Biology:DNA Polymerization-Triphosphate Nucleotide

Loving the enthusiasm!

05/08/2012

~ Lyndsay

I love the enthusiasm in this tutorial, it helped catch and keep my interest throughout. No mean feat at the moment as I am nursing a headcold and have the concentration of a goldfish, but this tutorial still managed to help me get my foggy head around the subject where my text book couldn't. The only reason it didn't score a five was because it left me hanging at the end...I hate movies that end on cliffhangers ;P ! Suppose I shall just have to purchase the next installment then, great sales technique!

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Molecular Genetics
Introduction to DNA Replication
DNA: Polymerization with Triphosphate Nucleotides

Meselson and Stahl, remember Meselson and Stahl? Remember the semi-conservative replication? Let’s just take a
real quick look at this. Remember that the big debate was how are we going to replicate this stuff? All right Watson
and Crick, you came up with this great crystallized description of this double helix, but how does it replicate? Because
we all know that cells replicate. We know that the genetic material has to have the ability to pass on its message.
And so what we saw was that, oh, okay we get it now. The DNA helix is going to split. And in splitting, one strand is
going to be a template of the other. So, for example, if this one has a sequence, oh we’ll just make it simple so I don’t
have to think, of four As and two Ts, well then this one would have had a sequence of four Ts and two As, as Watson
and Crick established. And then when a new strand comes in and is laid down, then the new strand is going to come
in here. And because of the rule that A and T always go together, and G and C always go together—I probably
should have put some Gs and Cs in here just so I didn’t seem like I was discriminating. What we have is two identical
strands. There’s one. And we’ll put this one right in here, A, A, A, A, T and T. Now look. Those two strands are
identical. And indeed if you were to pull out these purple strands and put those two back together, they would be
identical to the two daughter strands, if you will.
So this was the idea of semi-conservative replication. But there are some things that aren’t explained here. And
obviously there are some things that needed to be figured out. How is the polymerization of this new strand going to
happen? For those of you who don’t remember what polymerization means, don’t forget polymerization is making
polymers. It’s adding monomers, pieces on, so that you get a long chain. We know about, for example, that
carbohydrates, starches, are polymers of glucose or other monosaccharides. Well here are obviously polymerizing.
And that brings us to the whole dilemma. How are we going to polymerize this chain?
Well we have several dilemmas we have to solve. Number one, this is an endergonic process. Remember what that
means? If it’s endergonic, that means that there is no energy given off. This won’t happen spontaneously. Let me
show you what I mean by polymerization. I’m not just talking about base pair matching. Watch what I mean. If I am
going to go back to the strand, and I am adding onto the strand, let’s say that this is one of the parental strands. And
so I now need to add—and I’m going to catch it right like half way through. And say this is A, and I am starting to build
a new strand. Well let’s put a T here, but now let’s see what comes next. What comes next is not only do I have to
have a base pair match. That part is easy. That, in essence, can be spontaneous, or certainly more spontaneous
than what I’m talking about. By polymerization, I mean if there’s a G here, I don’t mean just matching up a C. That’s
good, but I want to know how I’m going to hook that C to that T. And now if there’s a T here, how am I going to hook
that A to that C? In other words, how am I going to form this new chain?
So if we take a look at, say, a strand of DNA lying down... So here’s my master strand, my template. And now I’m
going to start adding nucleotides in here. All right, cool, we’re going to put one there and I’m going to put one here.
But the real question is how are we going to link these two together, the polymerization. So the G-C and the A-T
matching have been explained by Watson and Crick. But what about the polymerization? What about the
lengthening of that strand? That’s where we want to go.
So number one, this is not going to be a spontaneous process. All right, we’ll make an enzyme. And we’ll name it
something real simple. We’ll name it DNA polymerase. Ase, that name of an enzyme; polymer, so it’s going to
polymerize DNA. And indeed, we’re going to find out that, in fact, here you go. Are you ready? You’re going to find
out. There are several different DNA polymerases that do several different jobs. But the key is enzymes, in and of
themselves, do not provide energy. Enzymes lower energy, but in and of themselves, they are not energy providers.
You know what energy providers are. You know that we have to form phosphorylated intermediates or we have to
