#42 Biochemistry DNA Replication II Lecture for Kevin Ahern's BB 451/551


Uploaded by oharow on 17.02.2012

Transcript:
Captioning provided by Disability Access Services
at Oregon State University.
Kevin Ahern: Some people like this.
Maybe I've got a bigger mouth.
Some people like this volume louder than I like.
Alright, so it's Friday.
Exam a week from today.
That didn't get a yay, did it?
As I said last time, I will not be here on Friday next week,
so Dr. Merrill will be here giving the exam.
And he knows how I like to do exams so give him your attention
and seat yourselves appropriate, as I've discussed before.
As I said I will do a review session in class on Wednesday
and material will stop with wherever I stop on Monday
which will likely be the end of this section.
So the material will stop on Monday
wherever I end up stopping there.
We've got a fair amount to cover
but actually it's quite doable
and I think we actually may slow down a little bit.
I know I'm going pretty fast for some of you
and I apologize for that.
A lot of material to cover this term.
Well last time when I finished
I was in fact talking about
the removal of Okazaki fragments
by DNA polymerase one of E.coli.
So let's go back and think about that.
So we're at the replication fork
and this is one representation of the replication fork.
I like it because it's simple,
and in reality the replication fork is actually
quite a bit bigger, or quite a bit more complicated.
This begins to show some of the complexity
of that replication fork and even this
doesn't do a very good job because it still shows
two separate polymerases when in fact it's one polymerase
with two heads that are doing this.
This figure illustrates
something called the "trombone model" of DNA replication.
And the trombone model arises primarily
because of the fact that leading strand synthesis
proceeds faster than lagging strand synthesis.
So what happens is the leading strand synthesis
gets zipping along here and the lagging strand synthesis,
remember, it has to make the primer,
it has to elongate the primer, it has to detach,
and it has to keep remaking that primer
and making new strand as it goes along.
So the lagging strand synthesis can't continue
as fast as the leading strand synthesis does.
So because of that, rather than stop everything
the cell sort of loops out a part of the lagging strand
as the leading strand is moving along.
Now the leading strand won't continue forever.
It will eventually slow down.
But for at least for a portion of the time
the leading strand is moving fairly fast
and the lagging strand is being looped out
in this sliding trombone model that's there.
If you look at the videos that I posted links for online
I think you'll see this trombone model shown reasonably well.
Again they're not perfectly accurate
because at least one of them shows two different polymerases
and again that's not exactly the right model,
but you get the idea.
That reminds me also.
Somebody sent me an email after class last time
saying that someone told them in,
I think I Micro class and I'm not sure what the story was,
and I've heard this before too,
that in the class they were taught
that the leading strand synthesis was Pol three
and the lagging strand synthesis was Pol one.
That's not correct.
That is definitely not correct.
Pol one participates in the replication of the lagging strand
but Pol three is still doing it.
In fact you can see it right here.
That's Pol three that's replicating the lagging strand.
So Pol one comes in and as I noted displaces the primers.
I'm going to explain that process to you
in a little bit more detail in the lecture today.
But to say that one is Pol one and the other is Pol three
is 100% wrong so forget that if they told you that.
Well let's think then about Pol one
because Pol one is pretty critical.
Pol one has the ability to remove these primers,
and that's single stranded binding protein there.
There's a primer right there.
We've got to remove this primer and only Pol one can do it.
Pol three cannot remove the primer.
So if we think about what happens
when we have Okazaki fragments being made.
Here are a series of Okazaki fragments.
And this is again representing what's happening.
In this case the leading strand is going from right to left
and the lagging strand is going from left to right.
And the head, that is the very leftmost portion
of each of these little blue fragments, has a primer.
That primer of course is RNA and that RNA must be removed.
So to remove that what happens is DNA polymerase one comes in
and starts at the 5 prime end of that fragment.
So this is the 5 prime end of the fragment right here.
3 prime.
5 prime, 3 prime.
5 prime, 3 prime.
So Pol one will come in and attach itself
to the 5 prime end of the Okazaki fragment
and it uses an enzymatic activity
that it has to remove that primer.
Now let me explain that activity to you a little bit.
So it turns out that DNA polymerase one has not one, not two,
but actually three different catalytic activities.
Three different catalytic activities.
One of them, well duh, it's got a DNA polymerase activity.
It makes DNA.
The second one I want to talk about here
is the catalytic activity that removes the primer.
