Leading and lagging strands in DNA replication
Roles of DNA polymerase, primase, ligase, helicase and topoisomerase in DNA replication. An explanation of leading and lagging strands.
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- A bit confused... When labeling the double-stranded DNA, didn't he draw the arrows wrong? Shouldn't the arrow on the left strand of DNA be going 5' --> 3', since phosphates would be continuously added to the 3' carbon of the deoxyribose??(70 votes)
- In other terms, the first part of what he said was the direction of deoxyribose (left the sugar is going down and right the sugar goes up,) the second part he states is that if you wanted to add any other phosphate group you would have to add from a 5' end to a 3' end, that is the only way you CAN add another.(13 votes)
- Why is it that the DNA polymerase can only add nucleotides on the 3' end?(13 votes)
- DNA Polymerase can only add nucleotides at the -OH group which is on the 3' end. This free -OH group is necessary because it can carry out a nucleophilic attack on phosphate group of the incoming deoxyribonucleoside triphosphate which would contain the base that is complementary to the template strand.(47 votes)
- I had understood that helicase unwinds DNA and then topoisomerase would reduce the strain caused by the unwinding by adding negative supercoils. In this video at5:08he says that the topoisomerase unwinds first then helicase comes in. What am I missing?(19 votes)
- That was my understanding from a class as well - helicase separates the hydrogen bonds between bases (e.g., A, T, C, and G), but thereby creates tension (because a coiled object is being held straight). To relieve this tension (and to keep the DNA from becoming knotted together), topoisomerase clips the DNA into shorter fragments.(19 votes)
- I have watched other videos regarding DNA replication. Some refer to an enzyme DNA gyrase. Is there any difference between DNA gyrase and topoisomerase?(10 votes)
- DNA gyrase is a subtype of Type 2 topoisomerase that is found in only plants and bacteria. Some people also say the DNA gyrase and topoisomerase 2 are the same thing. Gyrase relieves strain while double stranded DNA is being unwounded while topoisomerase Type 1 relaxes strain. Topoisomerase type 1 does not requires ATP while DNA gyrase does.(16 votes)
- Why is RNA Primer added to the lagging strand and not a DNA one? surely the RNA primer may be composed of uracil: how is this changed into DNA? also why is it DNA primase that adds RNA Primers?(4 votes)
- The RNA primer does contain uracil but it is actually removed and replaced by DNA by enzymes including a nuclease and a polymerase.
There are no DNA primers in humans. The evolutionary reason for this is not clear. DNA primers work fine in laboratory techniques such as PCR.
The enzyme is called 'DNA primase' referring to the thing being primed (the DNA) rather than the primer itself (RNA).(12 votes)
- In the beginning of the video you talk alot about the DNA going from 3' -> 5' but in the movie about the Antiparallel structure of DNA you say that it's going from 5' to 3'. I don't really understand!(5 votes)
- Take a look at the top comment—I think it addresses your question. At the start of the video, he isn't saying that DNA "goes" from 3'->5'. He just drew it that way to show you which end was the 3' end and which was the 5' end. The arrows their were arbitrary. If you listen closely, he always says that DNA is synthesized 5'->3', so he hasn't contradicted himself.(6 votes)
- Why can't you replicate from the 5' to the 3'?(3 votes)
- What do you mean? On the leading strand, replication occurs 5'->3' in a straightforward manner, while on the lagging strand, it occurs 5'->3' in a disjointed fashion via Okazaki fragments.(4 votes)
- How can people stay awake while doing science? Are there any tips?(3 votes)
- I like to doodle what he says in fancy script. So I stay on topic but can make my hands do something. Hope this helps!(4 votes)
- what are the blue things attached to the strands?(4 votes)
- They are called Single Strand Binding Proteins, or SSB proteins. They bind to single stranded DNA to keep the strands from re-annealing to each other during DNA replication.(1 vote)
- On the leading strand, how does primase know to start at the beginning of the fork? If it starts elsewhere, you have to wonder what happens to the split bases behind it.
