#41 Biochemistry DNA Replication I Lecture for Kevin Ahern's BB 451/551


Uploaded by oharow on 15.02.2012

Transcript:
Kevin Ahern: We have finished metabolism.
Yay, not yay, et cetera!
We get into new stuff.
Getting ready to talk about DNA replication,
recombination, and repair.
Most of what I will have to say will concern DNA replication,
a little bit of repair, and a very tiny amount
at the end of recombination.
This is the section of the term when we start
talking about molecular biology.
I'll remind you that we have an exam in this class
on Friday of next week.
I will not be here on Friday next week,
so one of my colleagues will be giving the exam.
I've given him instructions and so forth, so he'll be here.
He's Dr. Gary Merrill.
And exam format will be like the last exam that you had,
so there won't be any changes in that.
I have built into our schedule a review session on Wednesday.
And so I'm planning to use that as the review session
because I'm gonna be leaving on Wednesday night.
I'm leaving town on Wednesday night.
I won't be able to give a separate review session.
So my plan, unless I really get behind,
will be to have class period on Wednesday
be the review session for the final exam.
So keep that in mind.
That also means that if you have questions
for me about the material and so forth,
then you'll need to connect with me before Wednesday
because I'll be leaving town Wednesday night,
and I won't be back until the following Monday.
So keep those things in mind as we're going forward.
Well, DNA replication is a traditional place to start
talking about molecular biology because it was actually
our understanding about the structure of DNA
that led to the revolution in molecular biology
that occurred in the latter half of the 20th Century.
What we know about DNA structure
arose ultimately from the publication in 1953
of a paper by Watson and Crick in an issue of "Nature"
using data they stole from Rosalind Franklin.
They published what was described, what we now know
as the structure of the B form of DNA.
I like to say that that one page paper,
one single page in Nature, is probably the most impact
per word of any publication that's happened in science.
That's not a trivial thing.
More impact per word than any paper that's ever
been published in science.
When we look at the revolution in biology
and our revolution in our knowledge about
how living systems work, we can trace 95 to 99 percent
of that right back to that one paper.
That one paper told us things that were immediately apparent,
and they led to a revolution that we're still going
through today, The Golden Age of Biology.
Well, some of what I will have to say at the beginning
is very straightforward, you've seen before
in other classes, and you know.
And what will happen as I get going further along
as we do, get a little bit deeper into the material.
I'm gonna focus a lot today on the proteins
involved in DNA replication and also
some considerations about structure.
So I'm gonna start with DNA structure,
I'm gonna go to proteins, and then I'm gonna
come back to DNA structure.
So that's kind of the plan for the day.
Well, this schematically shows the structure of a strand of DNA.
We see that it has a phosphate linked to what would this be?
A sugar down here.
And sticking off of that sugar is a base.
And then a phosphate, sugar, base, et cetera.
And so the backbone of DNA consists of phosphate,
sugar, phosphate, sugar, phosphate, sugar.
Bases are internal.
And bases are linked to the sugars,
but the bases are not linked to each other.
It is the phosphate that's linked to the sugar.
The link of that phosphate to the sugar
is known as a phosphodiester bond.
And so you see one of those right there.
It's called a diester because it's an ester
in this direction and it's also an ester in this direction.
You know that, even freshman biology and high school
know that guanine pairs with cytosine
and adenine pairs with thymine.
And the forces that hold those together are hydrogen bonds.
So as I said, there's no linkage between the two.
The only thing that is there is a force,
that force being hydrogen bonds between
the individual bases that hold them together by attraction.
There's no covalent bond between the bases, only hydrogen bonds.
Well, we see of course that guanine-cytosine attractions,
there are three of them, three hydrogen bonds
between guanine and cytosine.
And there are only two between adenine and thymine.
And that turns out to have implications later
when we talk about transcription that I'll remind you
of at that time and also, for that matter,
for replication as well.
So those weaker bonds that hold together A-T base pairs
are important things for us to understand.
The DNA strands, as you know,
are arranged in a double helical form.
And the arrangement of that helix is what we call antiparallel,
meaning that one strand is oriented this way
and the other strand is oriented this way.
We'll see the polarity of those strands in just a second.
And they're intertwined to make those base pairs
that I've already described to you.
The DNA has what we call a major groove and a minor groove,
the major groove being this big gap out here,
the minor groove being that little space that's in there.
So as we look from bottom to top, we see minor groove,
major groove, minor groove, major groove, et cetera.
