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DNA Replication & Repair
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0:00
>> This week, our big theme is cellular replication, how cells make more cells. In order to get to that process of how cells divide, we first need to understand how DNA is replicated because we've got to replicate the DNA before cell division can occur. So, let's start there. Let's start with DNA replication. So, here, I'm expecting that we understand the enzymes and can explain what each enzyme does in DNA replication. You should be able to sketch out this process, and you should be able
0:40
to tell me what the leading and lagging strands are in DNA replication. At the end, after DNA replication, we'll talk about some general mechanisms for DNA repair. For that section, I'm just more worried, I want us to know that DNA can be repaired, there are different mechanisms to repair it, and we'll talk about a few of those. So, remember, DNA is present in ever single cell of a multicellular organism, every single one of your cells has the exact same set of DNA. Exact same sequence of DNA.
1:15
So, that means from the time that we're a single cell, just fertilized egg, a zygote, from that time forward, our DNA has been replicated and replicated and replicated so, that way, every single cell in our body can have a copy of that DNA. And this process of DNA replication happens before cell division. So, this whole process that we're talking about happens before the cell even starts to divide. So, we're going to talk about how the entire genome gets replicated.
1:47
Remember, our DNA strands are complementary, okay. Remember we spent a bunch of time last week talking about DNA structure, okay. If on one side, we have an A, on the other side, we have a T, so they're not exact copies. The strands of DNA are not exact copies, but they're complementary to each other. This allows us to use one strand as a template for building another strand because A and T will only bond together, and C and G will only bond together.
2:18
Okay, that complementary nature of our DNA strands means that we can use one strand as a template, which also helps DNA maintain its integrity as it is replicated. Remember, also, that DNA is antiparallel, right? If on one end, we have a three prime, the other end, that carbon that's facing up will be the five prime carbon of the sugar. And this is important because enzymes can only interact with that DNA nucleotide when it's in a certain orientation.
2:50
Okay, so some enzymes will only read the original DNA moving three prime to five prime.
2:57
So, this directionality of DNA becomes really important when we understand how DNA replication occurs, especially this directionality at this antiparallel of DNA explains why we have one strand that we call leading and one strand that we call lagging during DNA replication.
3:15
So, we say DNA replication is what we call semi-conservative. So, semi meaning, like, partially?
3:21
Right, so we have our two strands of DNA, so this is our two halves of one DNA molecule, right? What's going to happen during DNA replication is we will separate those strains and use each of the original strands as a template for building a new molecule of DNA. So, by the time DNA replication is finished, you will have two copies of that original DNA molecule, but each copy will have half of the old original DNA molecule making up one half of its strand.
3:59
The other will be newly synthesized DNA.
4:03
So, it's half old, half new DNA that results from DNA replication. So, we call this whole process semi-conservative because we use some of the old DNA, but we also create some new DNA, we synthesize some new DNA polymers.
4:20
So, DNA replication results in one strand splitting in half, and then, we get two strands each half old and each strand is half new DNA. Here's our general steps of DNA replication in eukaryotes. Let's go ahead and jot this down in your notes really quick.
4:48
Okay so, these are going to be our steps for explaining what's happening in the next image I'm going to pull up. So, we're going to start with DNA. It's going to get unzipped. We're going to break those hydrogen bonds by helicase. RNA primer comes in, leaves down, primase comes in leaves down an RNA primer because DNA polymerase, the enzyme that is used to build that new DNA can only bind to an RNA primer.
5:14
DNA polymerase then adds new bases to both strands of DNA. One strand will be leading, one strand will be lagging. And then, all those little pieces of DNA in the RNA primer are removed by ligase and then, the ligase enzyme connects together the individual pieces of DNA. Okay, this is our outline of what's happening in this next image. This next image is a representative, representation of how DNA replication occurs. Okay, a lot going on here. Let's start on this right-hand side over here.
5:49
So, let's ignore this left-hand side, I'm just going to cover that up.
