0:01 in this video we'll be talking about the
0:05 replication process in ukar in previous
0:06 video we have talked about replication
0:09 in procaryotes the video link would be
0:13 provided in the ey button anyway just
0:15 like procaryotic replication eukaryotic
0:18 replication is divided into three broad stages
0:19 stages
0:23 initiation elongation and lastly
0:26 termination now in this particular
0:29 context initiation and the termination
0:32 as aspect of eukaryotic replication is
0:36 quite different than procaryotes in this
0:38 video we would try to understand these
0:41 steps in sequential fashion and then try
0:43 to appreciate the difference with
0:46 respect to the procaryotic scenario so
0:48 let's begin with
0:52 initiation so in ukar the replication
0:56 initiation is pretty complex
0:59 because replication happens in a
1:02 restricted time point in the cell cycle
1:05 that means in the S phase of the cell
1:08 cycle in any other particular stage of
1:10 the cell cycle replication is not
1:13 happening but have you ever wondered why
1:16 replication only happens in the S phase
1:19 why not in other phase what prevents
1:21 replication to be restricted in S phase
1:24 you would get the answer
1:27 soon so let's look at how the
1:31 pre-initiation complex get ass assembled
1:32 and which phase of the cell cycle does
1:35 it get assembled on the DNA so the
1:38 pre-replication complex formation
1:40 happens in the G1 phase but it's
1:41 important to note that the
1:44 pre-replication complex is not activated
1:47 at this stage so the pre-replication
1:49 complex is composed of few
1:52 components most importantly the origin
1:55 of replication complex the MCM helicase
2:00 and the ctd1 and cdc6 proteins in a
2:01 moment it would be clear what are the
2:05 function of these proteins and then the
2:08 most importantly the pre- replication
2:09 complex get
2:12 activated in the S
2:15 phase but also at this particular point
2:18 no new replication complex is
2:22 formed so formation is prevented in the
2:26 S phase but it activates this prevents
2:29 two time replication in a cell cycle so
2:31 it's is a question why cell Cy while why
2:34 replication happens only once in one
2:37 cell cycle why not twice this answer has
2:41 to be understood very well so let's try
2:44 to understand so right now we are at G1
2:47 phase in this particular stage the
2:49 replication begins at a particular site
2:50 which is basically the origin of
2:52 replication equivalent this is known as
2:55 replicator site this is a particular
2:57 site on the DNA physically located on
3:01 the DNA now in this particular site
3:03 the origin of replication complex would
3:06 bind here the origin of replication
3:08 complex is examplified more simply but
3:10 it is a complex of several
3:12 proteins then there are two important proteins
3:14 proteins
3:16 cdt1 and
3:18 cdc6 these two proteins are really
3:20 important because they are the loader
3:24 proteins for the helicase so they load
3:29 the MCM 27 helicase proteins on the DNA
3:31 so if they liases are not loaded
3:33 properly then how the DNA strands would
3:36 be separated right that is why CDC 6 and
3:38 cdt1 is very
3:42 important now not only Association of
3:44 these protein to the orc is important
3:47 but also there is a licensing event that
3:50 has to happen so replication initiation
3:53 event is broadly differentiated into two
3:56 halves one is the replicator selection
3:58 where the portion of the DNA which would
4:00 be replicated is selected
4:02 and then origin activation where the
4:06 origin is activated with the help of
4:09 specific molecules such as
4:13 the uh such as the cyclan cdk complex so
4:15 this is a licensing event without
4:18 activating the PRC the replication
4:21 cannot start and the bubble cannot form
4:23 so how does it happen so in the S phase
4:26 we know the SAS cycling which is cyclan
4:29 e and cdk 2 is highly active so its
4:32 activity level is high in this a phase
4:35 so this particular cyclan and cdk
4:37 combination has the kyes activity so it
4:40 can phosphorate Downstream Target it
4:44 generally phosphorate cdt1 cdc6 and the
4:47 MCM helicases this phosphorilation event