So the catalytic activity has a name
and it's called 5 prime to 3 prime exonuclease.
E-X-O-N-U-C-L-E-A-S-E.
Well what does that mean?
Well let's imagine that I am an RNA polymerase-I'm sorry,
a DNA polymerase I that has attached itself to that
5 prime end and it's going to go sliding
from left to right.
It's going to start moving 5 prime to 3 prime.
The name "exonuclease" tells us something
about what the activity does.
"Exo-" means "outside of."
So this DNA polymerase I is starting on the outside of,
that is the end, of the fragment.
And the second part of the name, "nuclease,"
tells us it chews up nucleic acid.
So what it does is it starts at the 5 prime end
and it moves inwards destroying that RNA.
DNA polymerase III does not have a 5 prime
to 3 prime exonuclease.
Well if we look at this, what we can see if we started,
let's say, let's say instead of starting here
let's say I started here.
Imagine that I had a DNA polymerase that attached itself
here and said, "Oh look, I can replicate from
this 3 prime end because this is a primer."
This is DNA here and it's already primed because
it's already been made so all it has to do is attach itself
to the 3 prime end of this fragment.
If it attaches itself here and starts putting down
nucleotides it's got to come up here
and all the sudden it's going to hit its head
on this 5 prime end of the RNA of the next fragment
and the exonuclease says, "No problem.
I'll start chewing it up for you."
So at one end the polymerase is chewing up the RNA.
At the other end it's making DNA.
It's really cool.
So it's a Pac-Man at one end
and it's a polymerase at the other end.
Well the result of that is that eventually
the RNA gets all gobbled up, gets replaced by DNA,
and the only thing that's left is a slight gap
between there, and as I said in the lecture on Wednesday
the gap gets joined together by DNA ligase.
So the upshot of this is you have removed the RNA
and you've now made an intact DNA fragment
as a consequence of that.
Now this basic process that I've just described to you
works in human beings as well as in bacteria
and every living system on earth.
The enzymes have different names.
We're not going to worry about the names.
We're going to for the most part focus on
the names in the E. coli.
Well so that's what DNA polymerase I does.
DNA polymerase I you remember is very progressive meaning
that it doesn't stay on the DNA for very long.
It falls off.
So once it's gone here a little ways it gets into here,
it falls off.
Will it chew up DNA in a 5 prime to 3 prime direction?
The answer is yes it will.
So it doesn't have to stop exactly where
the RNA/DNA junction is.
It may go a little ways into the DNA but it's going to
replace it on its backside with more DNA
so it's not a problem.
So that's a unique activity of
the DNA polymerase I of E.coli.
That unique activity cannot be replaced.
If that is destroyed, the E. coli cell will die.
Yes question, Connie?
Student: What did you say the third activity was?
Kevin Ahern: I haven't said it yet.
So the questions back here were what was the third activity.
So I'm getting ready to the big climax to talk about
the third activity here in a second.
Now the third activity that we find in DNA polymerase I,
we also find in DNA polymerase III.
So this activity I'm getting ready to describe to you
is not unique to DNA polymerase I.
It's found in DNA polymerase III,
it's found in DNA polymerase I, and in fact,
it's found in most DNA polymerases that exist out there.
Not all of them, but most of them.
Most DNA polymerases have the activity I'm describing to you.
Well let's imagine, let's go back
to my better little replication fork here.
Actually that's not a better one.
Maybe I wanna go back here.
So let's say that I'm going down the leading strand.
I'm DNA polymerase and I'm moving along here.
In this case I'm on the leading strand.
I'm definitely a DNA polymerase III
because we don't see lagging strand on the,
or we don't see DNA polymerase I on the leading strand.
DNA polymerase I is going along and it sees an A
and it puts in a T, and it sees a G and it puts in a C,
and it sees a C and it puts in a G and it sees an A
and it puts in a T and it just keeps going along and along.
And remember it's going along doing this
at a thousand bases a second.
A thousand bases a second.
Well nothing is perfect and DNA polymerase
is pretty close to perfect but it's not perfect.
It goes along and it sees an A and it makes a mistake
and it puts in a C.
What happens?
Well DNA polymerases make mistakes and so it's going along,
everything's fine and dandy until all the sudden
it's got this C/A base pair.
Well as you might imagine a C/A base pair actually
doesn't form stable hydrogen bonds.
And remember how I said the dimensions are set up
perfectly for an A/T base pair and a G/C base pair?