5:56
Here we have DNA helicase. Helicase, most of these enzymes are named for what they do. Helicase opens the helix.
6:07
Helicase separates the two strands of DNA, which are shown here in this light blue ribbon, by breaking the hydrogen bonds between the bases. So, this enzyme, if we were to zoom back a few seconds, actually would have been back here.
6:23
And so, it moves forward and then creates what we call a replication fork. So, this fork is opening DNA, we call it a replication fork that is created by the enzyme DNA helicase. So, helicase is moving in this direction.
6:40
And separates the hydrogen bonds between the two halves of DNA bases. We're not actually seeing As and Ts and Cs and Gs in here, but know that they are there. That's what's represented by this blue ribbon, the entire strand of DNA. Okay, that's our first thing we've got to do, we've got to open up the DNA molecule, that's done by DNA helicase. Next, this enzyme isn't shown in here, but we can see it with these little green bits right here.
7:14
Those green pieces are RNA primers. This is a short sequence of RNA that is complementary to the sequences that we have in the DNA. The reason why we need this RNA primer, this short sequence of RNA, is because DNA polymerase, this enzyme right here, can only attach to that DNA molecule if there's an RNA primer there first.
7:44
So, I kind of like to think that this primer kind of like opening a jar, I don't know, [inaudible]. But you know, when you try to open a jar sometimes and like, you're like, ah, I can't quite get it, can't get it, give it to somebody else. And then, they can't quite get it, and then you, they hand it back to you and you open it up just fine, right? They're like, oh well, I loosened it for you.
8:05
I guess, I don't know. So, the RNA primer, which is created by the enzyme primase,
8:16
is necessary for DNA polymerase to attach to the DNA molecule.
8:22
So, DNA polymerase, we saw RNA polymerase last week in transcription and translation. RNA polymerase made an RNA copy of DNA. RNA polymerase is not involved in DNA replication, but it's the same basic type of enzyme. DNA polymerase, remember polymerases are just builders, they construct polymers. Guess what? DNA polymerase builds DNA, right.
8:53
So, DNA polymerase, this enzyme right here, if we zoom back in time, would have started right here. It would have attached first to this RNA primer and then, it can only read the DNA molecule, DNA polymerase can only read the original DNA moving from the three prime end over here, reading along that, moving towards the five prime end. DNA polymerase can only move in one direction. DNA polymerase can only read the original DNA moving from three prime to five prime.
9:32
This is important because this explains kind of a weird thing about DNA replication.
9:41
So, this DNA polymerase right here, kind of started basically at the same time, we zoom back, both helicase and DNA polymerase would have started right here. On this strand, this strand, this is our three prime end, this one over here is our five prime end, DNA polymerase can continue along following right behind helicase in one continuous stretch. So, because it is being synthesized in one continuous piece, on this strand right here,
10:12
we say this is our leading strand because it is synthesized much faster than this strand up here. So, because DNA polymerase reads the old DNA moving three prime to five prime,
10:28
it can follow right behind DNA helicase and creates DNA in one continuous piece, creates new DNA in one continuous strand.
10:38
Okay, so DNA polymerase reads old DNA three prime to five prime.
10:46
The DNA creates, remember, has to be antiparallel, so if it's reading three prime to five prime, the DNA it's building is in the five prime to three prime direction. Okay, but take a look at this strand up here. Remember, helicase would have started right here. If DNA polymerase were to attach on this strand right here and try and follow along right behind DNA helicase on this strand up here, it would be going in the opposite direction.
11:20
It can't read the DNA molecule, remember this is our five prime end right here of the old DNA. The old DNA five prime end is here, three prime end is here. DNA polymerase cannot move along the original DNA five prime to three prime. It can't because the nucleotides involve orientation, it doesn't sit in the active site of DNA polymerase in that orientation, in that direction.
