Eukaryotic DNA replication is a complex, tightly regulated process divided into initiation, elongation, and termination, with unique mechanisms for handling chromatin structure and chromosome ends (telomeres) to ensure accurate DNA duplication once per cell cycle.
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in this video we'll be talking about the
replication process in ukar in previous
video we have talked about replication
in procaryotes the video link would be
provided in the ey button anyway just
like procaryotic replication eukaryotic
replication is divided into three broad stages
stages
initiation elongation and lastly
termination now in this particular
context initiation and the termination
as aspect of eukaryotic replication is
quite different than procaryotes in this
video we would try to understand these
steps in sequential fashion and then try
to appreciate the difference with
respect to the procaryotic scenario so
let's begin with
initiation so in ukar the replication
initiation is pretty complex
because replication happens in a
restricted time point in the cell cycle
that means in the S phase of the cell
cycle in any other particular stage of
the cell cycle replication is not
happening but have you ever wondered why
replication only happens in the S phase
why not in other phase what prevents
replication to be restricted in S phase
you would get the answer
soon so let's look at how the
pre-initiation complex get ass assembled
and which phase of the cell cycle does
it get assembled on the DNA so the
pre-replication complex formation
happens in the G1 phase but it's
important to note that the
pre-replication complex is not activated
at this stage so the pre-replication
complex is composed of few
components most importantly the origin
of replication complex the MCM helicase
and the ctd1 and cdc6 proteins in a
moment it would be clear what are the
function of these proteins and then the
most importantly the pre- replication
complex get
activated in the S
phase but also at this particular point
no new replication complex is
formed so formation is prevented in the
S phase but it activates this prevents
two time replication in a cell cycle so
it's is a question why cell Cy while why
replication happens only once in one
cell cycle why not twice this answer has
to be understood very well so let's try
to understand so right now we are at G1
phase in this particular stage the
replication begins at a particular site
which is basically the origin of
replication equivalent this is known as
replicator site this is a particular
site on the DNA physically located on
the DNA now in this particular site
the origin of replication complex would
bind here the origin of replication
complex is examplified more simply but
it is a complex of several
proteins then there are two important proteins
proteins
cdt1 and
cdc6 these two proteins are really
important because they are the loader
proteins for the helicase so they load
the MCM 27 helicase proteins on the DNA
so if they liases are not loaded
properly then how the DNA strands would
be separated right that is why CDC 6 and
cdt1 is very
important now not only Association of
these protein to the orc is important
but also there is a licensing event that
has to happen so replication initiation
event is broadly differentiated into two
halves one is the replicator selection
where the portion of the DNA which would
be replicated is selected
and then origin activation where the
origin is activated with the help of
specific molecules such as
the uh such as the cyclan cdk complex so
this is a licensing event without
activating the PRC the replication
cannot start and the bubble cannot form
so how does it happen so in the S phase
we know the SAS cycling which is cyclan
e and cdk 2 is highly active so its
activity level is high in this a phase
so this particular cyclan and cdk
combination has the kyes activity so it
can phosphorate Downstream Target it
generally phosphorate cdt1 cdc6 and the
MCM helicases this phosphorilation event
is the licensing event so this ensure
that a uh the particular molecules uh
dislodge from the complex and the
helicases get activated so let's try to
understand why replication is happening
only once per
cycle so we understood the licensing
event it is happening in the S phase and
due to the licensing event the
replication bubble is starting to
form but why does licensing doesn't
happen in any other stage of the cell
cycle the answer is very simple the cc6
and cdt1 once phosphorilated would be
dislodged from the
complex now they cannot
reassemble if they are in a
phosphorilated state so phosphorilated
cdc6 and ctd1 cannot
reassemble that is why even if they are
present they cannot form an activated
PRC and who keeps them phosphorilated
this is basically the cycline B and cdk1
which is high in G2 and M phase end of
G2 and M phase so that is why in M phase
or in the end of G2 there is no repli
replication initi again it cannot happen
but at the end of M phase the cycling B
