now what we intend to talk about another
concept there is DNA polymerization
properties before going into the
detailed understanding of the DNA
replication process you need to know the
enzyme the key enzyme for DNA
replication in prokaryotes as well as in
eukaryotes the enzyme for DNA
replication is DNA polymerase
so this is the polymerase enzyme what
kind of enzyme it is a polymerase so
polymerase enzyme do polymerization okay
polymerase enzyme do DNA polymerization
polymerization of DNA means basically we
have
dntps
so dntps are
tagged together make a polymer of dntps
that is known as a DNA the polymer of
dntp is DN
now for this process of polymerization
the DNA polymer is which is involved
DNA polymerase is a multi-lobuild and
multiple structure containing enzyme
we'll see that in a moment but this
polymerase has some features the feature
is that let me write the first feature
that there is a Direction
of polymerase movement and that is 5
Prime to three prime Direction
that is the direction of movement of
polymerase polymers can only extend the
Strand and second important property and
drawback of DNA polymer is that no D no
row synthesis
what does that mean it means the DNA
polymerase enzyme cannot
start the process of DNA polymerization
it's a big disadvantage
it cannot initiate the process of DNA
polymerization
although RNA polymerase is a denovo
polymerase RNA polymerase can start the
process of polymerization of rntps not
dmtps
right that's why RNA polymerase I say is
more versatile RNA polymerase can
function all on its own with just the
help of a sigma Factor
to recognize the promoters
okay but DNA polymerase requires
multiple accessory enzymes in order to
continue the DNA replication process
now as we know the DNA polymer is not a
denovo type it requires a three prime
hydroxyl a free C Prime hydroxyl to
extend it
so basically
the DNA polymerase can extend the three
prime hydroxine so let me say that we
have this interaction phosphate and
DNA interaction like this
this is phosphodiester
backbone
and these are the hydrogen bonds
present
and what we can see let's say this is 5
Prime this is C
there is a phosphate free
and this is a template let's say
okay so DNA polymerase can only extend
this free Prime hydroxyl towards
so five towards three plane and this was
inverse
right I believe we all know the five
Prime three prime concept we were not
going to discuss that
so here the hydroxyl can be extended how
they extend it because we have D and TPS
coming in
and dntp is how many phosphates they
have let's say a and adenine they have
three phosphate groups are tests
and this hydroxyl
do a nucleophilic attack to the alpha
phosphate because imagine this is
adenine see phosphate groups attached
oh
this is known as Alpha Beta gamma
phosphate Alpha is the one closest to
the
base then greater than
so the hydroxyl attacks this Alpha this
bond is broken
beta gamma phosphate released as
PPI pyrophosphere
and this adenine with a phosphate group
is
attached to that position this is the
first four violation process I mean this
is a polymerization process
so DNA polymerase can only
polymerize an existing three prime
hydroxyl
okay
here we'll see the polymerization
properties of DNA
the polymerization properties is
explained here again you can see this is
the template stand template stand means
the Strand used
to code the other complementary DNA
strand so this is the template
and if a is there in template we will
have t if C is there we have G so a t g
c pairing a t with two hydrogen bond GCB
three hydrogen bonds and you can see
this is three prime hydroxyl
and this is this is the three prime
hydroxine this was the growing three
phosphate groups so hydroxyl attacks
here and this phosphodiester Bond will
be formed PPI will be released see the
bond formed here another free visible
hydroxyl is ready for catalyzing the
next step and this ppi is broken down
into two inorganic phosphate and this is
a high Del G negative reaction that
drives the whole DNA polymerase
forward Direction the whole DNA
polymerization to forward Direction
so for this DNA polymerization event
a polymerase enzyme requires at least
two phosphate group
it requires at least two phosphate
groups here how many phosphate groups
they had three if three phosphate group
is there the three prime hydroxyl easily
continue the process of nucleophilic
attack and the process is done
but if two phosphate is there still they
can manage to do that
but if one phosphate is present
then polymerase enzyme cannot polymerize
and there are situations during our cell
cycle cell growth where there is a
single phosphate Clips site present
those sites are known as Nyx
Nick in the DNA
n i c k so when I say the DNA Nick
a phosphodigester bond broken
because only one phosphate group is
present there
if one phosphate group is there
polymerase cannot polymerize it
so Nick cannot be sealed by DNA
polymerase
and we will see that in DNA replication
in lagging strength there are multiple
weak generators at the end
so those leaks need to be filled by a
separate dedicated enzyme known as
DNA
like is
DNA ligase seals the leak
in the DNA
try to understand this concept now how
exactly it's done we'll see that
but before that see how exactly polymers
work you can see in this picture how
exactly the polymer is function
[Music]
you can see in this this is the
catalytic site of DNA polymerase we have
substitute binding site
we have a active site
so this is the template DNA
right if C is there G if T is there a if
T is there a if C is there G
now this substrate binding side will
bind to the in this case is binding to G
the incoming
dntp
here the G
and how they position it due to the help