Also, if topoisomerase breaks the backbone of the mother DNA temporarily (in order to straighten it out), what causes it to get back together? Is that the the job of the Single Strand Binding Proteins (purple circles behind Ligase) or do they just keep the mother strand from reforming?(3 votes)
- Short RNA stretch made by primase is called primer. On handout (11-3) of events at the fork, RNA primer is positioned. Primase catalyzes the synthesis of primer, and then DNA polymerase adds on the 3' end of the RNA primer.
DNA helicases start unzipping DNA molecule propr to replication fork by hydrolyzing ATP and propelling themselves along the DNA strand. (1000np/sec).
Singel strand DAN binding proteins bind tightly and cooperatively to exposed single-stranded DNA strands without covering the bases, which therefore remain available for templating. You ma yay they stabilize fork and prevent from re-annealing.
A DNA topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves.
so, yes, you can say topoisomerase acts, but not to re-anneal DNA strands, but to untangle it.
Then why DAN strands re-anneal? No special enzyme or protein. SBS got removed, and hydrogen bonding helo bases get together again.
- [Voiceover] Let's talk a little bit in more depth about how DNA actually copies itself, how it actually replicates, and we're gonna talk about the actual actors in the process. Now, as I talk about it, I'm gonna talk a lot about the 3' and 5' ends of the DNA molecule, and if that is completely unfamiliar to you, I encourage you to watch the video on the antiparallel structure of DNA. And I'll give a little bit of a quick review here, just in case you saw it but it was a little while ago. This is a zoom-in of DNA, it's actually the zoom-in from that video, and when we talk about the 5' and 3' ends, we're referring to what's happening on the riboses that formed part of this phosphate sugar backbone. So we have ribose right over here, five-carbon sugar, and we can number the carbons; this is the 1' carbon, that's the 2' carbon, that's the 3' carbon, that's the 4' carbon, and that's the 5' carbon. So this side of the ladder, you could say, it is going in the ... it is going, let me draw a little line here, this is going in the 3' to 5' direction. So this end is 3' and then this end is 5'. It's going 3' to 5'. Notice three, this phosphate connects to the 3', then we go to the 5' connects to a phosphate, this connects to a 3', then it connects-- then we go to the 5' connects to a phosphate. Now on this end, as we said it's antiparallel. It's parallel, but it's oriented the other way. So this is the 3', this is the 5', this is the 3', this is the 5'. And so this is just what we're talking about when we talk about the antiparallel structure. These two backbones, these two strands are parallel to each other, but they're oriented in opposite directions. So this is the 3' end and this is the 5' end. And this is gonna be really important for understanding replication, because the DNA polymerase, the things that's adding more and more nucleotides to grow a DNA strand; it can only add nucleotides on the 3' end. So if we were talking about this right over here, we would only be able to add … We would only be able to add going that way. We wouldn't be able to add going … We wouldn't be able to add going that way. So one way to think about it is you can only add nucleotides on the 3' end or you can only extend … You can only extend DNA going from 5' to 3'. If you're only adding on the 3' end, then you're going from the 5' to the 3' direction. You can't go from the 3' to the 5' direction. You can't continue to add on the 5' side using polymerase. So what am I talking about with polymerase. Well let's look at this diagram right over here that really gives us an overview of all of the different actors. So here is just our of our DNA strand, and it's, you can imagine it's somewhat natural, in it's natural unreplicated form, and you could see we've labeled here the 3' and the 5' ends, and you could follow one of these backbones. This 3', if you follow it all the way over here, it goes, this is the corresponding 5' end. So this and this are the same strand, and this one, if you follow it along, if you go all the way over here, it's the same strand. So this is the 3' end, and 3' end of it and then this is the 5' end of it. Now the first thing, and we've talked about this in previous videos where we give an overview of replication, is the general idea is that the two sides of our helix, the two DNA, the double-helix needs to get split, and then we can build another, we can build another side of the ladder on each of those two split ends. You could really view this as if this is a zipper, you unzip it and then you put new zippers on either end. But there's a lot of-- in reality, it is far more complex than just saying "Oh, let's open the zipper and put new zippers on it." It involves a whole bunch of enzymes and all sorts of things and even in this diagram, we're not showing all of the different actors, but we're showing you the primary actors, at least the ones that you'll hear discussed when people talk