cdk1 activity would be down because it
would be degraded by the
APC at this point of time the cdt1 and
cdc6 would not be phosphorilated anymore
so that is why they can assemble back
onto the orc site and this is the
preliminary criteria for the origin to
fire at the S phase so now we understand
how selectively these cdt1 or cdc6 helas
loaders can assemble at the G1 phase and
can start its job at a phase
onset this ensures replication happens
only once in the cell cycle I hope this
concept is clear now let's move on so
right now we are at the initiation step
due to the phosphorilation the orc
complex fall off the cdt1 cdc6 all of
them disassemble
the hel started its job of unwinding the
DNA strands now two strands get
separated the hydrogen bonds be between
them is
broken and this is really important
because the replication bubble starts to
form now the first thing that happens
after Rubble formation is the Assembly
of several DNA polymerase enzyme in UK
carot DNA po Epsilon and Delta are the
key DNA polymerase enzymes so gets
assemble first eventually DNA Paul Alpha
which has the prim as activity that
means it can synthesize the primer get
assembled eventually last in the
sequence the clamp loader and the
sliding clamp which is also known as
pcna gets assembled to this replication
bubble now they Orient themsel into
these replication bubble and lastly the
single strand binding protein or RPA
proteins assemble which holds the uh
strands in a separated fashion it
prevents reeling of these
strands now these in the in in this
particular sequence the DNA Paul Alpha first
first
synthesize a primer this RNA primer
would be useful for elongating the or
extending that primer by polymerase
activity so the three prime hydroxy end
is important for the polymerous to start its
its
job so then this clamp loader the
Epsilon and the Delta polymerase align
itself properly in this replication
bubble it's important to note that DNA
Paul Delta is responsible for lagging
strand synthesis whereas Paul Epsilon is
responsible for leading strand
synthesis so the clamp loader and the
clamp is loaded onto the
uh in the front of the polymerase it
ensures the processivity is more in case
of eukariotic DNA replication because
eukariotic DNA is much longer in context
of procaryotes so obviously it has to do
this job in a processive manner highly
processive Manner and
precisely so now the replication bubble
extends on both the sides and it's
getting bigger you can see the lagging
and the leading strands are synthesized
but synthesized in a different way which
is pretty similar like the bacterial
scenario the lagging strand is uh
synthesized with the help of Paul Delta
in a back stitching mechanism that we
have described
earlier but anyway let's talk about the
polymer activity in bit more details so
if we zoom into this region we can see
the bases are added and this bases are
added in a complimentary fashion to the
strand so the Watson Creek B pairing
rule ensures that there is no wrong base
exonuclease the polymeris would shift
backward and try to synthesize it again
so DNA replication is quite a precise
mechanism so it doesn't incorporate
error that
much and it moves in a direction of 5
Prime to 3 Prime attaching new
nucleotides at the three prime free
hydroxy ends so this is the leading
strand so leading strand synth is pretty
straightforward whereas the lagging
strand is bit difficult because there
are discontinuity within the lagging
strand there are multiple primers which
has to be extended up to certain level
so several times the enzyme has to
engage and disengage in this process so
these fragments are obviously known as
okazaki fragments which we have already
looked at in context of procaryotic DNA
replication now eukariotic DNA
polymerase is diverse let us quickly
look at the most important ones the PA
Delta which is lagging strand
synthesizing polymerase and the Epsilon
which synthesize the leading strand
important to note that polymerase gamma
sorry polymerase gamma actually
responsible for mitochondrial DNA
replication in UK cariot Paul Alpha has
the primes activity Paul beta is
important for base exision repair there
are many other polymer such as
polymerous Theta mu Kapa all of them are
somehow associated with DNA repair procedures
now let's talk about the termination of
the uh eukaryotic replication so the UK
when we talk about the eukariotic
replication termination we have to think
about the telome which are the
specialized structures at the end of the
eukariotic chromosome the telier
replication poses a unique challenge to
the DNA replication machinery and this
is how it would be cleared I'll tell you
why so let's say we are at the end of the
the
replication bubble and the end of the
DNA cannot be replicated efficiently due
to a problem known as end replication
problem so here is the end of the
chromosome so we are right now at the
Tome look at the lagging and the leading
strand so once they are getting extended
we have to understand due to this back
stitching mechanism even if a primer is