They don't vary in dimensions?
If you try to put an A with a C it bulges.
It bulges.
Well the DNA polymerase recognizes that bulge.
It recognizes, "Uh-oh, I think I might have made a mistake."
And so it stops.
It stops right there.
And what it does is kind of cool.
The DNA polymerase III and the DNA polymerase I each have
a 3 prime to 5 prime exonuclease activity.
What's the difference?
5 prime to 3 prime goes one direction.
3 prime to 5 prime goes the other direction.
So let's think about this.
The polymerase is moving 5 to 3, 5 to 3, 5 to 3,
here's an A/C.
It says, "Oops," and it backs up.
When it backs up it's going 3 to 5.
The 3 prime to 5 prime exonuclease activity is kicked in
as soon as it recognizes it's made a mistake
and it starts chewing out bases.
It's chewing out, it's correcting the mistake that it made.
Really cool stuff.
That activity is also called proofreading.
Proofreading is the same as 3 prime
to 5 prime exonuclease activity.
They're the same thing.
So this guy backs up and chews it out,
and removes the mispaired base.
Now proofreading is an activity that is present
as I said in most DNA polymerases.
There are some DNA polymerases however
that do not have proofreading.
When we compare the error rates that they have
compared to the error rates of DNA polymerases
that have proofreading, no surprise,
we discover that ones without proofreading
make many more errors.
A really good example of an enzyme that does not
do proofreading is the enzyme that makes DNA for HIV.
HIV is very, its DNA polymerase does not have
a proofreading ability.
It makes errors at probably about a thousandfold greater
frequency than does a DNA polymerase with proofreading.
Once it makes a mistake, "Whoops, sorry.
I guess I'll just keep going along."
Well it turns out for a virus like HIV that actually works
because mutation in general allows for variance.
The more variance that you have the more likely you are
to succeed if something is trying to stop you.
If one of those things trying to stop you is an anti-HIV drug
you can evolve resistance around it by evolving changes
in the proteins of the virus.
It's one of the reasons, and there's many,
but it's one of the reasons that HIV is very difficult to treat
because it will usually mutate its way around a treatment
that kills it because it's got
this very error prone DNA polymerase.
A very error prone DNA polymerase doesn't work well
for an organism.
Why?
Well you're much more likely to activate an oncogene,
cause a cancer, kill the organism.
For a virus that's out there,
it doesn't have to worry about that sort of thing.
A very big difference between the two.
So as I said DNA polymerase III has that activity.
DNA polymerase I has that activity as well.
At the end of the lecture last time somebody said,
"Well you didn't say anything about DNA polymerase II."
You jumped from one to three, what happened to two?
Well two turns out to be a relatively minor polymerase
in the overall scheme in E. coli.
It's mostly involved in repair and repairing damage
and I really won't address any considerations
of it here in this class and it's really not a significant
polymerase in the cell at all.
I will slow down and stop there and take any questions
that you might have with respect to that.
Yes sir?
Student: Yeah, so when DNA is proofreading
itself it backs up?
Kevin Ahern: The DNA polymerase is proofreading, yes.
The DNA can't proofread.
Student: Right, when it backs up does it just delete everything
as it's backing up?
Kevin Ahern: His question is when it backs up,
when the DNA polymerase is proofreading and it backs up,
how far does it go, basically.
And the answer is it'll actually go back a few bases.
Student: And it just gets rid of every base then?
Kevin Ahern: For a few bases.
You know, five, ten bases.
And that means it's probably removing
some perfectly paired bases.
But that's fine because now the polymerase comes back in
and just fills them back in.
Yeah.
Yes, Jodie?
Student: So it back up over, or chews up the mispaired bases,
turns around and starts going forwards again.
But then it hits the DNA that was already there.
Does it fall off at that point until a ligase...
Kevin Ahern: Not on the leading, well in the case of
DNA polymerase III it's on the leading strand
so it's not hitting anything.
In the case of lagging strand,
when it hits something else that's there then of course yes
that's always going to be a cause for it to fall off.
Yes, Jared?
Student: If I remember correctly whenever HIV infects
it inserts its DNA into the host and then [inaudible]
code for the polymerase?
Kevin Ahern: Yeah, so when HIV infects the host
it puts its DNA in the host
and that includes the coding for the polymerase.
The HIV polymerase is a different kind of DNA polymerase
than we've talked about and since you've asked the question
I'll just very briefly mention what it is.
