- Let's take a look at a segment of DNA
that's in the process of being replicated.
I want to focus in particular
on the enzyme that replicates DNA.
That enzyme is DNA polymerase.
Actually there are a few different types
of DNA polymerases, and the one
that we're looking at right now is DNA polymerase III.
DNA polymerase III synthesizes new DNA,
and it also has the ability to proofread,
or kind of check the DNA it's putting together
and make sure there are no mistakes in it.
But before we get into that, let's just orient ourselves
and quickly summarize the diagram
that we're looking at.
This enzyme over here is DNA helicase.
That's the enzyme that unwinds
the double-stranded DNA,
so that DNA polymerase can then come in
and started replicating.
Right over here you can see
I drew the backbone in a different color.
That's the RNA primer.
Let's just label the DNA strand that's being synthesized,
it's synthesized from 5 prime to 3 prime.
Actually, the bottom strand of DNA
is synthesized in the same time as the top strand,
but I just left that out of the drawing
to keep things simple.
Let's say that the yellow bases represent
the nitrogen base thymine.
Let's say that the orange bases represent cytosine.
The green ones represent adenine.
And the blue ones represent guanine.
Thymine and cytosine are the pyramidines.
They are composed of a single ring structure,
so they're made up of one ring
that has six sides to it.
Then adenine and guanine are the purines.
They are a double-ringed structure.
They're composed of one ring with six sides to it,
and then that ring is attached to another ring
that has five sides to it.
Actually, these structures are a little bit more complex.
There are other atoms in it,
and there are some double bonds,
but we're just going to keep things simple for now
and leave it at that.
Let's get back to our DNA that's being replicated.
Right over here I left a space.
I didn't put the nucleotide in.
Let's say that by accident,
instead of it being paired up
with the proper base, which is adenine,
it accidentally gets paired up with a guanine.
That's a mistake.
DNA polymerase III actually has the ability
to sense if it made a mistake,
and if it does realize that it's going to go backwards.
It's going to actually remove the incorrect base
and replace it with the correct base.
So let's do that.
It's going to remove the incorrect base
and replace it with the correct one.
Of course, remember the nitrogen base
is attached to the sugar backbone.
This activity that I just described to you
is called exonuclease activity.
Nuclease, that tells us that means the ability
to remove a nucleotide.
Exo, just going to underline that.
Exo tells us that it can remove a nucleotide,
but only from the end of a DNA strand.
It was able to remove the nucleotide
because it was at the end of a strand.
This is in contrast to endonuclease activity.
An endonuclease can actually remove
a nucleotide from the middle of a DNA strand.
So it would be able to remove a nucleotide
from right over here, for example.
Just keep that in mind because we're going to
come across some endonucleases as well.
Anyway, back to our exonuclease activity.
If we want to be more specific,
the exonuclease activity of DNA polymerase III
is actually 3 prime to 5 prime
exonuclease activity.
The reason it's called 3 prime to 5 prime
exonuclease is because
when DNA polymerase 3 makes that correction
it has to move backwards in the
3 prime to 5 prime direction
in order to do that.
There's another enzyme, DNA polymerase I.
I'm just going to abbreviate polymerase with POL.
DNA polymerase I also has exonuclease activity.
DNA polymerase I is actually the enzyme
that will remove the RNA primer
at the end of replication.
Just as a side fact, the exonuclease activity
of DNA polymerase I is actually
in the 5 prime to 3 prime direction.
If you want, you can just keep that in mind.
DNA polymerase III and DNA polymerase I
are both able to repair or fix mistakes
that happen during DNA replication.
Just to give you some perspective
as to how often this occurs with and without repairs,
normally we'll have a mistake happening in replication
between 1 in 100,000 bases to 1 in 1 million bases.
That's normally the amount of mistakes that would occur.
But, with the repair mechanisms
of DNA polymerase III and DNA polymerase I,
this is reduced to a mistake that happens
once in about 100 million bases.
They are very, very effective at lowering
the error rate in DNA replication.
The next question I want to ask is
what if this mistake over here
was somehow not corrected during replication?
Maybe there was something wrong
with one of the enzymes, something happened
and that mistake was actually sustained.
Let's take a look at that.
Here is a piece of DNA with our mistake
incorporated into it.
Before we discuss if this mistake
can be corrected or not,
let's see what happens if this mistake is not corrected.
Right here we have our original DNA.
We're replicating it.
Let's just say that this strand over here
is the same as that strand.
Let's say that the bottom strand
in our original DNA is the same as this strand.
Let's look first at the newly replicated DNA on the left.
We have right over here a thymine base.
Assuming DNA was replicated properly,
it's going to have an adenine complementary to it.
Now let's take a look at the DNA on the right.
On the bottom we had a guanine,
and it's going to be paired up, hopefully,
with the correct base, which is a cytosine.
Now, let's just quickly look back at our original DNA.
We were supposed to have a thymine
with it's complementary adenine,
and actually, that's exactly what we got over here.
Just going to circle it.
So this DNA is actually in the correct sequence.
But look at the DNA over here on the right.
This is not correct.
This is a mutation.
This is an example of how mutations can occur
if the DNA repair mechanisms are not working properly.
Let's go back to our original question.
Can we fix the original mistake
so that this mutation does not occur.
The answer to that question is yes.
Fortunately, our cells have what's called
the mismatch repair mechanism.
The mismatch repair mechanism is composed
of a number of proteins.
The first thing these proteins are going to do is
they're going to recognize if there's a problem.
The reason that they're able to recognize the problem,
is that when you have a mismatch in DNA
it tends to distort the sugar backbone a little bit.
They are going to mark the area with a cut.
They are going to cut the incorrect base
or mark it with a cut.
The next thing that's going to happen
is an exonuclease is going to remove
the incorrect nucleotide.
So we're going to remove the incorrect nucleotide.
The next step is one of the DNA polymerases
is going to insert the correct nucleotide.
So we're going to pair our thymine up with adenine.
The last step is a DNA ligase is going to
connect the new nucleotide
to the nucleotides on its sides,
and also to its complementary nucleotide
on the other strand.
I'm actually going to just correct
that distorted sugar backbone.
Here's our repaired DNA.
Just to clarify, the mismatch repair mechanism
that we're talking about here
happens after replication.
The repairs done by DNA polymerase III
and DNA polymerase I that we discussed before,
that happens during replication
or at the end of replication.
One thing you might be wondering is
how does the mismatch repair mechanism
know to distinguish between the original parental strand
and the newly synthesized strand
that has the mistake on it?
In other words, how does it know
which base over here is correct,
in our case that's the thymine,
and which one is incorrect,
in our case, well, it was a guanine.
We know the answer to that question in bacteria.
In bacteria, the parental strand will have
adenines that are methylated.
I'm just going to draw some methyl groups
on all the adenines.
That allows the mismatch repair mechanism
to kind of recognize and distinguish between
the original strand that has the correct base on it,
and the new strand that has the incorrect base on it.
But we're not quite sure how
the mismatch repair mechanism in eukaryotic cells
and in other prokaryotic cells
knows to distinguish between
the strand that has the correct nucleotide
and the strand that has the incorrect nucleotide.