off the RNA primer, it still has a, what? Remember this is the three-prime end. What would this be here? Five-prime, three-prime. So remember, five-prime. This is a three-prime end right here. It still has a three-prime end that it can use to build off of and make DNA. At this point here. But watch what happens down here. Comes down to this end here. Plucks off these RNA primers. Uh oh. Do I have a three-prime end that I can add off of somewhere? I don't. Dudes, why? Because look, this is my five-prime end. I have no three-prime end here that that DNA polymerase can add on nucleotides to. So DNA replication won't occur at these points here. And that is problematic because guess what? This was the old DNA strand. You replicated and made a new DNA strand. This one's going to get shorter. It's shorter than the original one. Guess what happens when this one replicates? It'll get the new strain will be shorter than it. And then the next one, and the next one. And the DNA will continue to get shorter and shorter and shorter. And eventually start involving those genes. So thank goodness, in particular cells where we have a need, a lot of replication to occur, we have a special enzyme that comes in and says, "Hey, I'm going to elongate those telomeres for you." So that way, whenever DNA replication does occur, you don't start really taking away too much of the telomeres and involving these really important genes. So what is the name of this special enzyme that we should give great thanks to? This wonky looking enzyme here has two arms. And this enzyme is called telomerase. And telomerase is a really interesting kind of ribonucleoprotein. One arm comes here, okay? So remember, we're just looking at this portion here. So we're only zooming in on this lagging strand about right here, okay? And zooming in on it. So here we're on this portion where we didn't finish and synthesize the nucleotides because again, we didn't have that three-prime OH region here to add nucleotides off of the DNA polymerase. So what the telomerase does is it takes one arm and brings that arm out. And on this arm is something really, really cool. It expresses nucleotides. And a particular type of nucleotides that you guys really need to know. Okay? And so what are these nucleotides that it has? Well, what it expresses is complementary nucleotides that are commonly seen on the telomeres. Telomeres always have, and the easiest way, there's a mnemonic to remember them. Telomeres always have a particular repeat of nucleotides on them on their three-prime end. And the easy way you can remember that repeat is the mnemonic: "Tell them all genes gotta go." That is the repeat that you constantly see on that three-prime end of that parental DNA. So what telomeres does is it comes in and says, "Hey, I have all the complementary RNA nucleotides to this sequence that's commonly seen here." So let's kind of write down what would the complementary portion be. If it was T, it would be A. T would be A. U would be G. All the way across, it would be C, C, C. So it expresses that with its one arm. The other arm is really cool. The other arm will then use this RNA strand as a template to make DNA that's complementary to it. So if it does that, it's going to take this RNA, read it, and then what would be the complementary? T, T, A, G, G, G. This is really interesting. You want to know why I elongated my three-prime end? Okay, which is important. I elongated it so that way next time this DNA replicates, I won't really take too much of the DNA and involve those genes because the telomerase. But what I did is I used RNA and from that I made DNA. I need you guys to understand what that's called. What is that called when you go from RNA to DNA? Reverse transcription. This is called reverse transcription. And so what this kind of telomerase does, in a way, is it has, again, it's a protein with it, which expresses nucleotides. It expresses RNA. And then it has this other arm. This other arm that reads the RNA and says, "Oh, okay, this is A. I'm going to make T on the parental strand. That's A. I'm going to make T on the parental strand." So it can take RNA and make DNA, elongating the telomeres. Why would we want to elongate the telomeres? We obviously know to prevent gene loss and so we don't shorten those telomeres significantly. And what cells would you want there to be a lot of telomerase enzymes? A lot of high activity, highly replicating cells. Cells that are replicating so much that those telomeres would shorten if we didn't have it. So this is important. You need lots of telomerase enzyme. And what kind of cells primarily? In like stem cells. So if you're like, if you're a zygote and you're starting with one cell, you need those cells to have lots of telomerase activity to replicate and make the whole human body. Or your hematopoietic stem cells, which are making red blood cells, white blood cells, all those different things. You need those to be able to replicate and have enough telomerase enzymes so that it can replicate without hitting and losing those genes. That's important.

One last thing, clinical point. Telomerase. What if we figured out a way? Cells, certain damaging and really nasty cells, figure out a way to evade, uh, the cell replication where they can just continue to keep replicating without not being able to stop. What are that, what is that called? Cancer. Neoplasia. So cancer cells, you know what they can do? That we believe that they can do is they upregulate the activity of their telomerase enzymes. And if they upregulate the activity of their telomerase enzymes, they continue to elongate the ends of the DNA on the chromosomes, which allows for them to continue to keep replicating and replicating and replicating without shortening the telomeres enough that it starts to involve genes within that cell. That's really interesting. So really big thing I need you guys to take away. Telomeres shorten with every DNA replication. We can prevent that with telomerase enzymes, which perform what kind of process here? Reverse transcription. Use one arm, which is RNA, and build DNA on the parental strand to elongate that. And what types of cells normally would you see lots of telomerase activity? Normal stem cells, highly replicating cells in our body, or cancer cells, which dysregulate or upregulate the telomerase enzymes. All right, ninja nerds, that covers everything for DNA replication. All right, ninja nerds, so in this video, we talk about DNA replication. I hope it made sense and I hope that you guys did enjoy it. All right, engineers, as always, until next time. [Music]