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Cell Biology | DNA Replication 🧬 — Transcript

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0:14what's up ninja nerds in this video
0:15today we're going to be talking about
0:17dna replication but before we get
0:19started
0:19please continue to support us by hitting
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0:27go check that out all right ninja nerds
0:29let's get into it all right ninjas when
0:31we talk about dna replication the first
0:33thing that we need to talk about is a
0:34couple of fundamental points very
0:36important things that we're going to
0:37build on throughout the process of this
0:38lecture and so the first thing i need
0:39you guys to know is what in the heck do
0:41we do dna replication for
0:43and the whole point is is that in order
0:45for
0:46cells to be able to replicate and make
0:49more cells
0:50we need the dna within those cells to
0:53replicate
0:53because the dna is pretty much the
0:56the genetic portion of the cell it's
0:58what makes a cell
1:00what it is so in order for us to really
1:02understand dna replication i really want
1:04you to understand that the whole purpose
1:05of it
1:06is to allow for cell replication
1:09okay or sometimes we refer to this as
1:11the cell cycle
1:13okay so the cell cycle i know you guys
1:16know the cell cycle
1:17when in the cell cycle what's kind of
1:18the really quick part of it
1:20you start off you go g1 then you go
1:23s phase g2 phase and then you go into
1:26the mitosis part
1:28and then out of that you get two cells
1:30where you take one cell
1:32that cell enters into the g1 s g2 goes
1:34through mitosis and makes two cells
1:37the big thing i want you to know is that
1:38dna replication primarily occurs within
1:42a particular part of the cell cycle when
1:43a cell's replicating making more cells
1:45it primarily occurs within the s phase
1:49so in the s phase that is where dna
1:53replication is occurring so the first
1:57fundamental that you need to know is why
1:59do we perform dna replication
2:00in order for our cells to replicate make
2:03more
2:04cells so whenever they go through their
2:05cell cycle the particular point when the
2:08dna is actually replicating
2:10is in the s phase of the cell cycle now
2:13real quick what in the heck is cell
2:15replication it's really simple
2:16i'm taking this cell here which has 23
2:19maternal and 23 paternal chromosomes
2:23and all i'm doing is i'm making two
2:25identical cells that look
2:26just like this so i have to replicate
2:28the dna within this chromosome
2:30and the dna within this chromosome and
2:32i'll make two of this
2:33and two of this and that's going to give
2:35me these two identical daughter cells
2:37and that's the basic process of cell
2:38replication
2:40so that's the first thing i need you
2:41guys to know the second thing that i
2:43need you guys to know about dna
2:45replication is that it
2:46occurs in what's called a
2:48semi-conservative
2:49model what the heck does that mean zack
2:51i got you
2:52so the next thing you need to know is
2:54that dna replication is
2:57semi-conservative
3:00semi-conservative means let's take that
3:02a piece of dna here right
3:03so dna has two strands and we're going
3:05to call these we're going to give them
3:06two names
3:07we're going to call these blue dna
3:09strands parental strands
3:11or let's call them old strands
3:14what i'm going to do is when i want to
3:15replicate this dna i have to separate
3:18them and we'll talk about how we do that
3:20when you separate the dna into separate
3:22strands here
3:23i have two old parental strands
3:25separated
3:26what i'm going to do is i'm going to
3:28replicate
3:30the dna in a complementary
3:33fashion so what does that mean
3:35complementary
3:36that means if for example this
3:38nucleotide was adenine
3:40or a this would be t if this was t this
3:43would be a
3:44this was g this would be c if this was c
3:46this would be g
3:47you get the point i'm going to make
3:49nucleotides that are complementary on
3:51that
3:52but do you see how the color is
3:54different and i synthesized a
3:56new strand or a a daughter strand
4:00if you will that's one aspect of it i'm
4:01going to do the same thing to this other
4:03strand so i'm going to use this old
4:04parental strand
4:05and make a new strand with complementary
4:07nucleotides on the other
4:09old parental strand i'm going to again
4:12make dna that is complementary
4:16to this old or parental strand so the
4:19whole concept here is that i start with
4:21old if you will let's use the term old
4:25and i make new
4:28mixed with the old
4:33that's really the easiest way of
4:34understanding dna replication is i'm
4:37taking
4:38two old parental strands separating them
4:41and making two new dna strands that are
4:43complementary to them
4:45so what i did is i took this dna and
4:48made
4:48two new double-stranded dna molecules
4:51isn't that cool
4:52and when i did it i did it in this
4:54semi-conservative
4:55process the next really important thing
4:58that you guys need to know
5:00is that dna replication occurs in a very