And this shown in B is a view from the top down
of that same double helix.
And what you notice looking at that top down view
is that there are no bulges.
And that arises again from the fact that,
as I talked about the other day, that an A is a purine.
It's big.
It's paired with T, which is a pyrimidine, which is small.
So the dimensions of space that are taken up
by an A-T base pair are not different than
the dimensions in space taken up by a G-C base pair.
If they were, we would see bulges as we look
down there, but we don't.
So those dimensions are set by the geometry of the base pair
and the size of the bases involved in those.
There are other forms of DNA that are known.
And Rosalind Franklin, whose data was stolen
by Watson and Crick to publish Watson and Crick's paper,
actually published in the same issue
of Nature an alternative structure of DNA known as the A form.
And at the time that she published that, it was thought,
'Well yes, she has a structure of DNA,
'but it's not a very important one.'
Well, it turns out that the structure is not a trivial one.
And the structure, the reason it was thought
not to be important was that the only way
that that structure appeared in her work
was if she used very dry conditions.
She really had to dry this sample out
before this structure appeared.
And the thinking was that well of course,
DNA is never in those dry conditions,
so it's probably some sort of an artifact.
Well, we now know that even in aqueous conditions,
this structure, known as the A form,
can under some cases appear.
And one of the things that we see that
when it does appear is that the A form of DNA
causes a little bit of a bend.
We think of the B form as just going on,
and on, and on, and on, pretty much straight.
But A form DNA causes a little bit of a bend
in the DNA structure at that point.
Consequently, when we think about how DNA fits into cells,
we start thinking about how bends may actually
have some important roles.
If I have a linear strand of DNA that goes on,
and on, and on, and on, I've got a problem
because the DNA molecule is much longer
than the cell dimensions, let alone the nucleus
which is even smaller than that.
I think I've said in class before but I'll repeat if I haven't,
that if we take all the DNA in one single cell
and stretch it end to end, it goes seven feet.
So seven feet of DNA has to be coiled up,
and there's many ways of coiling it up,
and bends in the DNA may help to facilitate that coiling
and packing of DNA that is so important for it.
Well, the A form of DNA is relatively rare.
The B form is much more predominant.
Probably 99 percent of the time, the DNA is in the B form.
The A form, however, is not unknown in other systems.
The A form of DNA is in fact the form
that double stranded RNA forms.
RNA can exist in a double stranded form,
and it will always exist in the A form.
Now, there's a third form of DNA,
that when it was originally proposed, people said,
"No way.
"No way this makes any sense."
And it turned out that it made a lot of sense.
Let me just describe this to you a little bit.
If I take two strands and I intertwine them
like I have with DNA, it turns out that there's
two fundamentally different ways that I can intertwine them.
I can intertwine them one way going up.
And it turns out that I can intertwine the other way,
backwards as it were, going up.
And we think, 'Oh, that's just flipping it upside down,'
but it's not.
They're fundamentally different.
A good example is a phone cord.
If you want to see this in person,
come to my office and I'll show this to you.
But a phone cord is almost always oriented,
and this is a single strand, but it's still doing a helix.
It's almost always oriented in one way.
That one way is what's known as left-handed.
So we talk about the two different ways of putting
together strands as either left-handed
or right-handed depending upon how the coil goes.
So I can have a single strand that has a coil,
this guy has a coil, and I can determine if it's
left or right-handed.
Now I'm not going to require you on an exam
to look at something and tell me if it's right or left-handed.
So I'm just going to show you this, and if you want to see it,
I'll be happy to show it to you in person.
But the way that you tell if something
is left or right-handed for a helix, for a coil,
is to position your hand along the back side of it
and look how your fingers grip it relative to your thumb.
If they trace the way that the cord goes across the back,
then they are in fact consistent with that hand,
in this case being left-handed.
If, on the other hand, they trace going with the other hand
as I'm doing here, then they're right-handed.
Well, as I'm looking at this, this turns out to be
an unusual phone cord because it actually is right-handed.
My fingers are tracing up.
See up is there.
And I've got it.
If I try to do it with this, they cross.
If you look at the back strand, the strand is going
that way up and my finger going that way up.
It's making an X.
You probably won't see it unless you're sitting
in the very front row.
And don't worry about it if you don't,
because you don't have to do that.
But what I want to tell you is that DNA helices,
the B form of DNA, that helix is,
in fact, in a right-handed form.
The A form of DNA is a right-handed form.