of metal ions generally magnesium is
present there
and the Magnesium that are present here
with the help of the aspartic acid
residue which is negatively charged
aspartic acid means Co minus in the r
Loop and here we have metal ions that
stabilize this partic acid and also it
stabilizes the backbone the phosphate
that is present there I mean not exactly
the ribbon the phosphate the three
phosphate that are present here so that
the brain this is Alpha phosphate the
green color and this one beta phosphate
this is gamma phosphate and you can see
the beta gamma phosphate and Alpha
phosphate along with aspartic acid hold
together by magnesium ion here in the
center this is the Magnesium one when
they hold together so magnesium ion in
the center Four Hands is connected to
Beta gamma Alpha and this is aspartic
acid particularly the co
this cyto aspartic acid hold together by
the Magnesium here in the center
so it the primes are stick the G very
near to the substrate binding side where
the template DNA and introduction to the
substrate that is the G will be matched
so whether it's match or mismatch
depending upon that the polymers will
move forward if this is a match that is
C with G is of course Each G pairing is
correct then there is catalysis the
process moves to the next step
and if there is no match then there is a
structural change and this this
particular strand let's say a mismatch
is added then there is a structural
change and it will be transferred to
another site known as exonucleoside a
proofreading side of the polymerase
because the polymerase looks like a a
thumb like this and it has the catalytic
site and it also has a exonucleoside
which is a proofreading site
and how exactly DNA knows whether it's a
proofreading required or not because the
moment a miss or erroneous nucleotide is
bound let's say C should pair with G but
instead of G we put t
c t bond is there somehow in that case
the velocity velocity we can say that
the
speed of this polymerase process you can
say processivity not velocity the
processivity goes slow the moment you
add a wrongly floated the processivity
grows through the moment we add another
only possibility to further slows them
so it can withstand till four or five
consecutive mistakes
after that the DNA stalls I mean the
polymer installs in the DNA and of
course the DNA need to
go through the exonucleus domain of
polymerase and polymerase cuts the raw
or erroneous nucleotides out and a new
proper nucleotide is added to continue
the process so this is a natural
proofreading uh process on natural
proofreading idea of DNA polymerase but
what we are discussing here the
polymerization even particularly and
polymerization I told you that minimum
two phosphates are needed minimum two
phosphorus c-phosphates are good enough
but minimum two phosphates are needed
and
how exactly the polymer as I told you
that
two at least two phosphates are needed
if there are no two phosphate then there
is only one option that is the use of
DNA ligase to seal the knee because
ligase can attach
the phosphodestal Bond as it is like
before
without
the presence of CR2 phosphate
but the question is chemically it's not
possible
because it's a nucleophilic attack
hydroxyl attacks alpha alpha phosphate
beta gamma phosphate release so in this
case how is it chemically possible
ligase require the two extra phosphate
from some source
and that source is ATP
because ATP contains c-phosphates
so what like is do just look at this
picture very carefully
here you can see that this is DNA like
this is starting phase of DNA ligase
this is the lysine residue of the active
site at NH the amino group of the lysine
residues coming out here the active side
of the uh ligase now what happens is
that ATP Deno donates its amp form
okay amp form is donated adenosine
monophosphate
and
Pi is removed from there okay
so here inorganic phosphate two
phosphates are released and what we have
one phosphate is stacked with amp
addressing monophosphate which is linked
to the n-terminal side of the lysine
University
and now
you can see that the this is the growing
this was the Nick
means three plane hydroxyl which is free
and a five Prime single phosphate
right if there was a three phosphate
then this hydroxyl can attack the alpha
phosphate because of phosphate can be
released
by normal process of polymerization but
here
at this point this
phosphate this electron these are the O
minus that is here
it attacks this bond between NH and amp
so this electron is donated there as a
result of which what happens
lysine is now connected
lysine so from amp gets connected here
and lysine is free
lysine gets free so basically what is
the ligase gets free from lysine so what
is the job of life is ligase with the
help of Lysine associated with amp and
the moment this electron is donated amp
is attached to this phosphate and ligus
becomes free
so like this job is simply to take AMP
from ATP and transfer it to the five
Prime phosphate
of the Nick
so once that is done now how many
phosphate you got one phosphate
pre-existing green color another amp
phosphate from amp now this hydroxyl
this
see rearrangement amp gets
clipped out and a phosphodaster bond is
formed
clear
so normal process of phosphodestal bond
formation but it requires minimum to
phosphate I told you chemically also it
requires two phosphate in this case if
there is a Nick originated when there is
no availability of two phosphate then
what happens is simply
the like is enzyme utilizes ATP donates
amp from ATP where donating amb means
one phosphate is transferred existing
one phosphate was there so two
phosphates with amp and then the rest of
the attack continues and the amp slipped
out phosphodiester Bond forme