about DNA replication. So the first thing that needs to happen, right over here, it's all tightly, tightly wound. So let me write that, it is tightly, tightly wound. And it actually turns out, the more that we unwind it on one side, the more tightly wound it gets on this side. So in order for us to unzip the zipper, we need to have an enzyme that helps us unwind this tightly wound helix. And that enzyme is the topoisomerase. And the way that it actually works is it breaks up parts of the back bones temporarily, so that it can unwind and then they get back together, but the general high-level idea is it unwinds it, so then the helicase enzyme, and the helicase really doesn't look like this little triangle that's cutting things. These things are actually far more fascinating if you were to actually see a-- the molecular structure of helicase. But what helicase is doing is it's breaking those hydrogen bonds between our … Between our nitrogenous bases, in this case it's an adenine here, this is a thymine and it would break that hydrogen bond between these two. So, first you unwind it, then the helicase, the topoisomerase unwinds it, then the helicase breaks them up, and then we actually think about these two strands differently, because as I mentioned, you can only add nucleotides going from the 5' to 3' direction. So this strand on the bottom right over here which we will call our leading strand, this one actually has a pretty straightforward, remember this is the 5' end right over here, so it can add, it can add going in that direction, it can add going in that direction right over here. This is the 5' to 3', so what needs to happen here is to start the process, you need an RNA primer and the character that puts an RNA primer, that is DNA primase. We'll talk a little bit more about these characters up here in the lagging strand, but they'll add an RNA, let me do this in a color you can see, an RNA primer will be added here, and then once there's a primer, then DNA polymerase can just start adding nucleotides, it can start adding nucleotides at the 3' end. And the reason why the leading strand has it pretty easy is this DNA polymerase right over here, this polymerase, and once again, they aren't these perfect rectangles as on this diagram. They're actually much more fascinating than that. You see the polymerase up there, you also see you one over here, polymerase. This polymerase can just, you can kind of think of it as following the opened zipper and then just keep adding, keep adding nucleotides at the 3' end. And so this one seems pretty straightforward. Now, you might say wouldn't it be easy if we could just add nucleotides at a 5' end, because then we could say well this is going from 3' to 5', well maybe that polymerase or different polymerase could just keep adding nucleotides like that, and then everything would be easy. Well, it turns out that that is not the case. you cannot add nucleotides at the 5' end, and let me be clear, this 3' right over here, this, I'm talking about this strand. This strand right over here, this, let me do this in another color, this strand right over here, this is the 3' end, this is the 5' end, and so you can't, you can't just keep adding nucleotides just like that, and so how does biology handle this? Well it handles this by adding primers right as this opening happens, it'll add primers, and this diagram shows the primer is just one nucleotide but a primer is typically several nucleotides, roughly 10 nucleotides. So it'll add roughly 10 RNA nucleotides right over here, and that's done by the DNA primase. So the DNA primase is going along the lagging, is going along this side, I can say the top strand, and it's adding, it's adding the RNA primer, which won't be just one nucleotide, it tends to be several of them, and then once you have that RNA primer, then the polymerase can add in the 5' to 3' direction, it can add on the 3' end. So then it can just start adding, it can just start adding DNA like that. And so you can imagine this process, it's kind of, you add the primase, put some primer here, and then you start building from the 5' to 3' direction. You start building just like that, and then you skip a little bit and then that happens again. So you end up with all these fragments of DNA and those fragments are called Okazaki fragments. So, it's a Okazaki fragments, and so what you have happening here on the lagging strand, you can think of it as, why is it called the lagging strand? Well you have to do it in this kind of … it feels like a sub-optimal way where you have to keep creating these Okazaki fragments as you follow this opening, and so it lags, it's going to be a slower process, but then all of these strands can be put together using the DNA ligase. The DNA ligase; not only will the strands be put together, but then you also have the RNA being actually replaced with DNA and then when all is said done, you are going to have a strand of DNA being replicated, or being created right up here. So when it's all done, you're gonna have two double strands, one up here for on the lagging strand, and one down here on the leading strand.