So when somebody came along and said,
"Maybe we can have left-handed DNA,"
everybody said, "Well, you would have to interrupt
"the right-handed form of DNA to have that happen."
That's not gonna happen.
Well, Alex Rich, at MIT back in the late seventies,
crystallized a form of DNA.
And voila!
When he examined the crystals, it was a short stretch of DNA.
What he discovered was it was, in fact, in a left-handed form.
It has an unusual structure that he called a zigzag structure.
And that zigzag took hold and they call this the Z form of DNA.
Well again, it's one thing to take something
in the laboratory and create it
kind of like Rosalind Franklin did.
It is another thing to say it has biological meaning.
Because does this really happen inside of cells?
Well subsequent to that, it has been shown
that in fact, under certain conditions,
Z forms of DNA will form in the middle of a DNA
that's right-handed, flip back and left-handed,
and then go right-handed again.
Now some of those conditions as we will see include stress.
Torsional stress on DNA will cause unwinding
to occur to relieve that stress.
And it turns out that Z form of DNA
in some cases may play a very important role
in telling the cells something.
So I'll tell you a very brief story.
I'll make it brief, but I think it's instructive.
Back in about 2002, when the sequence
of the human genome became available,
at least the first chromosome was completely assembled.
It was chromosome 22, the shortest of the human
chromosomes other than the sex chromosome,
the shortest chromosome was sequenced.
I had a student in the HHMI program over the summer
who was working on a project with Dr. Shing Ho.
Dr. Shing Ho, who is no longer at the university,
had been a postdoctoral fellow with Alex Rich.
So he's very interested in Z DNA structures.
He was very interested also in A DNA structures.
And so Dr. Ho had created an algorithm
for determining the likelihood that a given
DNA sequence would appear in the A form,
the B form, or the Z form.
And so he told this student who's working,
and the student is a computer programmer,
he said, "Why don't we take this algorithm,
"you can write a computer program with it,
"and we'll analyze human chromosome twenty-two."
And so he did.
That was his summer project.
The student was a sophomore at the time.
By the end of the summer, he became the first human
being to ever characterize a human chromosome.
He was able to map on the entirety of chromosome 22
the likelihood that any given place in that chromosome
was in an A form, a B form, or a Z form.
Well, so what?
That's kind of fine and dandy.
That's kind of cool.
Well, the really cool thing was what happened
when they looked to see where exactly was a Z,
where exactly was a B, and all these things.
What he discovered was there was a pretty good
relationship between places that form Z
and places where genes started.
Now that's pretty darn cool because in a eukaryotic chromosome,
there's long stretches that appear to not have any function.
There's no genes in there.
So cells have to have a way of knowing where are the genes.
Well, if you have a nice flag,
which is a very different kind of structure that's there,
and the flag is going, "Hey, here.
"There's a gene right here, there's a gene right here,"
the cell has a very nice way of knowing that.
Needless to say this caused quite a stir.
The student got a first author publication
as a result of that, which was a very cool thing.
And he's, last I heard, was a graduate student
at MIT of all places.
So that's kind of cool.
That's a pretty neat thing.
It's one of those reasons I encourage students
to get out and do undergraduate research.
You can do amazing things even if you don't think
that you can by working with world-class researchers
and doing great things.
Many of you know I run the HHMI program at OSU,
and I'm happy to have students in that program.
So if you guys are interested in this summer's
HHMI program, please let me know.
We pay students 4000 dollars to work with a professor
11 weeks over the summer if we accept you into our program.
So there's a commercial advertisement paid for by the HHMI.
[class laughing]
Disclaimer.
So do undergraduate research.
I think you'll do great things if you do that.
Well, A form DNA, B form DNA, and Z form DNA,
A's and B's aren't too far apart.
Z is somewhat different.
I show you this figure not to give you a whole bunch
of numbers to memorize because I don't think
that's really important for you.
But I do want to show you a couple of numbers that are in there.
If we look, first of all, the very big thing I told you
right there was both A and the B form are right-handed
helices and the Z form is a left-handed helix.
The other thing I want to show you is that there
are differences in the number of base pairs per turn.
So if we go one turn around the helix,
how many base pairs are in that.
The B form, which is the most common form,
has about 10.5 base pairs per turn.
I'm gonna use that number later today
when I talk about strain on DNA.
Basically what we find is if we alter DNA composition
from 10.5 base pairs per turn to something else,
we create strain.