It's an enzyme known as a reverse transcriptase.
This is the DNA polymerase found in HIV.
And reverse transcriptase derives its name from the fact that
regular DNA polymerase has to have DNA that it copies.
A reverse transcriptase will copy RNA.
That is it will use an RNA template and make DNA from it.
A regular DNA polymerase will not do that.
Now I think the upshot of your question is,
well if this polymerase is present in the cell,
is it causing problems for the cell?
Is that the upshot?
And the answer is it may be to a small extent.
But the bigger problem of the integration into the cell
is that when the RNA is transcribed it transcribes
the whole genome and that whole genome now encodes
all the proteins for packaging and so forth.
So once that genome has been transcribed into the cell
the cell is pretty much hosed.
So it doesn't exist for long enough to really have much
of a reverse transcriptase as an issue.
Yes, Connie?
Student: When you say that DNA polymerase can do base pairs
at a thousand per second, does that include its proofreading?
Kevin Ahern: So yeah, it's pretty astonishing, isn't it?
Her question was, "You're telling me this thing goes
a thousand a second and that includes proofreading?"
And the answer is yes it does.
I mean it's pretty awesome, it's pretty darn awesome.
And it's not perfect.
So even proofreading-you know you've proofed,
you've turned in papers to professors, right?
You've read it a hundred times, "I'm sure it's perfect,"
and you get it back and you go,
"Oh man, I can't believe I said so and so."
Proofreading itself is not perfect but it improves
the accuracy of the DNA polymerase at least a hundredfold,
maybe a thousandfold.
Yes sir?
Student: Is there anything that inhibits
the proofreading capacity?
Kevin Ahern: Does anything inhibit the proofreading capacity?
In terms of drugs or something?
Student: Yeah.
Kevin Ahern: Not as far as I know, no.
Okay, yeah, one more.
Student: Is primase part of the whole polymerase complex?
Kevin Ahern: Yeah, a very good question.
Is primase part of the polymerase complex?
In fact it is.
So if we look at the replication fork that I showed here,
primase is actually a part of this overall complex, yes.
Because if it's not then you've got to wait for it
to come in and you've got this delay, etc., etc.
So the E. coli cells are very efficient.
They've got everything there that's needed to do this.
There are suggestions that in addition to having all
the proteins necessary to make the DNA,
and this is kind of a cool thing, that there are proteins,
that is enzymes here that are making nucleotides
right at this fork and shooting them at the fork.
Why would that be important?
Well there's a very odd thing.
And it's debated a little bit but there's a very odd thing
that suggests that the rate of diffusion of nucleotides
to the fork, if we assume that we have an equal concentration
of nucleotides throughout the cytoplasm,
that the rate of diffusion is not fast enough
to get them there at a thousand a second.
So then you have to account for, well how in the world,
if they can't diffuse there fast enough, does that happen?
There's some suggestions that some of the machinery actually
starts synthesizing nucleotides in that direction
so they have an artificially high concentration
at the replication fork.
Kind of cool stuff.
Student: So since those trinucleotides are sources
of energy themselves, what provide the energy
to synthesize new ones?
An excess of one to move to another?
Kevin Ahern: I'm not sure I understand your question.
Student: How would you synthesize new ones there?
What's the driving force?
Kevin Ahern: What would the driving force be?
The enzymes.
I'm not sure I understand.
Student: For the creation of the nucleotides,
because they're an activated intermediate
that actually goes into those, correct?
Kevin Ahern: So the nucleotides are made
from basic building blocks, yeah.
So your question is how do the basic building blocks get there?
Student: [inaudible]
Kevin Ahern: Okay that's fine.
We'll talk later.
Let's see, let's move on.
So that's basically what's up with DNA replication
in terms of the proteins.
There's one protein I haven't said much about
and I want to say a little bit about it because
it's really interesting also.
I think many of these proteins have really cool functions.
And this is the enzyme I called topoisomerase.
Now I want you to remember that topoisomerase
is a class of enzymes and there is a specific one
that's involved in E. coli called DNA gyrase.
DNA gyrase.
Well in order for me to make some sense of this for you
I have to tell you a little bit about
what are called topoisomers.
Question?
If we look at DNA, let's say I take some DNA from a cell
and I put it under an electron microscope
which is what's happened here, and I look at it,
these are both circular DNAs and as I said E. coli has
circular DNAs, I discover something interesting.
