- [Lecturer] DNA replication is a team effort.
It seems a little complicated at first to learn about
all the different entities or players
that have a role in replication.
But once you learn who they are and what they do,
I promise it gets a little easier.
And so here are all the players in action.
And I just wanted to show you this sort of up front
to give you an idea of the big picture
of where all these different molecular entities
sort of fit in the role of replication,
before going through each of them one by one.
And so I just want to point out to you here.
First is this enzyme helicase.
Depicted by this little blue triangle looking thing.
And it is responsible for actually unwinding
or unzipping the DNA double helix.
And once that's done you end up with the leading strand
down here and then the lagging strand right here.
And then DNA primase is an enzyme that comes in
and it synthesizes a little RNA primer
for DNA polymerase to use as sort of a starting point
to start laying down the new nucleotides.
Once that's done DNA ligase comes in,
ligates the little fragments on the lagging strand
called Okazaki fragments.
And then there are a few other players such as DNA gyrase,
single strand binding proteins and then another one
called clamp proteins,
that also play little sort of side roles
in this whole process.
Now DNA replication, like a lot of other molecular processes
proceeds in three enzymatically catalyzed
and coordinated steps.
Those being initiation, elongation and termination.
And now comes the first major idea to really take note of
and that's the fact that DNA is read in a three to five
prime direction.
So DNA is read in a three to five prime direction.
And it is synthesized in the opposite direction,
from five prime to three prime.
So it's synthesized from five prime to three prime.
So let's see what this looks like in the leading strand.
Here we have DNA polymerase that is adding each
of the nucleotides one by one.
And here we have the template strand,
and I'm trying to trace over that right here.
And you can see that the direction
that DNA polymerase is moving is from the three prime end
to the five prime end here.
So it's reading the template three to five.
And you can see that the new strand
that is being synthesized is being synthesized
in the opposite direction.
It is being synthesized from five prime
to the three prime end.
And this often gets confused and so it's a favorite concept
to be tested.
And the way I like to remember it is if you were
to draw out some numbers say from one to five,
you would naturally read it in
the direction of three to five.
So if you just remember that DNA is read three to five,
then you'll remember that the synthesis has to happen
in the opposite direction from five to three.
So now that we've got that down we can move on
and talk about initiation first.
So replication begins at specific locations in the genome.
And those are called origins of replication.
So this here would be say, draw it out here,
an origin of replication.
And so back to this enzyme, DNA helicase.
Well helicase breaks the hydrogen bonds
holding the two DNA strands together,
and unwinds this helix.
And in the human genome there are multiple origins
of replication that go on in a single strand of DNA.
And these regions tend to be A-T rich.
Or just rich in adenine and thymine bases.
And that's because if you remember just the basics
of DNA structure,
the bonds between adenine and thymine,
there are just two hydrogen bonds,
rather than the three formed in say
a cytosine to guanine pair.
And so it's easier for DNA helicase to unzip
just the two hydrogen bonds between
adenine and thymine base pairs.
Now the resulting structure once helicase
has done its unzipping has these two prongs
which are made up of a single strand of DNA.
And one of the strands is called the leading strand
and the other one is called the lagging strand.
And each of these strands forms the template
for DNA polymerase to synthesize the new strand of DNA.
And this whole complex here is known
as the replication fork.
Which kind of makes sense with the leading
and lagging strand serving as the prongs
if you will for this fork.
Now next the enzyme DNA primase, shown here,
this little blue donut looking thing,
synthesizes a short fragment of RNA called an RNA primer,
which is then paired with the template DNA
before the main workhorse of replication,
DNA polymerase can synthesize a new strand of DNA
by extending the three prime end,
of this existing nucleotide chain.
And it adds new nucleotides to the template strand
one at a time via the creation of phosodiester bonds.
Now the energy for this process of DNA polymerization,
comes from hydrolysis of the high energy phosphate bonds
between the three phosphates attached to each
unincorporated base.
So let me show you what this looks like.
Say we have a nucleotide and here you can see
the triphosphate group, the sugar and the base.
And when a nucleotide is being
added to the growing DNA strand,
the formation of a phosphodiester bond
between the proximal phosphate to the nucleotide,
so this phosphate right here, to the growing chain,
is accompanied by the hydrolysis of this very high energy
phosphate bond here.
Let me draw it with a color that's maybe easier to see.
And so once this bond is hydrolyzed,
you have the release of these two distal phosphate groups.
So they kind of go off on their own.
And known as a pyrophosphate group.
And then the enzymatic hydrolysis
of this pyrophosphate group of this bond here,
consumes a second high energy phosphate bond
and renders the reaction effectively irreversible.
Now in general DNA polymerase is really highly accurate.
It has an intrinsic error rate of less than one mistake
for every 10 to seventh nucleotides added.
And in addition, some DNA polymerases
also have proof reading capabilities.
There are also post replication mismatch repair mechanisms
that can fix the errors that DNA polymerase makes.
And together all of these steps enable replication
fidelity of less than one mistake for every
10 to the ninth nucleotides added.
Now since the leading and lagging strand templates
are oriented in opposite directions
at this replication fork,
there is a major issue of how to achieve synthesis
in the nascent or new lagging strand of the DNA,
who's direction of synthesis is the opposite direction
of the growing replication fork.
So you can see that the replication fork is opening
in this direction and yet the synthesis of this
lagging strand is in the opposite direction.
So let's take a closer look at this lagging strand.
Now because of the way the lagging strand is oriented
to the rest of the DNA double helix
which has yet to be unwound,
the lagging strand is synthesized
in these short separate fragments.
As you can see here the primase reads the template DNA
and initiates synthesis
of the short complementary RNA primer.
Then DNA pol a, which is different from pol delta
in the leading strand, comes in and extends
this primed fragment.
Which then forms these short fragments of new synthesized
DNA called Okazaki fragments,
which you only find in the lagging strand.
The RNA primers are then removed, replaced with DNA,
and the fragments of DNA are joined together
by this enzyme here, DNA ligase.
Now let's move on to the other sort
of special team players in replication.
The first being DNA gyrase here,
which is a type of topoisomerase.
As helicase unwinds the DNA of the replication fork,
the DNA up ahead is forced to rotate.
And this process results in a build up of twists
in the DNA head.
The build up forms this torsional resistance,
this twisting resistance that would eventually
halt the progress of this replication fork.
And that's where DNA gyrase comes into play.
DNA gyrase comes into play.
This enzyme temporarily breaks the strands of DNA,
which relieves the tension in the super coils,
and it achieves this by adding negative super coils
to the DNA helix.
Now another set of special teams players
are the single strand binding proteins.
Once helicase has unwound the double helix,
bare single stranded DNA tends to fold back on itself
forming secondary structures.
And these structures can interfere with the movement
of DNA polymerase.
To prevent this, single strand binding proteins
bind to the unwound DNA until the second strand
is synthesized.
And then finally clamp proteins form a slagging clamp
around DNA helping the DNA polymerase
maintain contact with its template.
And thereby assisting with proccessivity,
or processing the DNA template to make new DNA.
And finally we can talk about termination.
Now termination and bacteria because they have circular
chromosomes, ends when the two replication forks
meet each other on the opposite
end of the parental chromosome.
And because eukaryotes have linear chromosomes,
DNA replication's unable to reach
the very end of the chromosome but instead ends
at a telomere region of repetitive DNA close to the end.
And shortening of the telomeres occurs each time
DNA is replicated, which is a normal process
that occurs in cells and is further discussed
in another video.