Just like we take a rubber band
and we start twisting it, we create strain.
So, too, if we alter this number, we create strain.
DNA molecules will work to relieve that strain.
Those are the basics of structure.
I'm going to come back as I said and talk about structure more.
But now I want to turn our attention
to talking about the replication of DNA.
In a very simple scheme, we look at the screen
and we see how replication proceeds.
And you probably have looked at this a hundred times before,
and you went, "Duh!
"You start with two strands, you get four.
"You go with four strands, you get eight, et cetera."
There's something else you should see on this
that you probably didn't pay attention to before.
And that is this replication is proceeding
by a what's called a semiconservative model.
What does a semiconservative model mean?
And it means that each strand in the progeny
is copied from the other one.
That means therefore that when I have two original
strands up here, the daughter strands will each
have one original and one new.
Well, that makes sense if we're copying
one strand off of the other.
At the time that people first started studying DNA,
they didn't know that.
They didn't know how DNA was replicated,
and so there was a very famous experiment performed
by two researchers known as Meselson and Stahl
who were able to demonstrate this before
any of the proteins that I'm getting ready to tell
you about were known.
Before anything was known about
the way in which replication occurred, they were able
to demonstrate that the semiconservative model was real.
Well, we follow this through, we see as we go down
here the second generation, only two have the original strands,
and then we have new strands being made here.
Well, the other thing that you notice on this figure
that's important is that at every round, I have a doubling.
I start with one DNA molecule.
I have two DNA molecules.
Then I have four DNA molecules.
Then I have eight.
Well, that makes sense because cells are that way.
If I start with one cell and it divides, I have two.
If I have those two cells that divide, I have four.
If I have those four cells divide, I have eight.
This is a progression.
It's a doubling each time.
And that doubling each time leads to really cool things
if we replicate DNA in the laboratory in the same way.
So cells are doing this automatically.
We wish to do this in the laboratory,
and I'll talk a little bit about that later.
Well, in order to replicate DNA,
we need to understand what the proteins are that
are involved in that process.
And I'm gonna start talking about proteins.
In fact, most of what I will have to say
will actually be about proteins from E. coli,
but I will later talk about eukaryotic DNA replication
because there's some really interesting
things out of that as well.
Before we can understand eukaryotic replication,
we need to understand the basics of prokaryotic
replication because the overall process is very similar.
We look at the structure of DNA polymerase,
and by the way, the enzyme that catalyzes the formation
or the copying of the DNA is known as a DNA polymerase.
We see, first of all, that it has a structure not unlike a hand.
I'm holding my hand up here in a rough approximation
of the way that DNA polymerase's structure actually exists.
You see my thumb on the left is this red guy over here.
You see my fingers, I guess on your right,
my left, I'm not saying it the right way there,
on your left, you see the fingers.
And in the middle, I've got the palm.
And it's in that palm where the DNA double helix resides.
It's in the palm where these new bases are being added.
So we've said from day one in this class
that structure is necessary for function.
Structure implies function.
Here is a hand that says, "I'm gonna hold onto DNA,
"and I'm gonna have all the action going on here in my palm."
That structure is really important.
When we compare the structures of different DNA polymerases,
they have this same general structure of a hand.
And RNA polymerase as well,
which makes RNA has a similar structure.
Not identical but a similar structure in terms of a hand.
DNA polymerase is interesting.
Now there are many, many, many, many DNA
polymerases that are known.
And essentially every one of them obeys
the following rule I'm going to tell you.
They cannot start synthesis of a strand
without there being a preexisting strand.
Whoa, what does that mean?
Let's say I've got a double helix all along here.
And I decide, okay, I'm gonna pull this double helix apart.
I'll put one strand up here.
I'll put one strand down over here.
That's this thing that I've got right here.
I've got one strand in isolation.
If I take that one strand and I add DNA polymerase,
and I add DATP, DGTP, DCTP, and DTTP,
and I mix all those guys together,
exactly nothing will happen.
Nothing.
DNA polymerase cannot start a strand on its own.
You want an absolute rule in biology, there you go.
DNA polymerase cannot start a strand on its own.
It can only extend an existing strand.
Well, if it can't start a strand on its own,
that means that the starting material can't be a DNA.
Because how would the DNA get started
if there's nothing that can start it?
Well, cells solve that problem.
Cells solve the problem by using RNA as the starter material.
The RNA is placed on the DNA strand as a result
of action of this enzyme that's called primase.
