Well let's think a little bit about this.
Let's talk first about the DNA polymerase.
Now a DNA polymerase is an enzyme that
catalyzes the formation of the strand
and you can see that shown schematically
at the bottom of this figure.
You can see that the DNA polymerase
is sitting on top of the strand
at the end of a strand which is labelled 5 prime
end. So it's sitting on the 3 prime end
and you see that the direction
of replication is to the right.
All DNA replication, no exceptions, occurs
in the 5 prime to 3 prime direction.
As I said, it reads the base on one strands and then
it knows what to put into the strand that it's building.
So for example, if the DNA polymerase sees a G in the
opposite strand, it will incorporate a C in the strand
that it's building; because, C is complimentary to the G.
The DNA polymerase selects the proper bases by
knowledge of the base pairing rules which say
the G goes with C and A goes with T for example.
So the role of the DNA polymerase is to
grab the new base, as I said, and join it in
a phosphodiester bond in the growing chain.
Now the DNA polymerase is
an interesting enzyme in that
many DNA polymerases especially ones found in cells
has another catalytic activity associated
with it called proofreading.
Now proofreading is, you can
think of like an editor.
DNA polymerase is really good at finding the
right base across from the opposite strand.
But you have to remember that the DNA polymerase
is working at an incredible rate.
Some DNA polymerases are incorporating the growth
of a strand by a 1000 nucleotides a second.
So proofreading and checking for errors
is very important; because, this
DNA molecule that's made will become the genetic
information for the next cell and you wanna have
as few of errors as possible.
The 3 prime to 5 prime exonuclease is an
activity of DNA polymerase that checks for errors.
So what it does is it reads every base
pair not only before it puts it in
but also after it has put it in and it checks for correctness.
Now this is actually a little complicated
but in simple terms what happens
is that if there is a bulge that it is at the
wrong base was put in, it will bulge. It won't
fit the same ways as if the proper base is put in.
So if the polymerase senses a bulge it knows
it has made an error and then it backs up.
So it does this with a 3 prime
to 5 prime exonuclease, as you
can see in this schematic here, and
that literally is a backing up
and a chewing out. It is literally
taking out what it put in.
And if that happens then the correct base can then
be put in by advancing in the proper direction.
Now proofreading we know improves the fidelity that is
the accuracy with which the DNA is copied by a 100-fold.
That's a pretty remarkable again as a result
of proofreading, especially, for a process that
is going as fast as DNA replication.
Now DNA polymerases have an interesting
structure and you can see the structure here.
This is actually the ribbons part of
this is actually the DNA polymerase.
And the stick figure in the middle, it is
little harder to see is the DNA being replicated.
So I have drawn in green here a figure to
show that what the DNA polymerase has
is something that looks like a hand.
And in the middle of the hand we have
the DNA strand actually being held.
Now that hand is common to many many DNA polymerases
and you can see that the DNA, as I said, being held here.
Here is a DNA polymerase known as DNA polymerase 1.
And you can see the different
regions of it that are marked here.
Now the one on the left is a modified DNA
polymerase 1 in which the proofreading
part of it is being retained but the primer removal
part, that I will talk about later, has not.
I show this figure to show you again the claw that you
can see and you can see the claw in the figure on the right
and that's the place where the DNA molecule is
held and where the synthesis actually is occurring.
Now E-coli cells have 3 DNA polymerases
that are labelled as 1, 2 and 3.
Now many cells have multiple DNA polymerases
and each polymerase tend to have
its own function. There are quite a few DNA
polymerases in human cells for example.
In E-coli, the polymerase known as
polymerase 3 replicates most of the genome.
The polymerase 1 that just I showed you
replicates fairly short fragments
and the polymerase 2 doesn't
have a major role in E-coli
but it’s thought it may play some
roles in repair the damage of the DNA.
Now polymerases differ in their fidelity and
their processivity. So let me explain those terms.
Fidelity is, again, the accuracy with which
the polymerase is copying the DNA.
This is affected by the 3 prime to 5 prime
exonuclease or the proofreading function.
Not all DNA polymerases have a proofreading
function but most cellular ones do.
Viral DNA polymerases frequently lack a
3 prime to 5 prime exonuclease and that means
that they are much more prone to making errors.
And making errors for a virus is actually a
pretty good thing; because, it helps it
to evolve faster and that's why we see difficulty
with viruses becoming resistant to
drugs sometimes for example.
Processivity is another important
concept to get a hold off.
Processivity is a description
of how long a DNA polymerase
will get on to a DNA and stick
with it as its replicating.
Now that might seem a little odd to think about.
But in fact, there is some big differences in the
way that DNA polymerases do this.
DNA polymerase 3, the one that replicates
an E-coli, is highly processive.
It gets on to a DNA molecule and will stay
on and replicating for thousands or millions
of nucleotides. Whereas DNA polymerase 1
is not highly processive.
It goes on and it comes off.
It goes on and it comes off.
Well how does it do this?
DNA polymerase 3 gets onto
and stays on the DNA; because, it uses a protein that
you see in the figure here on the right called the clamp.
And the clamp, not surprisingly, helps it to hold on to the
DNA even better than the claw of the hand that I showed you,
and it makes a little ring around
it as you can see like this.
Well with the DNA in the middle there is no
place for the polymerase to go flying off.
Polymerase 3 uses a clamp called a beta clamp.
Polymerase 1 does not use a clamp. So polymerase 1
will go on to a DNA molecule, it will replicate
and then it will fall off.
And it will go on to another DNA molecule
and it will replicate and then fall off.
Now as we will see that that falling off is built into
the types of replication that polymerase 1 is performing
and the staying on is built into the types of
replication that polymerase 3 is performing.
Now, as I said, there are some viruses that have DNA
polymerases that don't copy things very accurately.
Retroviruses are very a good example. HIV
is a virus that makes a lot
of mutations very quickly.
And it does that because it doesn't copy
the nucleic acid very accurately.
They have a DNA polymerase that's
called a reverse transcriptase
and reverse transcriptase copies RNA and makes DNA.
It's one of those exceptions to the central dogma.
The other thing associated with that DNA polymerase is odd
is that it has low fidelity; because, it has no proofreading.
And the lack of proofreading allows the retrovirus
to wildly change and so that's why
maintaining HIV with a drug
regime can be a problem;
because, the virus has built into ways of
evolving itself very quickly through mutation.