attached at the Extreme 3 Prime hydroxy
end and a polymerase can extend that
three prime hydroxy of that primer still
when the primer is removed a gap would
be remaining in the Extreme 3 Prime
hydroxy end that is why the end would
not be replicated
efficiently that is the problem in
Progressive cell division cycle this end
becomes shorter and shorter making the
Tome short this is Tome shortening
problem that happens now how Tas can
handle these problem let's try to
understand that so teleras is an enzyme
which has protein subunit along with it
it has an RNA component the RNA
component is complimentary to the end of
that DNA molecule and also there is a t
subunit which has reverse transcripts like
like
activity so it can basically synthesize
temp synthesize the DNA without any
requirement of an exogenous template the
template is inbuilt the RNA molecule the
RNA component serve as an internal template
template
now basically this particular enzyme
sits on the end of that Strand and you
can see there is a complimentarity
already now it can synthesize the Strand
and extend it once extended it would do
this process several time and extend
the particular lagging
strand in a uh Direction eventually what
happens this particular enzyme dislodges
and then there is a
synthesis of the other strand this would
eventually help the telome to be
replicated properly now the question is
how the Tome length is regulated because
the telomere cannot be too short or too
long it turns out there are different
proteins known as Tome binding protein
which regulates the length rap one riff
one and riff 2 complexes are known
telome uh binding protein in sacom misis
so basically this inhibits the teleras
activity if there are to if the telome
length is small then less amount of
proteins would be found in the in the
bound condition so obviously the
inhibition would be less that is how
there would be more
elongation but if the length is long
there are too many of these telome
binding protein posing a very strong
inhibition on the t's activity that's
how no further elongation would happen
and the telome elongation would stop at
that point this is how a stringent
length of the tome is maintained but the
end of this particular teloma still has
a small overhang that can be understood
as a potential DNA Break by the overall
DNA repair machineries so how does cell
overcome this problem it's very easy
don't allow the enzymes to recognize
this and this is basically achieved by
folding the particular uh end in format
of a loop eventually The Strand Invasion
happens and a t-loop formation occurs at
at the end which makes the overall
overhang totally inaccessible to any
enzymes which is going to repair or any
exonucleases are actually
protected so telome shortening is
actually a big problem during eukaryotic
eukariotic cell division because each
subsequent cell division round can
shorten the teloma significantly and
scientists believe that teloma
shortening is one of the key cause of
cellular cence which is a state where
replication pause occurs cell lose its
capability to grow or divide and this is
a key method by which cells undergo
aging so our body under goes aging with
the help of replicative
senance now the problem is one might
think that replicative cin can be
overcomed if we can increase the teleras
activity and here is another problem
teleras activity is quite high in cancer
cells so if the telas activity is high
the cells become immortal and that's 2
is not very good for the body cancer
cell has the unique ability to maintain
the telome length over the subsequent
number of cell divisions so too much
telome length or too less of the telome
length is bad question is how does a
cell decide what is the optimal length
still this question is unanswered but
this is how the eukariotic replication
Ends by replicating the telomers of the
chromosome but before we end this video
let me complicate the things a little
bit more because in UK carot the DNA is
not linear it's not only DNA because
inside the nucleus the DNA is organized
in form of
nucleosomes and whenever replication or
transcription need to happen these
nucleosomes are acting as a roadblock so
nucleosomes has to be removed or slided
in order for the replication Machinery
to access these
regions and it's a huge challenge
because the correct correct scenario
exactly looks like this where the helas
has to unwind the strands while the
nucleosomes are already there the
polymeris has to polymerize when the
nucleosomes are there so basically they
have to loen up the chromatin first and
then replicate the entire eukariotic
chromosome and this is not a easy task
how difficult it is to navigate through
a road when there is a huge amount of
traffic or or roadblock exactly that is
the scenario in case of eukariotic DNA
replication so I hope this video give
you detailed insight about the
eukariotic replication
scenario so there are many such videos
in my Channel watch these videos for
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