It's making a primer that does that.
Now what's beautiful is that once that primer is on there,
and notice the antiparallel nature.
Here's 3' to 5' in this direction.
Here's 5' to 3' going parallel with that.
The fives and the threes come from the numbering
on the deoxyribose, which I've talked about before.
And the RNA primer can be extended by the DNA polymerase.
So now if I take this situation,
I've got a strand of DNA.
I've got a strand of RNA that's complementary.
A paired with U, and G paired with C, etc., throughout here.
The DNA polymerase, when I dump in the DATP,
the DGTP, the DCTP, and the DTTP,
now it's gonna make DNA.
Bang!
That's cool.
It tells us that a primer is absolutely necessary.
It also tells us that we've something that's a hybrid.
Look at this.
This is part RNA.
This is part DNA.
But you know that chromosomes, in fact, are only DNA.
What does that tell us?
It tells us that the RNA has got to be removed
and it's got to be replaced if it can be.
Well, let's imagine that this guy,
we're only seeing part of the picture,
let's imagine that this is a circle,
that this circle extends all the way around.
It's not a linear piece, but it's actually a circular DNA.
And this guy goes all the way around
and is reconnected over here.
What's gonna happen when DNA synthesis occurs,
copying that strand that goes all the way around?
Well, it's gonna get all the way over here,
and it's gonna go head on with that RNA.
Everybody picture that?
So we've got a circular DNA.
And by the way, this is not unusual because bacteria
have as their chromosomal material circular DNAs.
This is what happens in bacteria.
They go all the way around.
It gets over here.
And then something has to deal with that RNA.
It has to chop out that RNA.
But now we've got a primer of DNA.
We have a DNA that's been copied all the way over here.
The polymerase simply has to continue, and it's filled the gap.
So the circle allows the cell to come back,
get rid of this thing, and everybody is fine and dandy.
Now I'll show you a little bit more of the mechanics of that.
But in principle, that's what happens
in the replication of an RNA.
I'll give you a little clue about something
that's really interesting when we look in our chromosomes.
Our chromosomes are not circular.
They are linear.
They have ends.
They're not unlike what we see right here.
They don't have a way of coming back around.
Once they have started and gone inwards,
when they're done, what are they left with?
They are left with this guy right here.
They're left with this guy right here.
This, as we shall see, leads to a shortening of the chromosomes.
Every time our cells replicate, because there's RNA
that's on the end that can't be removed,
our chromosomes get shorter, and shorter,
and shorter, and shorter.
And that has some very big implications for longevity.
We'll say more about that later.
But I want you to understand at a structural level
what's happening with replication of a linear chromosome.
It causes problems.
Well, we are not quite at that point yet.
So I need to fill in some of the blanks
about prokaryotic DNA replication.
Well, I didn't tell you something.
What I didn't tell you was if we go back
all the way around on that circle, I didn't tell you
first of all how we removed that RNA.
And further, what I didn't tell you
was once I've removed it and once I've replaced it with DNA,
I actually have a gap.
These two pieces haven't been attached to each other.
We haven't formed covalent bonds.
They just butt up into each other
and create what's called a gap or a knick.
That gap has to be joined.
A covalent bond has to be made between
the new strand and the old strand.
And that gap is closed by catalysis
of an enzyme known as DNA ligase.
Very, very, very important enzyme.
Not just for the cell because DNA ligase is necessary
to make sure we've got a fully intact DNA molecule.
But it's also important in a biotechnology laboratory.
Because one of the revolutions that's happened
in molecular biology in the past 40 years
has been due to the fact that enzymes like DNA ligases
can be used to join together different DNAs
that didn't start together.
I've got a double strand over here.
I've got a double strand over here.
I use DNA ligase.
They get glued together.
That means I can take a gene for human growth hormone
that I can isolate from a human being very readily
and I can join it to a DNA that goes into a bacterial cell,
and the bacterium will start making
human growth hormone that I can sell.
DNA ligase is one of the most important enzymes
that's been discovered, and it's known for creating
recombinant DNA molecules.
It's almost impossible to do that without it.
So DNA ligase does this.
If we look at one strand, we're only looking at one strand now,
and DNA ligase will actually work on both strands if necessary.
Here's an existing nucleotide.
It's joined to the rest of the DNA.
Here is the other side.
This could be a single nucleotide.
This could be a chain as well.
DNA ligase catalyzes the formation
of a phosphodiester bond.
Here's a phosphate.
