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M.Ed., Stanford University
Winner of multiple teaching awards
Patrick has been teaching AP Biology for 14 years and is the winner of multiple teaching awards.
When you look at a person, you’re actually looking at a creature made of about a trillion cloned cells, all working together to do things like walk, grow skin, or think up new reality TV shows. Now how is it that each of those cells has almost identical DNA? The answer is DNA replication, the process of DNA synthesis.
Now DNA is an important topic in Biology. So there’s going to be questions on DNA replication both in multiple choices, and the essay portion, most likely. So what I’m going to do is, I’m going to start off by going over quickly the overview of DNA synthesis, and its importance in the cell cycle. Then I’ll get in depth how does this whole process start. Then I’ll go with how does it continue, and then much like this intro, I’ll tell you how it ends.
As I said, DNA replication is important for cell division. So when does that happen? It turns out most of the cell’s life is spent in a stage called interphase. You should know that if you’ve studied cell division. So that stage of the life cycle, this interphase is when the cell is growing and going through its normal processes. When the cell’s ready to begin cell division though, it needs to make sure that it has the DNA for the two daughter cells that are being created. That’s when it begins DNA replication.
Before then, the DNA was in a form called Chromatin. Chromatin is the loosely organized DNA that’s wrapped around Histone protein’s pools. When it’s ready to do DNA synthesis however, it has to get ready to begin copying.
Remember, floating around in the cytoplasm and nuclearplasm of the cell there’s a whole bunch of nucleotides. Where do they come from? Well, when you eat something that used to be alive, you can chop up its DNA into the individual nucleotides. Plus you’ve got some enzymes, if needed, that can build the raw materials that you’re going to use later on in DNA replication.
Let’s look closer at an overview of how DNA replication begins. First, you’ve got to open up the helix if you’re going to copy it. That happens and creates these openings called replication bubbles. Now for a prokaryote a thing like a bacteria, they’ve only got one molecule of DNA. It’s a circle, and it’s not even wrapped around those Histone proteins. So the whole process of DNA replication, while it behaves at molecular level very much like eukaryotic DNA replication, prokaryotic DNA replication only begins with one replication bubble. That happens at something called the 'point of origin'. That one bubble expands until it goes all the way around the circle, and you wind up with two new circular molecules of DNA.
With eukaryotes like you and me, we have so much more DNA. You actually have about 6 feet worth of DNA in one of your cells. So if you just started in one place, or on the 46 chromosomes, if you just started there, one per chromosome, it would take way too long. So instead, you have many replication bubbles open up on your DNA.
What happens is that you begin copying following the Standard Base Pairing rules of Adenine joining to Thymines, Guanines joining to Cytosines. That’s called Chargaff’s Rules. As it goes along, you’ll see that our replication bubbles are going to expand, until they finally merge. And then their efforts are joined together. We wind up with two new molecules of DNA. Something to notice, our original DNA molecule was red, our new one’s being build are made out of blue. You’ll see that each of the two molecules, each of the two double helices that are made, one strand is old, one strand is new.
This is called Semi-conservative. It would be a good idea in case they ask an essay question about this. That you read about the experiment that was used to demonstrate the DNA replication was indeed done by Semi-conservative replication. I will just give you a quick little hint.
What they did is they marked the old original template DNA using an isotope that made that strand heavier than normal. Then by allowing the new ones to be built using different isotopes, that made the new strands made out of lighter materials.
Now in that overview, DNA Replication sounded pretty simple. And at its heart it’s actually a very simple process, one that James Watson and Francis Crick were able to just figure out, just by looking at the structure of DNA. For a lot of introductory Biology course, the overview that I just did is good enough. But the AP Biology people and some Biology teachers, are going to expect you to know a lot more. So let’s start looking at it in depth.
Now the first thing that you’ve got to know, is that we need some way to open up the helix. So let’s try and figure out how that would work. It’s just like if you were going to copy your textbook, you need to open it up first. That’s one of the things that your teachers keep telling you. If they want you to read and take notes from your textbook, you’ve got to open it up. So let’s take a look at a DNA molecule.
Here is a model of one. Now, I need to get this open. How can I get this open? I need an enzyme. What would you call an enzyme that opens up a helix? Helicase. Remember –ase means enzyme. So I’m now going to represent the helicase enzyme. What I’m doing each of these wooden things represents a base, where the little wooden dowels represent the Hydrogen bonds that are keeping the two sides attracted to each other.