One here on the upper left looks all kinky and all tight
and the one on the right looks, oh, it's really great big.
Well in fact these two DNAs have exactly the same number
of base pairs in them.
They have the same number of base pairs in them.
The one up here in the upper left has been all kinked up.
Well again, if you played with rubber bands you know
if you start twisting and twisting and twisting you'll see
the rubber band what it's going to do is it all coils up.
That's called supercoiling and that's what's happened
to this guy here.
Supercoiling plays several functions in cells,
one of which is making a DNA smaller.
There are other functions that supercoiling has.
But supercoiling arises because we have a closed circle,
and if we take a strand and we make a little break,
and we untwist, untwist, untwist, untwist
and we twist it back, what have we done?
Well we've just changed the number of times
that the DNA wraps around itself,
and since I said that 10.5 base pairs per turn
is the magic number, I've just changed the number of turns.
What's going to happen to that ratio?
It's not going to be 10.5 anymore.
That molecule is going to be under stress
and that stress is relieved by this.
It's relieved by kinking up.
The kinking up happens as a way of releasing that stress.
What the kinking up is trying to do is artificially,
I shouldn't say artificially, but re-put turns back into it
so that it is 10.5 base pairs per turn.
And by putting turns back into itself
it kinks up in the process.
Now I've got a better figure to show you that
so you don't have to try and envision it so much.
Let's think about this.
So there's three parameters we need to think about with DNA.
It's called linking number, twisting, and writhing.
Linking, twisting, and writhing.
For our purposes twisting and turns
are going to be the same thing.
Twisting and turns are going to be the same thing.
There's a relationship between the linking number,
the twisting number, and the writhing number.
And it's very simple.
Linking equals twist plus writhe.
L equals T plus W.
Now I'm going to tell you what linking is in a minute,
but turns are basically the number of twists
that we have of this guy.
Let's imagine I have a linear DNA.
It's got 25 turns in it.
And for our purposes of keeping this simple
I'm going to say this is at 10 base pairs per turn.
Instead of 10.5 to be relaxed we're going to say 10
to make things simple.
So this guy is 250 base pairs long.
It's got 10 base pairs per turn.
It's relaxed.
It's like you are watching the tube, right?
You're sitting there.
If I take that DNA, I lay it out on the table.
It's just going to relax
and it's going to be at 10 base pairs per turn.
If I take that DNA and I join the ends,
I join this end to this end, I'm not going to change anything.
It's going to remain relaxed and it's going to look like this.
It's still got 25 turns, twists, same thing.
It has 0 writhes and it has 25 linking number.
Now what does that mean?
Well in order to understand linking number and writhes
we have to understand what happens if we change things.
Let's say I take that same DNA.
It had 25 turns, it's got the same number of bases,
but no I unravel it a couple of times.
So now instead of having 25 turns or 25 twists it now has 23.
I have the same number of base pairs.
Same number of base pairs.
So when I put this guy together into a circle
I've got 23 base pairs but I still have 250,
I'm sorry, 23 turns but I have 250 base pairs.
This guy's going to be under strain
because it's no longer 10 base pairs per turn.
Well it's a closed circle.
It can't relax all by itself.
If I have it, lay it out here linearly it'll just kind of go,
"Vrooop," it'll kink back up.
It'll put a couple more turns in itself and it's fine.
Well if it tries to put a couple turns in itself,
let's say this strand decides,
"Okay I'm going to wrap around there once,
"then I'm going to wrap around there again," it can do it.
But it does it at the cost of what's known as the writhes.
Look at this.
The twists are 23.
The links, the linking number is 23.
Now this thing is going to say,
"Well I really want to have 25, let's get 25."
So it wraps itself around a couple times but that changes
the conformation of the molecule.
The linking number does not change because again,
I haven't opened this guy up.
In order to make this equation be equal,
the linking number equals the twisting plus writhes,
I had to put in what's called 2 negative writhes.
This is supercoiling.
When the writhe is anything but 0,
the molecule will be supercoiled.
Yes?
Student: So is it back to 10 base pairs?
Kevin Ahern: It is back to 10 base pairs per turn, exactly.
By putting these two turns in there it's happy.
But the molecule overall has a distinctly different shape.
Well where does the linking number fit in?
It turns out the linking number is the number of times
that the strand crosses over itself,
just like the number of turns,
plus the number of times the double helix crosses itself.
We see that it crosses itself twice.
In this case the crossings are negative.
