Now there are other replication proteins
and I wanna say a
few words and illustrate to you
the concepts about how they work.
If I take a DNA polymerase and I take
the 4 deoxyribonucletides necessary to make DNA,
and I take a strand of DNA, as I have
seen right here on the screen.
When I try to replicate that, what I
discover is it will not replicate.
Now you might say well that's the function of
DNA polymerase but it turns out DNA polymerases
have built into them
another need. And the other need they
have is that that they will only extend
a strand that has already started.
So you can see on this figure
that the top red is a strand
that has already gotten started.
And if I put those together with this and by the
way this strand that got started is called the primer,
okay? And the primer is an RNA
molecule not a DNA molecule.
So the polymerase will extend
from an RNA primer forwards
and when I put those things together I
make the molecule that you see above.
Now as you might imagine that RNA
primer on there is gonna have to be
removed at some point in order
for replication to be complete.
And we will see how that is different in a
prokaryotic system versus a eukaryotic system and that
has some very interesting implications.
Another molecule involved in replication
is single strand binding protein.
As its name suggest its role
is binding to single strands
and it turns out that single strands
are pretty hazardous things.
They are hazards is because a break in a
single strands, lets every thing fly away.
There is no information on the other strand
because there is no other strand
to copy and repair and replace it.
So single strand binding protein
helps maintain the integrity
and hopefully protect that single strand
during the time the replication is occurring.
So we can see this occurring right here.
If we have a replicating DNA in a cell and there
is a bare single strand that usually
it will have that single strand binding protein on there.
As the replication proceeds, the single
strand binding proteins will be released
and go to bind other single strands.
Now this helicase enzyme is
a very interesting enzyme.
This enzyme has the role of peeling
apart the strands of a DNA duplex.
Now that is pretty cool. This helicase has to do this
so that it is not slowing down the DNA polymerase;
because, the strand have to be pulled apart
in order for the polymerase to replicate.
Now here is the amazing thing.
The polymerase replicates at the
rate of 1000 nucleotides a second.
A 1000 nucleotides a second is about
100 turns of the DNA per second.
And if you multiply that out, that means that
DNA has to be unwinding at the rate of 6000 rpm.
That's faster than the engine in your car. It's really
remarkable process and it's facilitated by helicase.
Now the helicase does this seemingly without effort,
although, it does use ATP in the process of making this happen.
Well as you might imagine that if we
peel things apart really quickly
with the helicase, then ahead of that
place we are peelings things hard
what we may be doing are creating overwinding.
So you can imagine if you pull something
apart like that that has two strands
ahead of place for you pulling it apart it's
gonna very very tight and very very overwhelmed.
This tension that's created by peeling apart
of the strands has to be relieved;
because, if it's not relieved the DNA
will break, the DNA will get knotted
and any of those circumstances will be
very very detrimental to the cell.
Well fortunately the cell has an
enzyme called the topoisomerase
that specializes in reliving tension.
So, by relieving the tension
ahead of the helicase
the DNA doesn’t get overwhelmed, the replication can proceed
and everything is surprisingly smooth.
Now this does this by a couple of different mechanisms.
They are called topoisomerase 1 that work with
1 strand at a time or the topoisomerase 2
that work with both strands at the same time.
Now the topoisomerase 2 is called gyrase
and that's the enzyme that is mostly
involved in the E-coli replication system.
Completing the DNA replication, as I said,
requires the removal of RNA primers.
So how is it that RNA primers
are removed and replaced?
Well this requires the action of two additional
proteins and a DNA polymerase. So let's take a
look at how that happens.
Let's imagine we have a bacterial
DNA. Now bacterial DNAs
are circular. They are not linear like the DNAs
in our cells are. Bacterial DNAs are circular.
If the cell would to take and
start replication of this circle
we could imagine that it might start with
an RNA primer, as seen here, and in fact would
wind its way around the circle
until it got back up to the top.
And when it gets to the top, we can zoom it
a little bit and see what's here,
on the incoming strand on the left that's
the 3 prime end of that growing strand
and the place where the primer was
where was the 5 prime end was,
okay? The primer gets removed
by an RNA-cutting enzyme.
What is an RNA-cutting enzyme?
Well in E-coli it turns out
that the RNA-cutting enzyme is an
activity within DNA polymerase 1.
DNA polymerase 1 does three
things. It replicates DNA
It proofreads read DNA and it removes RNA primers.
And we can see that that's already happened here.
It's called a 5'-3' exonuclease.
And it's found again in the
DNA polymerase of E-Coli.
In our cells, in eukaryotic cells, we have a
separate protein that performs that function.
But nonetheless the RNA
primers have to be removed.
The gap then is filled by DNA
replications. So DNA polymerase 1
fills in that little gap and you will see there is
a notch up there where the two strands aren't joined.
The two strands have to be joined
in order to finish the duplex
and the joining of those is made
by an enzyme called DNA ligase.
A DNA ligase's job is to join the pieces.
Now there is only one piece here to join but as we
will see in the replication fork, there are many.