Helicase enzymes sit there and opens this up. So it’s gone and, crap it closed by itself. What could I do? Remember Hydrogen bonds are these in credibly weak attractions in slightly positive and slightly negative portions of the Nitrogenous bases. They’re like molecular Velcro. If you’ve ever opened up the strap on a Velcro sneaker, and you let go on the strap, it’ll fall down and reclose. So what we need, we need some kind of protein that’ll bind or stick to each of these single stands, to keep them as single strands.
What would you call a protein that binds to a single strand? I’d call it single strand-binding proteins. You see that word? It tells you exactly what it is. That’s one of the tricks to doing well in Science, is really figure out what do the names mean. You think about it, you could have made that name up. During an AP Biology essay, go ahead make names up. You might be able to trick the reader into thinking you know more than he does, or he’ll think oh crap that’s what they call it on the West Coast, and I’m an East Coast professor or vice versa.
So let’s take a look at how this would look. Here we see our DNA double helix, and we’re going to open it up. Now there’s a problem here. Let’s do a quick review of the structure of DNA, because you need to know this stuff in order to get a good grade on DNA Replication. We look at this and the blue guy is labelled with a 5 there. What does that refer to? If you remember, DNA nucleotides have three components. There is the phosphate, the 5-Carbon sugar, and the Nitrogenous base.
Now the Deoxyribose sugar is what forms the backbone of the nucleotide. The Phosphate is attached to that 5th Carbon going around the ring of the Deoxyribose. So this 5 here refers to the fact that, this is the Phosphate end of the sugar Phosphate backbone of the DNA molecule. So if this is the 5'-Phosphate end, then it should be 5'-Phosphate. Then the 3' refers to one of the Carbons that’s on that sugar, the Deoxyribose sugar.
The next DNA nucleotide will join its 5'-Phosphate to the original DNA nucleotides 3'-Carbon. So it should be 5'-Phosphate, 3' Sugar. 5 to 3, 5 to 3, 5 to 3 sugar, Phosphate, sugar, Phosphate, sugar, Phosphate, sugar, Phosphate, sugar. That should be a 3. So let’s go ahead and make it 1. You need to know how to do that. You need to know. There is the number 3. At one end of a DNA strand, should be the number 5, or 5 prime, just put a little apostrophe on it. At the other end should be a 3.
Now you know that one of the things that James Watson, Francis Crick figured out, is that the two sides of a DNA double helix, aren’t going in the same direction. They’re actually going in opposite directions. So if this is the 5' end here, and 3' end of the blue, then if that’s 3' end of the red, this right here should be the 5' end of the red.
Now this seems like it’s a nit-picky detail, but these are kinds of details of nit-picky details that will appear on the AP Biology exam. Especially, if you can include them in the essays, you’ve got yourself one more point. Again, you’re only trying to pile up 10 points per essay. If you can get 3, or 4 you’ve got an average or normally a passing grade.
Now that we’ve got the helix open, let’s see how it gets started. What happens is that, the enzyme that actually build DNA, which is called DNA polymerase, it’s not allowed to begin building new strands of DNA. That’s a protection against mutation, because the first several bases in any strand of DNA, or any kind of nucleic acid, those first few bases that are added, are more likely to be mistakes or mutations.
So as a protection against this, because remember you’re trying to copy your DNA without any mistakes. Because remember, your DNA holds the instructions on how to build and operate your cell. So it will begin using not DNA, but RNA. That marks the first, maybe 10 or so nucleotides as these might be mistakes. You’ll go back later with proofreading enzymes and you’ll remove the RNA and replace them very carefully with DNA. So these beginning portions here, I’m using white to indicate where I’m using RNA.
Now since I told you that DNA polymerase is the enzyme that builds DNA, if I’m making this out of RNA, you know it must be a kind of RNA polymerase. And it is.
Now there’s many different kinds of RNA Polymerases. In this case, because we’re building the RNA primer, which is what this short segment is called during DNA Replication. Since we’re building a primer, we’re going to use an enzyme called primase. So it will build 5'to 3'. What does that mean? That means there’s a Phosphate here of a nucleotide, and a sugar or 3' end over here. You can think of new DNA or RNA strands as conga lines. Where there’s a 5' end that’s where your fingers are, and then the 3' end is like your head and shoulders 1, 2, 3, and this is 5.