It could also be positive.
Right?
What if I had put in 2 more and made 27
in the twists instead of 25?
Well it's going to uncoil a couple times
and when I uncoil a couple times
I've got to make this be a positive 2.
You're not going to have to look at a structure and tell me,
is this negative writhe or is this positive writhe.
But I think that you should know that the linking number
is equal to the twists plus the writhes.
You'll always be able to figure out what the writhe is.
You should always know that if we have supercoiling,
the writhe is something other than 0.
Now I know that is a little complicated
and I know that is a little hard to get around your head
so I'll stop there and take questions for that.
Connie?
Student: [inaudible]
Kevin Ahern: The twisting number is just simply
the number of times that the twists go around
and around and around and around.
Student: Oh okay.
[inaudible]
Kevin Ahern: Yes, that's the writhe.
So the crossing, any time the double helix crosses itself
we get a writhe.
If we look back, now let's think about this,
we go back to that picture
that I showed you to start this process.
Look at this guy.
Do you see any writhes there?
Do you see the thing crossing itself at all?
You don't.
Do you see writhes up here?
Yes you do.
The helix, in fact it's a little hard to see how many times
its writhing but you can see that there's,
that double helix is crossing itself many times.
This guy is very supercoiled.
Supercoiling can be positive or negative
and they both have the same effect.
They both will cause the molecule to kink up.
Jerry?
Student: [inaudible]
Kevin Ahern: Yeah, so his question is
will this affect gene expression?
The answer is absolutely, absolutely.
Some genes get turned on by this.
Some genes get turned off by this, yep.
Student: So is this integrated as part of, like,
chromatin expansion and contraction?
Kevin Ahern: So his question is this somehow related
to chromatin and in fact this is a consideration
in chromatin structure.
Then you say, "Well DNA in eukaryotic cells is linear!
"Why don't the ends just ravel or unravel like that thing
"I laid out on the thing?"
They're so long that you get local areas of tension
that don't get relieved in that way.
So yes it's a factor for chromatin, indeed.
Yes sir?
Student: Is the ultimate reason for this thermodynamics?
Is it more energetically favorable to be twisted up like that?
Kevin Ahern: Yeah, so his question is
is this rooted in thermodynamics,
and the answer is absolutely yes, absolutely.
Do you want to see the thermodynamics?
[Kevin laughing]
Kevin Ahern: We're not going to do that.
Student: [inaudible]
Kevin Ahern: Yeah.
Student: [inaudible]
Kevin Ahern: Okay, so let's look at what happens.
I'll show you the answer to the question, okay?
So here's what we had.
We had a relaxed circle, right?
We took the relaxed circle
and we untwisted it a couple of times
and when we put that back together we had this.
Notice that in this strained state these two guys are equal.
Notice the writhes are 0.
The double helix is not crossing itself yet.
But this is not stable.
It's not going to sit like this for very long.
It tries to re-establish its balance and when it re-establishes
its balance what we see is that the twists goes
to what made it stable in the first place.
The writhe, I mean the linking number does not change.
That means that the writhe had to change.
So a guiding principle here is if the DNA
is at something other than 10, or actually 10.5,
but it's something other than 10 base pairs per turn
it's going to have strain on it and it's going to do something
to relieve that strain, and the something it's going to do
to relieve that strain is going to be to try
to get back to 10 base pairs per turn.
In doing so it's going to have to writhe
in order to make that happen.
Other questions about that?
Well why do I tell you all this?
The reason I tell you all this is what
topoisomerases are doing.
They're affecting this twisting that's there.
They're affecting that twisting that's there.
Let's think about what's happening at that replication fork.
Remember we have a helicase ripping things apart at 6000 rpm.
The strands are coming apart and you said that ahead
of where that helicase was, if we don't relieve that strain
that the DNA's going to turn into a knot.
So I said ahead of that strain we've got,
thinking about this strain now in a different way, right?
We've changed base pairs per turn, right?
We start pulling.
If the other end doesn't give we're going to have
more base pairs per turn
and it's going to cause some supercoiling.
We're going to see some kinking.
And you've seen examples of kinking.
It can get really ugly.
The topoisomerase ahead of that is known as DNA gyrase
and DNA gyrase, in order to alleviate that strain
has got to basically open up the DNA
and let it unwind at the other end.
So what you're putting in as strain at this end
has got to be relieved at the other end.