Here's an OH.
Here is a phosphodiester bond.
DNA ligase is putting those together.
There's different DNA ligases.
Some of them have different requirements.
We're not going to worry about the requirements.
The important thing being that DNA ligase
is really good at creating phosphodiester bonds
and joining these two guys together.
Now the DNA is whole.
Now I'm gonna skip down to the replication fork
because I need to show you some things.
If we look at replication as it occurs inside of a cell,
this is a pretty good schematic representation of it.
It's not perfect, but it's pretty good.
DNA polymerase is catalyzing the synthesis
of the new strand by copying the old strand.
There's that hand that's there.
Well, what's really interesting when we look
at how this process occurs, to understand the importance
of all these different proteins that are in there.
I've told you a couple of them already.
One was primase.
The RNA primer got in there by action
of the enzyme known as primase.
Primase started that.
The second is the DNA polymerase.
In addition to not starting a strand on its own,
that is always requiring a primer, in addition to that fact,
all, underline this, all DNA polymerases only work
in one direction, synthesizing DNA
in the 5' to 3' direction.
They will absolutely not make DNA in the 3' to 5' direction.
They can only make it in the 5' to 3'.
That's absolute across all of biology.
There are very few absolutes in biology,
and you've heard two of them today.
DNA polymerases will not start a strand on their own.
And DNA polymerases will only work 5' to 3'.
Now because they only work 5' to 3',
it means that replicating these two strands
has to occur in a different way, one strand versus the other.
Let's focus on the one on the left.
The one on the left is starting.
And what's being replicated is the red,
so the new strand is the blue one.
We see the blue moving this way.
What's the direction of the blue strand?
It's going 5' to 3'.
So all the DNA polymerase had to do was get on this thing
and start copying red, and it's moving up,
and it's going in the right direction.
It just starts here.
It will go forever until it runs out of things to copy.
This strand is what's called the leading strand.
It's very simple synthesis.
Very simple synthesis.
It starts, it makes one piece, and that's it.
It will go all the way around if it's a circle
and come all the way back to the end.
And it's only made one piece.
The other strand, on the other hand,
is copied in a very different fashion.
Because remember that DNA polymerase has to also go 5' to 3'.
But that means that the synthesis
has to occur from top to bottom.
It has to occur from here downwards.
The starting material has to be high and move low.
And what does that mean?
It means that to start high, as this guy keeps moving upwards,
upwards, upwards, more new high regions keep appearing,
and synthesis has to start over, and over, and over.
Synthesis of the other strand is what's called
lagging strand synthesis.
And instead of occurring in one long piece,
it occurs in hundreds of pieces.
Little, short pieces.
Because each one has to start with an RNA.
So now we see this inaction.
Here is a piece that's being replicated.
Here is a piece that's been replicated before.
What's gonna happen?
Polymerase is gonna come along here,
and it's gonna come along here, it's gonna come along here,
and it's gonna hit this, which is?
An RNA primer.
The same thing's gonna happen over, and over,
and over as this strand is being replicated.
These little pieces have a name.
They're called Okazaki fragments.
O-k-a-z-a-k-i.
Okazaki fragments are produced during lagging strand synthesis.
Each Okazaki fragment has an RNA primer
followed by a DNA that's made by DNA polymerase.
Now I haven't told you yet how those RNA primers get removed,
but I'm gonna tell you that in a minute.
So before this strand is completed,
the RNA will have to be removed and replaced by DNA.
And DNA ligase is gonna have to tie
all of those pieces together.
Well, I'm getting a little ahead of myself.
There's other proteins here that I want to tell you
about before I tell you how the RNA primers are removed.
You see some of them on the screen.
SSB.
What is SSB?
SSB stands for single stranded binding protein.
We have them as well, but why does E. coli
have a single stranded binding protein?
Look at this right here.
There is one strand sitting out there facing the world.
It is in a much more tenuous state
then these two strands are up here.
If I damage the blue strand up here,
I know how to replace it by copying the red strand.
If I damage the blue strand right here,
what's gonna happen?
Everybody's gonna fall apart.
To protect that single strand and keep it as much
as possible from damage, single stranded binding
protein is essential to cover it up.
And it turns out single stranded binding protein
works with a DNA polymerase nicely.
It helps the DNA polymerase to do what it does,
which is copy that strand.
So single stranded binding protein
has a very important protective function.
Primase, I've already said primase.
Ligase, I've already said what ligase does.