When you’re doing a conga line, one person is at the beginning, and then everybody adds on their 3' end. It’s very bad manners to jump to the front of a conga line. We’re going to make a conga line out of nucleic acids here. Makes sense?
Now you notice I’m building a primer here, and here. That’s to represent what’s going on at this replication, for this branching event here. So these will allow me to copy this side. I’ve got some primers, because I also want to copy what’s going on at that Replication Fork. So let’s take a look now and see what happens.
Remember that DNA polymerase, I said that’s the one that actually can build DNA? It comes along and says, "Oh look a primer. I can add to pre-existing strands." And it does. It goes and starts extending the primer. But now, instead of using the white RNA, we’re using various colors of DNA.
I’m adding DNA nucleotides on the end of this. You can think of the primer as the guy at the party who starts the conga line. Most of us are too shy to do that, because we’d look like total docks. But once a conga line’s gone, hey you can hop on in. That’s what people are doing here. Nucleotides are coming in, extending here, there. So we’re making our conga lines our DNA strands.
So now the problem is however, we’re hitting that Replication Fork. So far, things have been pretty easy. Yeah, the primer-primase stuff has been a little weird, but so far, things have been going okay, I think. What you’ve had so far would do pretty well. In most Biology classes, it’s this next step that causes kids problems. So if you have any issues, just raise your hand, and I’ll call on you, because we’re taping this.
Let’s go ahead and see what happens when that DNA polymerase hits the Replication Forks. So what do we do? What happens is our budyy helicase just opens up more the replication level. So we open up the fork here, and we open up the fork there. Remember, what’s happening in this side is happening on this side. So what happens? Well, down here, this growing strand, this growing conga line, no problem. You just keep adding more and more nucleotides. Over here, this strand is also growing in the direction of this opening Replication Fork. So we can add in to our growing strand there.
Over here though, this strand is growing away from the Replication Fork. Down here, this strand is growing away from the Replication Fork. So what are we going to do? We have exposed DNA nucleotides here. How are we going to ever get them copied? It’s our friend primase. It comes in and says, "Hey DNA polymerase, you need to begin another conga line over here." Primase builds an RNA primer, and that will be the signal for our DNA polymerase to come along and start adding in.
Now because this side is growing continuously, in the direction of this Replication Fork, from here to there, this is called the Leading Strand.
Over here though, because we have to build a new primer here with our primase, we wind up having short segments of nucleotides being built, because we have to keep building these over and over again and again. This is called the Lagging strand. Now, the data the evidence for this model of how DNA replication happens, scientists had come up with their theories and ideas. But the guy who actually found these, and proved definitely that this was the way things worked, his name was of course Doctor Okazaki. How do I know that? Because we call these short segments Okazaki fragments.
So this strand here, because it has to keep being started over and over, is the Lagging strand. This strand here is the Leading strand. Similarly, because of the anti-parallel structure, over here, this portion is called Lagging, this portion is called Leading.
Now this is really complicated for a lot of kids, and they still think, "I don’t quite get it." So let me give you an analogy that might help you understand what’s going on at one of these Replication Forks. What I’ll do is I’ll use a textbook to represent our DNA molecule.
Here’s a textbook, pretty weighty. Let’s suppose your teacher says we need a copy of this. Now what you and your friend decide to do is, you decide okay we’re each going to copy half of the textbook. So what you do is you open it up. That’s like helicase opening up the helix. We’re only going to do one of our Replication Forks.
So let’s suppose your friend decides I’ll copy this side and you guys just decide we’ll begin right in the middle of the textbook, and each of you will begin copying towards the end that you are responsible for. So your friend will be copying from this page, all the way to the end. You’ll begin copying from this page going towards the beginning. It’s just like we’re copying this blue and red strand.
Now the problem that you’re going to face, and this is why your friend outsmarted you. Is remember, just like DNA, polymerase can only build in the 5 to 3 prime direction, you only read and write in the left to right direction. So your friend, he’s got no problem. He begins copying at his beginning which is right here and he begins copying. Now as he works on finishing up his half, he’s got to go pretty far. As he begins going along, he continues turning the page. You on the other hand, you begin here. So far so good and it’s like doing this, then oh no you’ve run into what your buddy’s doing over here. Remember you’re not trying to copy his half, you’re trying to copy your half. So what do you have to do? You have to turn the page the other direction.