11:44
So, instead, what's going to happen, see all these short pieces right here,
11:52
on the opposite side, on this side right here, DNA is constructed in short tiny little pieces. DNA polymerase can move in this direction on the strand. It can move from three prime to five prime.
12:05
So, what happens is helicase is going to move a little bit forward, that creates a little space.
12:14
RNA primase comes in, lays down an RNA primer, DNA polymerase grabs on, creates a short piece until it reaches the end of that replication fork. Then it'll fall off, DNA polymerase will fall off. Helicase moves forward a little bit more, exposes a few more bases,
12:36
primase comes in, adds the primer.
12:39
DNA polymerase attaches and creates a short sequence of DNA, again, until it hits that previous primer.
12:48
Helicase moves forward a little bit more, exposes a few more bases on this strand,
12:54
primase DNA polymerase creates a short strand. So, it's synthesized in short pieces because this strand, the orientation of this strand is basically running opposite to the direction that DNA polymerase can read.
13:09
So, this strand is synthesized much, much lower compared to this leading strand down here.
13:17
This process of opening up a few more bases, adding a primer, making your short piece of DNA, falling off, opening up a few more bases, adding a primer, making a short pieces of DNA, falling off, that process takes a lot more time, so this strand is actually synthesized quite slowly, relatively, compared to the leading strand.
13:38
So, the strand that's synthesized in short pieces, we call this the lagging strand in DNA replication. And we give these synthesized pieces of DNA, we call them Okazaki fragments after the scientist that discovered that this is how DNA replication happens in the lagging strand, these short pieces of DNA. So, DNA polymerase can only read three prime to five prime, so on one end, it can follow right behind DNA helicase.
14:12
On the other side of that original DNA molecule, it kind of has to go backwards. It has to move in the opposite direction the DNA helicase is working. So, it synthesizes that DNA in short pieces, in fragments.
14:27
But we're still replicating DNA, we're still making progress, right? So, this will continue on, helicase will continue on all the way down the line through the chromosome. And then, we've got to connect all these pieces together, we don't want any gaps in our DNA molecule. We want it to be one continuous molecule, right.
14:46
So, there's going to be another enzyme that comes in called ligase
14:52
that will connect together these pieces. It will remove the RNA primer and lay down a short sequence of DNA to connect together these fragments of DNA. So, ligase stitches everything together to make one continuous piece of DNA. So, we don't have any of these RNA primers hanging out there and then, all of those phosphodiester bonds between the sugar and the phosphate groups, they're all connected into one beautiful continuous piece.
15:21
So, our key players, again, helicase opens up those hydrogen bonds between the two strands of DNA.
15:29
Primase creates an RNA primer which allows DNA polymerase to attach. DNA polymerase can read the old DNA strand moving three to five prime direction.
15:44
So, one strand, DNA helicase can follow right behind, sorry, in one strand, DNA polymerase can follow right behind DNA helicase.
15:53
On the opposite strand, DNA polymerase has to be synthesized, these sections, in small pieces. We can call that the lagging strand. The strand that's synthesized in one continuous piece is the leading strand.
16:10
So, now let's remove this block on our left-hand side and let's look at, also, what's going on over here. It's the same thing. So, here's helicase, moving in this direction.
16:24
Here's polymerase following right behind it, so this would be our leading strand.
16:29
And then, this strand now down here is our lagging strand.
16:34
Okay, it's all about that orientation in the molecule, polymerase can move towards the five prime end. Polymerase has to move towards the five prime end.
16:42
So, these two pieces right here together, DNA helicase will move in both directions, open up the DNA strand because we've got a lot of DNA to replicate, 3.2 billion base pairs. So, just moving one direction, it's going to be pretty slow. So, we start in the middle and we move both directions.
17:02
And actually on a chromosome, there will be several replication forks like this. This is what we call this, a replication fork where DNA helicase pops in and then unzips that DNA by breaking those hydrogen bonds.
17:16
Okay, that was a lot going on there. So, refresh your memory of what each of these enzymes do and be able to explain why do we have a leading strand and why do we have a lagging strand during DNA replication.