Now one of the most remarkable proteins is right here.
It's called helicase.
Helicase is absolutely awesome, especially in E. coli.
What does helicase do?
Well, think about what has to happen in order
for replication to occur.
You see this single strand here?
That meant that this duplex had to be pulled apart.
If you're gonna make a single strand,
you've gotta pull apart two strands.
DNA polymerase doesn't pull it apart.
DNA polymerase is good at copying things.
It's not so good at pulling things apart.
A separate protein is needed to pull things apart,
and that protein is called helicase.
Helicase unravels DNA, and it unravels
DNA at a remarkable rate.
A remarkable rate.
DNA replication in E. coli occurs at the rate
of 1000 base pairs per second.
A thousand base pairs per second.
That's pretty mind-boggling, especially when you consider
that it makes one error in about every 10,000,000 base pairs.
Imagine typing at 1,000 characters a second
and making one error every 10,000,000
times you type something.
Pretty hard to do.
Polymerase is really good.
But in order for polymerase to work that fast,
the helicase has gotta be unraveling things.
And if I have a 1000 base pairs,
that means I have roughly 100 turns of DNA
that have to be unwound because it's about
ten base pairs per turn, 10.5.
About 100 turns per second get unwound.
That means that the helicase has to be spinning
at 100 turns per second.
If you translate that to minutes,
that means that the helicase is operating at 6000 rpm.
Your car would be in trouble when it hits 6000 rpm.
Here is a nanomachine that does it without even blinking.
Nanomachines are pretty incredible.
Helicase is unraveling DNA
because it's spinning at 6000 rpm.
That's really remarkable.
Well, if this guy is unspinning DNA at 6000 rpm,
what do you suppose is happening to DNA
as I pull the strands apart up here?
I'm stressing the heck out of it.
You got it.
This DNA is gonna get really stressed.
And needless to say, if I go for any period of time
without relieving that stress, what's gonna happen
is DNA is gonna end up wound in a knot.
Well, I don't want my DNA in a knot.
Ahead of the polymerase is another enzyme
that helps to relieve that stress, and that enzyme ahead
of the replication fork is called a topoisomerase.
T-o-p-o-i-s-o-m-e-r-a-s-e.
A topoisomerase relieves that stress.
In fact, in E. coli they have a specific
topoisomerase that does that.
It's called DNA gyrase.
G-y-r-a-s-e.
So DNA gyrase is a specific type of topoisomerase.
We'll see that there are several types.
And DNA gyrase is what E. coli uses to relieve
that stress ahead of the replication fork.
Yeah, a lot of proteins.
I'll stop and take questions while you guys catch your breathe.
Yes, Karen.
Karen: That single stranded binding protein,
is that only found on the lagging strand or just primarily?
Kevin Ahern: Her question is, is single stranded binding protein
only found on the lagging strand.
It's primarily found on the lagging strand.
But anyplace in the cell where single stranded DNA appears,
single stranded binding protein has
a very good affinity for it.
Yes, sir.
Male student: For DNA polymerase, where does it
know where to start and stop?
Kevin Ahern: Oh, good question.
So his question is how does DNA polymerase
know where to start and stop.
It turns out that to start, it takes assembly
of a complex in the cell.
And I'm gonna talk about that when I talk about
replication initiation later.
But that's a very important point.
It does not start in a random place.
Yes?
Female student: Where is the helicase getting the energy?
Kevin Ahern: Oh, good question.
Where is helicase getting the energy?
What's the gasoline of cells?
Kevin Ahern: ATP.
It takes a lot of ATP to replicate DNA.
A lot of ATP to replicate DNA.
So when I talk about cells having to get ready
for division and I say there is a big commitment
of energy that's there, think about how much DNA
it takes to spin a motor at 6000 rpm.
That energy has to come from a lot of DNA.
Absolutely.
Back here.
Jerry.
Jerry: [inaudible]
Kevin Ahern: So his question is are there multiple
topoisomerases that are helping this to happen,
and this can happen with one topoisomerase off in the distance.
Yeah, it's a pretty remarkable enzyme itself.
Yes, sir.
Did you have a question?
Now this is pretty remarkable.
I have to tell you.
[clanging]
There we go.
Hopefully nothing important there.
There is one other protein I want to tell you here.
One other protein.
This starts to get into the different
kinds of DNA polymerases that are there.
You only see one DNA polymerase here.
And in fact the polymerase that you see
isn't completely accurate because it turns out
there's not a polymerase here
and a separate polymerase here.