So a topoisomerase is changing the topoisomeric configuration,
that is the way that the DNA lays is changing that twisting,
writhing phenomenon.
It's changing how the strands are wound around each other.
There are two types of topoisomerases
in general that we will consider.
One type is called Type I.
The other type is called Type II.
DNA gyrase turns out to be a Type II but it's a little harder
to understand so I'm going to start with Type I.
Type I topoisomerase.
Let's imagine I'm back here with this guy right here.
This guy is all kinked up.
It's got 10.5 base pairs per turn but that writhing
has really made the overall molecule
be in a very different configuration than this guy.
How did we go from this guy to this guy?
It turns out there's a very simple way to do it.
Imagine, if you will, that I'm an enzyme
and I break one bond between two nucleotides on one strand.
All of this energy that's stored up in here,
what do you suppose is going to happen?
Well once, so I have a double helix,
one strand-that's a little harder to do.
Okay let's see, how am I going to do that?
I've got a double helix and they're sitting here
as we see and an enzyme comes along and it makes a break
in this one strand on top but not the strand on the bottom.
Everybody understand that?
This top strand, I can have half of the thing,
actually is free to do this around the bottom strand.
Alright?
It's free to do it like crazy.
There's nothing to stop it from doing that and look at all
the energy that's stored up in this kinky molecule.
What's going to happen when I cut one strand?
Well it's going to be like a spring.
It's going to go, "Sproing!" and it's going to go
[buzzing sound]
and unwind.
It's going to relieve that strain in a different way.
What I've just described to you
is how a topoisomerase I works.
It cuts one strand to alleviate the strain
and the strand basically uses its own tension to just wrap,
wrap, wrap until finally everybody's set.
Connie?
Student: Does that mean it breaks off the hydrogen bonds...
[inaudible]
Kevin Ahern: No.
So her question is does this break the hydrogen bonds.
It does not because all that's happening is the other strand
is swiveling around its phosphodiester bond.
It's a swivel.
So the hydrogen bonds are not broken.
The base pairs are not broken.
The base pairs are still there.
But we have one phosphodiester bond
that's holding that thing together.
That's how a topo I works.
A topo I will always work by nicking one strand
and letting this guy do its thing.
Jodie?
Student: Do these attack one particular site
or is it just when the tension gets high enough for it?
Kevin Ahern: Yeah, they don't have site specificity, no.
So his question was does it attack a specific site
and the answer is they don't.
How does a topo II work?
Topo II's are pretty wild.
Topo II's work by cutting both strands.
So instead of cutting just the top strand here,
a topo II cuts both of these strands
and lets the same thing happen.
"Vriiiing!" It lets go.
Do you like the sound effects?
It lets go.
But the problem is you've got to keep the strands together.
You can't just let it go flying off in space and unwinding.
In the first case you had one strand
that was holding everybody together.
Now you've cut both strands but you still want
to hold them together.
You want to hold them so that they don't fly apart
because you want to put them back together when they're done.
And that's what DNA gyrase does.
It's a Type II topoisomerase.
Type II's cut both strands but basically hang onto both ends
and let them do their thing without flying away.
That's logistically hard for me to get my head around.
But that's what they do and that's how they work.
DNA gyrase requires ATP to do it and it's partly because
of the need to try to maintain some control here
in keeping those strands together.
Yes sir?
Student: Could these re-ligate the phosphodiester backbone-
Kevin Ahern: Oh, very good question.
His question is we've broken the bonds.
Do these enzymes put the bonds back together?
That is the phosphodiester bonds that were broken?
The answer is yes they do.
And that's part of the reason they need the ATP energy as well.
Yes sir?
Student: [inaudible]
advantageous to upregulate expression of DNA gyrase?
Kevin Ahern: Okay so his question is,
medically does this have any significance?
I'll change your question a little bit.
Medically does this have significance.
The answer is yes.
Let's think about this.
Topoisomerase is essential for DNA replication, right?
Are there differences between E. coli's topoisomerases
and human topoisomerases,
or bacterial topoisomerases and human?
And that's how you look for any antibiotic is looking
for a difference between the two.
And the answer is yes there are.
And you can find and identify drugs that will target
bacterial but not human and they're used in antibiotics.
Ciprofloxacin will do that.
Ciprofloxacin will do that.
I always like to tell the story.
No actually I won't tell the story.
I'll hold off on that story.
But there are enzymes that are very specific
for topoisomerases in E. coli that will make that happen.
There are inhibitors there.