5:03very specific direction dna replication
5:07has to occur okay the direction
5:10okay replication direction
5:14is very important it's kind of annoying
5:17we'll mention it a lot
5:19throughout the process of this lecture
5:21but dna direction always has to occur
5:23from the
5:24five prime end to the three prime
5:27end i can't stress that enough super
5:29important it's going to come up a lot
5:32what the heck does that mean really
5:33quickly do you guys remember from the
5:35dna structure video
5:36what was on the five prime end of a
5:37nucleotide the phosphate group
5:40what was on the three prime end of the
5:41nucleotide the oh group
5:44so when i'm adding nucleotides i'm
5:47adding
5:47a phosphate group onto a three prime
5:50group of the preceding nucleotide let's
5:52show you an example of that
5:53so let's say that we took this old dna
5:55strand we're gonna do replication
5:56following the semiconservative model
5:58here i have two old parental dna strands
6:02separate them i'm going to replicate it
6:04via the semiconservative model
6:06but i'm also going to follow this
6:07process where i have to replicate five
6:09to three
6:10so let's say on one end of this old dna
6:13strand i have a three prime end here
6:14what does that mean
6:16that means there's an o h group here on
6:18the five prime end of this dna strand
6:20i have a phosphate group here okay same
6:23thing here
6:23oh group and then the phosphate group
6:27right here so that's basically what the
6:29five prime three prime end is
6:31and you guys remember that dna is
6:32antiparallel so if it's three
6:34prime on one end five prime on the other
6:36end the other dna strain has to be
6:38flipped
6:39so it's five prime on the same and then
6:40it's three prime and three prime on the
6:42same end that it was five prime that's
6:44important
6:45so when dna replication occurs
6:49it has to occur five to three so here's
6:52the three prime end
6:53when i make a new dna strand it has to
6:55be
6:56five prime first three prime end here
7:00and then what will i do i'll add another
7:01nucleotide
7:03and then this connection here will be
7:06between what
7:07i'll have a three prime end here and a
7:09five prime end of this next nucleotide
7:11i'll have a three prime end here i'm
7:13gonna add another nucleotide
7:15so when i make nucleotides i make them
7:17and synthesize them from
7:18five to three okay
7:22same thing if i'm going to do it off of
7:23this strand here's the five
7:25end here's the three end of the parental
7:26strand if i want to make the new strand
7:29this is the five prime end so i'm going
7:31to be synthesizing dna in which
7:33direction
7:34in this direction here right and again
7:37doing this according to the
7:38complementarity rule
7:40okay so that's the important thing i
7:42need you guys to remember is that
7:44dna replication occurs in a five to
7:46three direction
7:47the last fundamental thing that is
7:49really important here
7:50is that dna replication
7:54is bi-directional
7:58and you're like why the heck do i need
7:59to know that i think oftentimes when
8:01we're looking in textbooks
8:04we only focus on one end where dna
8:06replication is occurring
8:08but what's really important is we're
8:09going to talk about this in a second but
8:10what we do is we take the dna and we
8:12already have an idea that we're going to
8:13separate the two older parental strands
8:15away from one another
8:16so that we can create new dna from that
8:19when we do that we create these little
8:21ends here these little y-shaped regions
8:23called replication forks
8:26okay so it's called the replication fork
8:28and you have two of them one on this end
8:30one on this end
8:31there's going to be enzymes called
8:33helicases which are going to come in and
8:34unwind the dna
8:35on both sides moving it in this
8:38direction
8:39and moving it in this direction
8:42and then enzymes called dna polymerases
8:45which we'll talk about they're also
8:46going to move into these areas
8:48and follow the helicase synthesizing new
8:51dna
8:51off of that parental strand in a
8:53bi-directional fashion
8:55so the big fundamentals i need you guys
8:56to take away is that why do we do it
8:58dna replication in order for cells to
9:01replicate and make more cells
9:02it occurs in a semi-conservative fashion
9:05taking old
9:06making a mixed old and new double two
9:08double-stranded dna molecules
9:10it occurs in a five to three direction
9:13and it occurs
9:13bi-directionally from what's called the
9:16origin of replication or where these
9:18replication forks are
9:19okay now that we have the fundamentals
9:22let's now talk about the steps of dna
9:24replication
9:25all right so the first thing that we
9:26have to talk about when we're talking
9:28about the stages of dna replication
9:30there's three stages of dna replication
9:32initiation elongation and termination
9:34okay initiation elongation and
9:36termination
9:37initiation is really an easy process
9:39it's not too hard to remember
9:41so what happens is let's say that here
9:44i have my double-stranded dna okay and
9:47let's say there's a particular