4:51 is the licensing event so this ensure
4:56 that a uh the particular molecules uh
4:58 dislodge from the complex and the
5:02 helicases get activated so let's try to
5:03 understand why replication is happening
5:05 only once per
5:08 cycle so we understood the licensing
5:11 event it is happening in the S phase and
5:13 due to the licensing event the
5:15 replication bubble is starting to
5:18 form but why does licensing doesn't
5:20 happen in any other stage of the cell
5:23 cycle the answer is very simple the cc6
5:27 and cdt1 once phosphorilated would be
5:28 dislodged from the
5:30 complex now they cannot
5:32 reassemble if they are in a
5:35 phosphorilated state so phosphorilated
5:37 cdc6 and ctd1 cannot
5:39 reassemble that is why even if they are
5:43 present they cannot form an activated
5:47 PRC and who keeps them phosphorilated
5:50 this is basically the cycline B and cdk1
5:53 which is high in G2 and M phase end of
5:56 G2 and M phase so that is why in M phase
5:58 or in the end of G2 there is no repli
6:01 replication initi again it cannot happen
6:04 but at the end of M phase the cycling B
6:06 cdk1 activity would be down because it
6:08 would be degraded by the
6:12 APC at this point of time the cdt1 and
6:15 cdc6 would not be phosphorilated anymore
6:18 so that is why they can assemble back
6:22 onto the orc site and this is the
6:26 preliminary criteria for the origin to
6:30 fire at the S phase so now we understand
6:34 how selectively these cdt1 or cdc6 helas
6:37 loaders can assemble at the G1 phase and
6:40 can start its job at a phase
6:43 onset this ensures replication happens
6:46 only once in the cell cycle I hope this
6:49 concept is clear now let's move on so
6:52 right now we are at the initiation step
6:55 due to the phosphorilation the orc
6:58 complex fall off the cdt1 cdc6 all of
6:59 them disassemble
7:02 the hel started its job of unwinding the
7:04 DNA strands now two strands get
7:06 separated the hydrogen bonds be between
7:08 them is
7:10 broken and this is really important
7:14 because the replication bubble starts to
7:17 form now the first thing that happens
7:19 after Rubble formation is the Assembly
7:23 of several DNA polymerase enzyme in UK
7:26 carot DNA po Epsilon and Delta are the
7:29 key DNA polymerase enzymes so gets
7:34 assemble first eventually DNA Paul Alpha
7:36 which has the prim as activity that
7:38 means it can synthesize the primer get
7:40 assembled eventually last in the
7:43 sequence the clamp loader and the
7:45 sliding clamp which is also known as
7:48 pcna gets assembled to this replication
7:51 bubble now they Orient themsel into
7:53 these replication bubble and lastly the
7:56 single strand binding protein or RPA
8:00 proteins assemble which holds the uh
8:01 strands in a separated fashion it
8:04 prevents reeling of these
8:08 strands now these in the in in this
8:10 particular sequence the DNA Paul Alpha first
8:12 first
8:15 synthesize a primer this RNA primer
8:19 would be useful for elongating the or
8:21 extending that primer by polymerase
8:23 activity so the three prime hydroxy end
8:25 is important for the polymerous to start its
8:26 its
8:30 job so then this clamp loader the
8:33 Epsilon and the Delta polymerase align
8:36 itself properly in this replication
8:39 bubble it's important to note that DNA
8:41 Paul Delta is responsible for lagging
8:44 strand synthesis whereas Paul Epsilon is
8:47 responsible for leading strand
8:50 synthesis so the clamp loader and the
8:53 clamp is loaded onto the
8:57 uh in the front of the polymerase it
8:59 ensures the processivity is more in case
9:03 of eukariotic DNA replication because
9:06 eukariotic DNA is much longer in context
9:09 of procaryotes so obviously it has to do
9:12 this job in a processive manner highly
9:14 processive Manner and
9:17 precisely so now the replication bubble
9:19 extends on both the sides and it's
9:20 getting bigger you can see the lagging
9:22 and the leading strands are synthesized
9:24 but synthesized in a different way which
9:25 is pretty similar like the bacterial
9:29 scenario the lagging strand is uh
9:31 synthesized with the help of Paul Delta
9:33 in a back stitching mechanism that we
9:35 have described
9:39 earlier but anyway let's talk about the