There's nalidixic acid.
That's more of a general one.
And there's ciprofloxacin which is an antimicrobial.
What do you guys say to a song?
I said at the beginning this one's going to make me a little nervous.
I've never sung this song before.
You know I don't sing well.
And this is a really hard one to sing.
I've never sung it in class before so I really want your help.
It's a Beatles song.
Another Beatles song and it's called
I Wanna Hold Your Strands.
[class laughing]
[singing I Wanna Hold Your Strands]
Lyrics: Oh yeah, I'll tell you something
It helps to understand
Pol three decrees
To meet the cell's demands
It's gotta hold the strands
It's gotta hold the strands
The key, Pol III
Acts mostly processively
'Cause see, Pol III
Has beta clamping hands
It uses beta's bands
To hold onto the strands
As it starts replicating a DNA
The unwinding requires a lone
Helicase, helicase, helicase!
Primase starts the primer
That Pol one can erase
Pol three takes the primer
And starts the DNAs
It starts the DNAs
By using RNA
And when a fragment Okazaki displays
There must be joining
That requires
A ligase, a ligase, a ligase!
Thanks to all the factors
And all of their ligands
The cell has what matters
To replicate the strands
It replicates the strands
It replicates the strands
[singing ends]
Oh that's enough, okay.
[class laughing]
That was fun.
[applause]
Thank you.
I've never sung that one in class and with good reason.
I think you heard why.
I don't do those high notes.
I don't do any of the notes very well
but the high ones are even worse.
We've got just a little bit of time left
and in that time that's left I want to say something
about how the process starts.
And I'll probably finish with that today.
So somebody asked me last time,
"Well does DNA replication just start randomly or what?"
Well it turns out that the overall process
always starts at a specific place.
It starts at a specific sequence in a DNA
called the origin of replication.
The overall process starts at something called
the origin of replication.
E. coli has one of these.
We hope it does.
There we go.
The process starts with the binding of a protein called DnaA.
So DnaA is a protein that recognizes
and binds to some specific sequences in the E. coli genome.
In this case you see five of those sequences that are present
and we are looking right now in a region
of the overall chromosome called the origin.
In the case of E. coli it's called oriC,
O-R-I and then a capital letter C.
OriC.
You'll notice that next to these binding sites for
the DnaA there are regions of DNA that are A/T rich.
That turns out to be important.
Now when DnaA binds, it turns out that it causes DNA to coil.
It causes it to wrap.
What has it done?
It's just changed its writhing.
That has put some stress on the DNA
and the DNA will act to relieve that stress.
And how does it relieve that stress?
Well we've seen winding and unwinding.
This guy's going to unwind and it's going to unwind
at the weakest place, the place where the strands
can most easily come apart, and that's where
the A/T base pairs are because that's where
the fewest hydrogen bonds are between the bases.
This region right here will open up.
The strands-we're not breaking bonds now.
I mean we're not breaking covalent bonds.
We're breaking hydrogen bonds.
The strands, we see a loop that goes up here
and we see a loop that goes down here,
and that big opening is a place for a complex
known as DnaBC to bind.
So we get DnaA binding, opening of the DNA,
and then DnaBC binding.
Now DnaBC has something very cool associated with it.
It's a helicase.
It's a helicase.
Well what's a helicase going to do when it gets in there?
There's a single stranded binding protein
helping to hold it open.
There's B and actually C has left by this point,
but DnaBC binds, C leaves, and then we're left with this guy.
This guy is a helicase.
Well a helicase is going to take
and it's going to start peeling strands, right?
And isn't that exactly what we need to do
in order to get replication started?
You got it.
Primase is going to come in.
It's going to make an RNA primer.
And we've started the process.
The whole process happened because DnaA bound
to specific sequences next to this A/T rich region.
Yes sir?
Student: Is that just a standard TATA box?
Kevin Ahern: No it's not a standard TATA box.
TATA box is used in transcription.
So it's an A/T rich region
but it's not a standard TATA box, no.
Yes, Jerry?
Student: Is there regulation of this process?
Kevin Ahern: Is there regulation of this process?
You betcha.
There is regulation of this process.
Cells have to coordinate replication of this
and virtually every cell on earth has
some sort of control over that.
In our cells we have a very intricate set of controls
that I'll talk a little bit about in the lecture next time.
Okay I think that's a good place to stop.
Let's call it a day and I'll see you guys on Monday.
[END]