dephosphorylate ATP to get energy passed. And that, my friends, brings us back to something you’ve heard of
before, triphosphate nucleotides.
Let’s talk about triphosphate nucleotides. Triphosphate, you’ve seen one before. Let’s talk about a nucleotide. A
nucleotide, the whole analogy of the house with the driveway and a swimming pool, well in our cells, particularly in the
nucleus of our cells is what I’m going to refer to as the nucleotide pool. And I’m going to make it a little bit bigger. In
our cells is a triphosphate nucleotide pool. Now first let me show you where that comes from, or why we call it
triphosphate nucleotide pool. Simply stated, what we have are these nucleotides, but not in the form that they appear
on a polymer of DNA, with their single phosphate groups in the backbone of the ladder. But instead we have these
floating freely throughout the nucleus. And we can have A, in fact this one, wow, looks an awful lot like ATP to me,
which pretty much it is ATP. Do you know the only difference between the triphosphate nucleotide pool A and ATP?
Right here, in fact the nucleotides we’re going to use to make DNA are going to be triphosphate deoxyribonucleotides,
because remember DNA stands for deoxyribonucleic acid. And what that simply means is that right here we have
some bonds that are going to be a little bit different. This one is missing an oxygen. This one has OH. Okay? So
this is really a deoxyribonucleotide, just a little bit of semantics there between you and I so you know that. So this
triphosphate deoxyribonucleotide pool, they’re all floating around.
So now let’s not lose track of the forest for the trees here. What are we looking to look for? We’re looking for
something that, as it polymerizes, can provide energy. Don’t forget that. That was the original question. How can we
do this endergonic bonding when we don’t have an energy source? And now we see it. The nucleotides, the
deoxyribonucleotides are found in the triply phosphorylated stage. So therefore we must have not just ATP, but we
also must have TTP, GTP and CTP. Yah, we do. So now when we start polymerizing these things, we can use that
triple phosphorylation there and actually break it. And what are you going to break it to form? Well think about it. If
you break this right here, and remove those two phosphates, and form what is called a pyrophosphate, the double
phosphate bond, you will release energy. And you can use that energy to polymerize that DNA. And it is the energy
that is released by breaking that bond that is going to be used to polymerize this DNA.
And we have one last problem we have to discuss here. And we go back to that whole idea of anti-parallel structure.
Very briefly, let’s look at this. Remember this. Here we go. Remember that one end of the DNA molecule has what
we’re going to call the five prime end. That simply means that the number five carbon is at the top, and I’m going to
draw this thing in this direction. So let’s just say this DNA just split. So we’ve got to do the other side. And remember
the other side is opposite. And remember something that I taught you guys. And I know you all know, even if I didn’t
teach it to you, you know something about enzymes. And what do you know about enzymes? There are three
important words when it comes to enzymes. What are they? Shape, shape, and shape. All right, that being said, I
have this molecule now that I just split open right here. And now I want to polymerize. So I’m going to pop an
enzyme on here. Boom! DNA polymerase lands on here. And its job is, if this is an A, to take a triphosphate
nucleotide and pop it in there. And if this is a T, to take a triphosphate nucleotide and pop it in there. What’s my
dilemma? My dilemma is this. If shape means everything, how can an enzyme read this strand, which is the upside
down version of this strand, if shape is everything? Do you see what I’m getting at? So the bottom line is it looks like
our enzyme is going to have to make a choice. Do I read this side? Or do I read this side? Or is there a way I can
read them both? Well I’m not going to tell you the answer to that yet, but you’re starting to get the idea that this is not
going to be as simple as my little drawing.
So before we go, let’s just take one quick look at what this looks like. Here’s one strand right here. And here’s what I
really want you to see. Here comes my triphosphate nucleotide, TTP. This one happens to be thiamin triphosphate.
It’s coming in. There’s its OH group. And it’s adding onto this side. In order to do this, we have to break the bond.
We’re going to break the bond right here. And now we can take this T and hook it right here. And look what we end
up with. We’ve given off my pyrophosphate. My other P hooked right here. And there is my polymerization. So
you’re saying, “Oh great, now I get DNA. Now I know how it’s polymerized.” We’re not even close, but you have the
basics. Phosphates, pyrophosphates released; polymerization, a line begins. But now we have to see how are we
going to read both sides of the strand, and what enzymes are involved. That’s coming up next.