But instead of just continuing on that same piece of paper, that wouldn’t make any sense if you start adding this. Instead you need to start a brand new piece of paper. Oh no we’ve got to start yet another piece of paper, because remember your job is to copy the front half of the book.
You would be the equivalent of the Lagging Strand, with each of these pieces of paper that represents one page’s worth of effort, being those Okazaki fragments. Your buddy who’s growing in the direction of this opening Replication Fork, he’s just going to have a continual scroll of paper that may be hundreds of feet long. But he’s just going to keep going, until he reaches the end. He’s leading, you’re lagging, because you have to keep starting over, because you’re opening in the opposite direction of what you’re copying. Does that make sense to you?
Now you know what we’re trying to build is not one complete molecule of DNA, and then a bunch of little chunks of those Okazaki fragments. We’re trying to make two complete molecules of DNA. So how do we do that? Or just like when we were trying to copy the textbook? How do you take all of the efforts of your friend and your little pieces of binder paper, and assemble those into a new textbook? Let’s take a look at what your cell does.
So what happens is that, remember, this used to be a short Okazaki fragment. This was that leading strand over here. When the DNA polymerase, that’s building that Okazaki fragment runs into the previous RNA primer, it stops and says somebody’s here. What is this? Another enzyme, a kind of RNA, which means it chops up RNA comes along and says, "Out of here primer." It removes the RNA nucleotides. Then a different kind of DNA polymerase, but it’s the same kind of molecule comes in. And as each RNA nucleotide is removed, a new DNA is put in.
This time we can use various copy editing functions of these RNA or DNA polymerases. They can go through and they can very carefully make sure that we’re putting in the correct A wherever there’s a T, the correct G wherever a C and so on. But still, we’ll have a DNA molecule right up next to a DNA molecule; two DNA fragments.
How do we join them together? That’s where one final enzyme comes in called Ligase. DNA ligase or just ligase for short, is the enzyme that actually joins the Okazaki fragments together. You need to know about DNA ligase. Not only for DNA replication, but later on if you ever read about genetic engineering, scientists have said, "Hey an enzyme that can join DNA fragments together?" Because what that allows us to do is we can join DNA fragments from different creatures. We can add in new DNA molecules using this DNA ligase.
Now some of you maybe going ligase, that’s a weird word. Actually, you know what it means. –Ase of course means enzyme, that’s right. What does liga- mean? Do you know any other word that begins with liga? Yes you do. What do you have in your joints? What holds your joints together? Ligaments. Liga means to tie or bind or join. So they could have called it ‘joinase’, but then everybody would know what they mean, and nobody would have to pay a scientist to explain these things.
So instead the scientists said hey let’s all call it ligase, and that way nobody will know what we’re talking about. So ligase joins our Okazaki fragments together. That’s pretty much how this whole finishes up.
The DNA polymerase keeps going on. As we open up more of the bubble, we have our primase build our primer. Then DNA polymerase comes along and says, "Oh that’s where I’m supposed to begin," and it goes. And we just keep going, until finally we reach the end of the DNA molecule. That’s what we see here. Once it’s free, now these new strands, notice we have two strands being formed here. It starts to coil up into the double helix. We have a helix being formed here and here. Once it’s complete, we have two separate individual molecules. That’s DNA Replication.
Now to review, DNA Replication is all about making identical copies of the original DNA molecule. So let’s go through that really quick.
The first thing you have to do is you open up the helix. Now luckily, because of that Base Pairing rule, we can just add our As to Ts, Gs to Cs. The enzyme that opened up the helix, that was helicase. Then we use single stranded binding proteins to keep it single stranded.
Next up, that RNA polymerase enzyme called primase, builds the primer. That gets it started with DNA polymerase continuing to elongate those strands. As we open up more of the helix, on the Lagging side, you have to start again with that primer building the beginning of the strand with DNA polymerase coming along to extend that. On the leading side, it’s growing towards the opening fork. So it doesn’t have to do that.
Eventually, DNA polymerase comes a long and replaces the primer RNA nucleotides with DNA. Then our ligase enzyme joins fragments together, those Okazaki fragments. Eventually you wind up with two new molecules of DNA that both wrap up. That’s it.
I really strongly recommend you go through this video again and get used to the names of the enzymes. But once you figure out why they’re named what they are, it gets a lot simpler. But that’s it, DNA Replication.
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