17:31
So, DNA replication, this process, we get about 1,000 nucleotide base pairs per second. Per second. That is so fast, 1,000 base pairs are created in this process of DNA replication every second.
17:47
Whoa, that's a lot, right?
17:50
So, DNA polymerase, this enzyme is not perfect. It might make an error. It might accidentally bond a G with a T, instead of a C. Right, it might bond guanine with a thiamine instead of a cytosine. So, it's not perfect. It does do some proofreading, DNA polymerase does proofread to make sure that it's connecting these bases correctly as it's creating its new DNA molecule. When you're laying to 1,000 bases per second, you're going to make some errors, you're going to mess up.
18:23
And that's okay, right. Generally, that's okay. For the most part, that's not a huge problem. We have mechanisms for DNA repair, okay. So, let's say we have some damaged DNA, or like this might be the wrong base that was added on by DNA polymerase and it didn't see that in its proofreading process.
18:44
So, we can actually take out this chunk of DNA and basically repair it. So, there's another type of DNA polymerase that can come along and repair sections that are damaged. So, even though proofreading does occur, we might get either this bond happened incorrectly, the wrong parts of the sugar and phosphate were bonded together which creates like a little bump in the DNA molecule. Maybe we put a cytosine instead of a guanine or instead of adding a thiamine,
19:14
the wrong base could be in here, right? So, basically what's going to happen, we're going to trim out that section of DNA, that's the error has, a few bases on either side, trim it out. It's an enzyme called an endonuclease, basically, nuclease is just enzymes that can, boop, act like scissors to kind of cut open the DNA.
19:34
A different type of DNA polymerase comes in, fixes that sequence that was cut out, and then ligase comes in and seals it all up.
19:45
So, there's many other mechanisms of DNA repair. We can sometimes use, also, the homologous chromosomes, so if there's a big section of DNA that's damaged, we can cut out that entire section of the chromosome, and then use the sequence from the homologous chromosome to repair or to determine what sequence was supposed to be there in that damaged DNA.
20:12
So, usually, we can fix most of those errors. But DNA replication is happening all the time, our cells are dividing all the time. There's going to be some errors that are not caught. There's maybe some errors that are not caught by DNA polymerase. There are going to be some errors that are not caught by the enzymes that just keep your DNA from having errors, right. Not all enzymes are perfect.
20:36
So, sometimes mutations might arise. And a mutation is when a DNA base pair is either damaged or the wrong base pair gets inserted.
20:49
Right? So, let's say we had a sequence of DNA, originally, that was supposed to be ATG.
20:58
The original sequence was ATG.
21:01
So, let's say the sequence, again, ATG. What would the mRNA codon be for that?
21:08
UAC right? If we look at our codon chart of UAC, we would have the amino acid tyrosine.
21:25
So, DNA ATG, RNA UAC gives us the amino acid tyrosine.
21:36
But now, let's say we switched that last letter of DNA, instead of ATG, we have ATC. The mRNA codon of that would be UAG. UAG which would result, instead of having tyrosine there, that's a stop codon. That protein, that polypeptide chain would stop at that point, even if there's more information, more amino acids that should be added on, ribosome doesn't know that, all it sees is stop codon. So, that polypeptide chain ends right there. That could be hugely detrimental.
22:24
Right? So, bases can be inserted on accident, extra bases added, bases can be accidentally removed, or we can mix up the bases. All of those are mutations that could result in a change to the final polypeptide sequence.
22:45
And we call those mutations.
22:49
But mutations aren't always bad, aren't always good, most of the time, they're neutral, but mutations are how we get genetic variation. Mutations are how you and I look different, how everybody in the world looks different. Mutations are how we have so many species on our planet, right.
23:07
But generally, mutations, typically say we don't want them to happen in living organisms because they can have detrimental effects. This is how we cause - cancer is often caused by mutations.
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