9:49region in that double-stranded dna which
9:52is
9:52really really a nice little area that i
9:54want to go and i want to separate the
9:56dna so that i can
9:57create two separate dna strands parental
9:59strands that i can use as templates to
10:01make new dna
10:03that area is going to become our origin
10:06of replication
10:07all right so this area is really what's
10:09going to be our origin
10:13of replication and why do we need to
10:17know this okay
10:18so whenever we need we're picking this
10:20spot how do we determine what that
10:22we're picking that spot like okay i know
10:24that there's a bunch of regions on the
10:25dna
10:26why is the this point here the
10:28particular origin
10:30and there's a really cool reason why
10:32there's particular
10:34nucleotides in this region which are
10:37really highly concentrated with
10:40adenine and thymine so it's an adenine
10:44and thymine
10:45rich area now let's talk about this for
10:48a quick second
10:49why would i pick adenine and thymine as
10:52the area that i
10:53really want to target as compared to
10:55guanine and cytosine do you guys know
10:57why
10:58so adenine and thymine how many hydrogen
11:00bonds are there between them
11:02two how many hydrogen bonds are there
11:03between guanine and cytosine
11:05three it's going to be easier to break
11:09two hydrogen bonds and it is going to be
11:11able to break three hydrogen bonds
11:13so in this area there's going to be
11:15particular areas which are really
11:16concentrated with adenine and thymine
11:19nitrogenous bases or nucleotides and
11:21that's going to be
11:22better suited as the area we want to
11:24kind of separate
11:26why because there's only two
11:29hydrogen bonds and that's going to
11:32require
11:34less energy
11:39okay to break those hydrogen bonds as
11:41compared to
11:42guanine and cytosine okay so that's the
11:45first thing we have particular areas now
11:47the next thing i need you guys to
11:48understand is
11:49in eukaryotic cells is there only one
11:52origin that okay there's just one area
11:54here
11:55where there's a lot of adenine and
11:56thymine i just have enzymes bind to that
11:58portion and separate it
11:59and the enzymes just work from the
12:00center and go to the ends
12:02no what's really important that you guys
12:05need to know is that in eukaryotic cells
12:08there is multiple
12:12origins sometimes represented or e c
12:15origins of replication
12:19so that's really really important so for
12:21example i may just be representing this
12:22one portion but there may be another
12:24origin of replication right here
12:26and another origin of replication here
12:28which is really rich in adenine thymine
12:31nucleotides okay so that's one important
12:33thing
12:34the next thing is what type of structure
12:36is going to bind onto these areas
12:38and help to break the bonds between the
12:40adenine and thymine
12:41what do we have there's a really
12:43interesting protein
12:45it's got one heck of a name they always
12:47do don't they
12:49and this protein here is called the
12:50pre-replication
12:52pro pre-replication protein complex one
12:54heck of a name
12:55so here we're going to draw a cute
12:57little enzyme here okay this is a cute
12:59little enzyme
13:00and this enzyme is going to come in and
13:02bind
13:03onto these areas and when they bind onto
13:05the areas they separate
13:06the adenine and thymine nucleotides in
13:08that area to separate the dna
13:10what is this protein here called that
13:12separates them it's called a pre
13:15replication
13:18protein complex
13:22so again it binds to the origin of
13:24replication and separates the
13:26adenothymine
13:27uh nitrogenous bases now once it does
13:29that what's that going to look like well
13:31let's take the next step
13:32we had this protein bind on to the
13:33origin of replication where there's a
13:35lot of adenine and thymine nucleotides
13:38we separated the bonds between them the
13:40two hydrogen bonds
13:42and we create this little like bubble if
13:44you will you know what we call this
13:46it's not hard it's called the
13:48replication bubble that's literally what
13:49it's called i know it sounds crazy
13:51but now we kind of form this little
13:52bubble due to this whole process and
13:54it's called the replication
13:58bubble now once we form this replication
14:02bubble there's a couple things that i
14:03need you guys to know we have separated
14:05the nucleotides so if you imagine here
14:07we're not showing them but let's say
14:08that here i show a couple
14:09you know here's my nitrogenous bases
14:11that are coming off this
14:12sugar phosphate backbone right which is
14:14made up of again the
14:16deoxyribose sugars the phosphate groups
14:18all that
14:19and these are just my nitrogenous bases
14:21that are popping out off of it right
14:23so i'm going to have these exposed now
14:26so these were once
14:28really connected nicely together like in
14:30these regions i really separated them
14:32so they're really kind of like
14:33vulnerable right now and you know what's
14:35really important