These two guys are the same polymerase.
This is what is known as DNA polymerase III,
and polymerase III has two hands,
one hand working here, one hand working over here.
The hand over here has a very different
kind of a job than the hand over here.
But they're joined kind of like this.
It has to rotate back and forth doing its thing.
So these two guys shown as separate polymerases
are the same polymerase.
Now there is another DNA polymerase that's involved.
It's not shown in this figure,
but I'm gonna describe it to you in a second.
But before I do that, I need to tell you
something about DNA polymerases.
When DNA polymerases were first discovered,
Arthur Kornberg, a researcher at Stanford,
purified out of something like 30 pounds of E. coli mass.
And by the way, E. coli is the stuff
in your poop that really stinks.
So you can imagine what 30 pounds of E. coli
was like to work with.
Tell your graduate student, "You go purify that enzyme."
They purified out of that a DNA polymerase that they said,
"Oh, we've figured out what replicates DNA in the cell."
And they called it DNA polymerase.
And they discovered that well, it didn't work very well
because they knew how fast DNA had to replicate
in order to support cell division.
They knew it was about 1000 base pairs per second.
But this DNA polymerase poked along at maybe
ten or 20 base pairs per second.
Kind of odd.
Do we have hundreds of these working at the same time?
It turns out that they knew
that there was only two working at any given time.
So they said, "Well, you've worked with 30 pounds of poop,
"and we didn't find anything.
"You're gonna go back and you're gonna find
"what really is there."
So they went back and found another polymerase.
And by this point they said, "Okay, this is DNA polymerase II."
And when they analyzed it, they discovered
it didn't work very well at all either.
Well, by this point the graduate students
are getting a little wound, a little unhappy
with having to dig through this stuff.
And they convince them to make an even bigger mass of poop.
And it's not poop.
It's the bacteria of the poop.
After a long search, they finally found
in very, very trace quantities,
they found another polymerase they called DNA polymerase III.
So the first one they called DNA polymerase had to be renamed.
It was DNA polymerase I.
The second was DNA polymerase II.
And the third one was DNA polymerase III.
Well, the reason they hadn't found
the DNA polymerase III originally was if you only
need a couple of them to replicate a chromosome,
you only need a couple of copies per cell.
The DNA polymerase I, on the other hand,
was present in a few thousand copies per cell.
Well, what the heck?
It was much more abundant, but it had a fundamentally
different way of replicating.
DNA polymerase I will replicate DNA,
but it does the following thing.
It gets onto a DNA, and it falls off.
It will replicate for maybe 100 base pairs,
and then it falls off.
Not a very useful quality if you want
to replicate a whole chromosome.
They found on the other hand,
that DNA polymerase III would in fact,
once it got onto the DNA, it wouldn't let go.
It was very, what we call processive.
DNA polymerase III was very processive,
meaning it would get on and stay on for the ride.
DNA polymerase I was very progressive,
meaning it would make something
and then it would progress to something else.
It would leave.
Well, what was the fundamental difference between the two?
Structurally both had sort of hand-like structures.
It wasn't the hand, the ability to hold on.
It turned out it was another protein.
And the other protein is seen right here.
It's called a sliding clamp.
In E. coli, that sliding clamp is called a beta clamp.
Now the beta clamp is really neat.
The beta clamp is like a ring.
The ring goes around the DNA, and the ring
attaches to a DNA polymerase.
So imagine I've got this ring,
and there's the DNA right there,
and my polymerase is attached
where my hand holding the remote is here.
It's not gonna come off.
DNA polymerase III uses the beta clamp.
DNA polymerase I doesn't.
And so it became immediately apparent
why polymerase I was falling off.
It would replicate for awhile,
but there was nothing to hold it on.
"Okay, I'll go play in the cytoplasm."
You guys want to have fun, go out and play in the cytoplasm.
I still have another minute and a half.
Let me finish this.
DNA polymerase I does have a role in this structure.
DNA polymerase I has the ability to remove RNA primers.
DNA polymerase III does not have that ability.
Ah!
So we need polymerase III to get everything started,
but E. coli needs polymerase I to remove the primers
and fill in those little tiny gaps.
And once it's filled something in,
it's okay for it to fall off.
Its structure and function are matched.
That clear as mud?
When I come back, I'm gonna say more
about both of those on Friday.
Have fun.
Captioning provided by Disability Access Services
at Oregon State University
[END]