9:42 polymer activity in bit more details so
9:45 if we zoom into this region we can see
9:48 the bases are added and this bases are
9:50 added in a complimentary fashion to the
9:57 strand so the Watson Creek B pairing
9:59 rule ensures that there is no wrong base
10:06 exonuclease the polymeris would shift
10:09 backward and try to synthesize it again
10:12 so DNA replication is quite a precise
10:14 mechanism so it doesn't incorporate
10:16 error that
10:19 much and it moves in a direction of 5
10:22 Prime to 3 Prime attaching new
10:24 nucleotides at the three prime free
10:27 hydroxy ends so this is the leading
10:29 strand so leading strand synth is pretty
10:31 straightforward whereas the lagging
10:33 strand is bit difficult because there
10:35 are discontinuity within the lagging
10:37 strand there are multiple primers which
10:40 has to be extended up to certain level
10:43 so several times the enzyme has to
10:46 engage and disengage in this process so
10:47 these fragments are obviously known as
10:50 okazaki fragments which we have already
10:53 looked at in context of procaryotic DNA
10:55 replication now eukariotic DNA
10:58 polymerase is diverse let us quickly
11:00 look at the most important ones the PA
11:01 Delta which is lagging strand
11:04 synthesizing polymerase and the Epsilon
11:06 which synthesize the leading strand
11:09 important to note that polymerase gamma
11:11 sorry polymerase gamma actually
11:13 responsible for mitochondrial DNA
11:16 replication in UK cariot Paul Alpha has
11:18 the primes activity Paul beta is
11:21 important for base exision repair there
11:22 are many other polymer such as
11:26 polymerous Theta mu Kapa all of them are
11:27 somehow associated with DNA repair procedures
11:34 now let's talk about the termination of
11:38 the uh eukaryotic replication so the UK
11:40 when we talk about the eukariotic
11:42 replication termination we have to think
11:44 about the telome which are the
11:45 specialized structures at the end of the
11:48 eukariotic chromosome the telier
11:51 replication poses a unique challenge to
11:53 the DNA replication machinery and this
11:55 is how it would be cleared I'll tell you
11:58 why so let's say we are at the end of the
11:59 the
12:02 replication bubble and the end of the
12:05 DNA cannot be replicated efficiently due
12:07 to a problem known as end replication
12:10 problem so here is the end of the
12:12 chromosome so we are right now at the
12:14 Tome look at the lagging and the leading
12:17 strand so once they are getting extended
12:19 we have to understand due to this back
12:21 stitching mechanism even if a primer is
12:23 attached at the Extreme 3 Prime hydroxy
12:27 end and a polymerase can extend that
12:30 three prime hydroxy of that primer still
12:32 when the primer is removed a gap would
12:35 be remaining in the Extreme 3 Prime
12:38 hydroxy end that is why the end would
12:40 not be replicated
12:43 efficiently that is the problem in
12:45 Progressive cell division cycle this end
12:47 becomes shorter and shorter making the
12:50 Tome short this is Tome shortening
12:53 problem that happens now how Tas can
12:55 handle these problem let's try to
12:58 understand that so teleras is an enzyme
13:01 which has protein subunit along with it
13:03 it has an RNA component the RNA
13:06 component is complimentary to the end of
13:10 that DNA molecule and also there is a t
13:13 subunit which has reverse transcripts like
13:14 like
13:17 activity so it can basically synthesize
13:21 temp synthesize the DNA without any
13:23 requirement of an exogenous template the
13:26 template is inbuilt the RNA molecule the
13:28 RNA component serve as an internal template
13:29 template
13:32 now basically this particular enzyme
13:35 sits on the end of that Strand and you
13:36 can see there is a complimentarity
13:39 already now it can synthesize the Strand
13:42 and extend it once extended it would do
13:46 this process several time and extend
13:48 the particular lagging
13:53 strand in a uh Direction eventually what
13:55 happens this particular enzyme dislodges
13:57 and then there is a
14:01 synthesis of the other strand this would
14:03 eventually help the telome to be
14:06 replicated properly now the question is
14:09 how the Tome length is regulated because
14:11 the telomere cannot be too short or too
14:12 long it turns out there are different
14:14 proteins known as Tome binding protein
14:17 which regulates the length rap one riff
14:20 one and riff 2 complexes are known
14:24 telome uh binding protein in sacom misis
14:26 so basically this inhibits the teleras
14:30 activity if there are to if the telome
14:32 length is small then less amount of
14:35 proteins would be found in the in the
14:37 bound condition so obviously the
14:40 inhibition would be less that is how
14:41 there would be more
14:44 elongation but if the length is long
14:45 there are too many of these telome
14:47 binding protein posing a very strong
14:51 inhibition on the t's activity that's
14:53 how no further elongation would happen
14:55 and the telome elongation would stop at
14:57 that point this is how a stringent
15:00 length of the tome is maintained but the
15:02 end of this particular teloma still has
15:05 a small overhang that can be understood
15:08 as a potential DNA Break by the overall
15:10 DNA repair machineries so how does cell
15:12 overcome this problem it's very easy
15:15 don't allow the enzymes to recognize
15:18 this and this is basically achieved by
15:22 folding the particular uh end in format
15:25 of a loop eventually The Strand Invasion
15:28 happens and a t-loop formation occurs at
15:31 at the end which makes the overall
15:34 overhang totally inaccessible to any
15:36 enzymes which is going to repair or any
15:41 exonucleases are actually
15:44 protected so telome shortening is
15:46 actually a big problem during eukaryotic
15:48 eukariotic cell division because each
15:50 subsequent cell division round can
15:53 shorten the teloma significantly and
15:54 scientists believe that teloma
15:56 shortening is one of the key cause of
15:59 cellular cence which is a state where
16:01 replication pause occurs cell lose its
16:04 capability to grow or divide and this is
16:07 a key method by which cells undergo
16:09 aging so our body under goes aging with
16:12 the help of replicative
16:14 senance now the problem is one might
16:16 think that replicative cin can be
16:18 overcomed if we can increase the teleras
16:20 activity and here is another problem
16:23 teleras activity is quite high in cancer
16:26 cells so if the telas activity is high
16:28 the cells become immortal and that's 2
16:31 is not very good for the body cancer
16:34 cell has the unique ability to maintain
16:37 the telome length over the subsequent
16:40 number of cell divisions so too much
16:42 telome length or too less of the telome
16:45 length is bad question is how does a
16:48 cell decide what is the optimal length
16:50 still this question is unanswered but
16:53 this is how the eukariotic replication
16:55 Ends by replicating the telomers of the
16:57 chromosome but before we end this video
17:00 let me complicate the things a little
17:03 bit more because in UK carot the DNA is
17:07 not linear it's not only DNA because
17:10 inside the nucleus the DNA is organized
17:11 in form of
17:13 nucleosomes and whenever replication or
17:16 transcription need to happen these
17:19 nucleosomes are acting as a roadblock so
17:22 nucleosomes has to be removed or slided
17:24 in order for the replication Machinery
17:26 to access these
17:29 regions and it's a huge challenge
17:31 because the correct correct scenario
17:34 exactly looks like this where the helas
17:37 has to unwind the strands while the
17:39 nucleosomes are already there the
17:41 polymeris has to polymerize when the
17:43 nucleosomes are there so basically they
17:46 have to loen up the chromatin first and
17:49 then replicate the entire eukariotic
17:53 chromosome and this is not a easy task
17:56 how difficult it is to navigate through
17:57 a road when there is a huge amount of
18:00 traffic or or roadblock exactly that is
18:02 the scenario in case of eukariotic DNA
18:04 replication so I hope this video give
18:05 you detailed insight about the
18:07 eukariotic replication
18:10 scenario so there are many such videos
18:12 in my Channel watch these videos for
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