14:36these when you separate them they want
14:39so badly
14:40to re-anneal with one another all right
14:42so what is this
14:44protein that really helps to protect
14:47these
14:47vulnerable separated parental dna
14:50strands
14:51you know it's really ironic uh you know
14:53sometimes
14:54science is there's a big old protein
14:56that comes and binds to this end it
14:58would do the same thing on this one
15:00and this protein you know what it's
15:02called it's called the single
15:04stranded binding protein i'm not even
15:08joking that's literally the name of it
15:10and it's perfect it's not hard to
15:12remember why because
15:14it's a protein binding to a single
15:16parental strand so you would have one
15:18over here i'll just draw a portion of it
15:20but you'd have the same thing over here
15:22another single strand of binding protein
15:24binding onto this parental strand and
15:26what is the purpose of these single
15:28stranded binding proteins that's what i
15:29really want you to remember
15:30one of the functions is that it prevents
15:36the parental strands from re-annealing
15:41what the heck does that mean what's that
15:43term re-annealing from reconnecting to
15:45one another
15:46right so that's important so it present
15:48prevents the parental strands from
15:50re-annealing
15:52to one another because they honestly
15:54they really want to click back to one
15:56another
15:57the other thing that they do is that
15:59when you have
16:00these uh parental strands separated as
16:02kind of these single strands if you will
16:04they're very vulnerable to very nasty
16:07little enzymes there's some nasty little
16:09pac-man-like
16:10enzymes that want to come to the area
16:12and break the
16:13phosphodiester bonds these are called
16:17nucleases so what these single-stranded
16:20binding proteins do is they kind of act
16:22as a barrier
16:23and protect these single strands from
16:26exonucleases or endonucleases
16:28so again it prevents it protects
16:33from nucleases
16:37okay so so far free replication complex
16:40binds to the origin of replication or
16:42the at rich area separates it forms a
16:44replication bubble
16:45single strand of binding proteins bind
16:47to the single strands prevent them from
16:49kneeling and protect them from nucleases
16:52the next thing is once you form a
16:53replication bubble you form these two
16:56ends here
16:57okay and these two ends we already kind
16:59of mentioned it a little bit
17:01this end here where it kind of like
17:02makes like a y shape if you will
17:06this right here is called your
17:08replication fork
17:11okay there's going to be an enzyme that
17:14we're going to talk about in just a
17:15second
17:15ah frickin we'll talk about them now he
17:18hops in here
17:19this little enzyme is like the energizer
17:22bunny
17:22he's got so much energy as long as you
17:24keep feeding them
17:26and he hops in here look at this cute
17:28little enzyme look at this guy
17:31this enzyme will come in at this point
17:35here
17:36and really unwind the dna at
17:39both replication forks
17:43what is the name of this enzyme that
17:45really works
17:46in these replication forks unwinding the
17:48dna in front of them
17:50this enzyme is called helicase
17:56and the big thing i really want you to
17:57know about the helicase enzyme
17:59is that he requires a ton of atp
18:03in order to perform this process okay
18:07so step by step again real quick
18:09pre-replication
18:10complex binds to the origin of
18:11replication at rich area separates it
18:14single-stranded binding proteins bind
18:15protect them from exonucleases and
18:17endonucleases
18:18prevent re-annealing whenever you do
18:20that separating them you create a
18:22replication
18:22bubble at the ends of them you have
18:24replication forks
18:26helicases highly atp dependent hop in
18:28there and start unwinding the dna
18:30in front of them after they do that
18:33something happens which is really
18:35important
18:35that we definitely need to know let's
18:37come down here and this is a really
18:39important area that i really really need
18:41you guys to understand
18:43so let's say that we come back to this
18:44point here
18:46again what we have binding right here to
18:48these two ends
18:50single stranded binding proteins we're
18:52not going to show it on the top but the
18:53same thing here
18:54and then again what enzyme let's just
18:56focus on this area right here just on
18:58this replication fork
19:00what enzyme would be in this area here
19:05really working and unwinding the dna in
19:07front of it
19:08again that's called helicase
19:12what happens is as helicase continues to
19:14unwind the dna the whole purpose of that
19:16as it unwinds the dna separates the
19:18strands so that enzymes would be able to
19:20use those parental strands to make new
19:22dna
19:22okay but there's a problem that happens
19:25as dna helicases just you know going
19:27through those mofos and unwinding the
19:29dna constantly in front of it
19:31it bunches up the dna in front of it
19:35distal to it okay from that replication
19:37fork so distal to the replication for
19:39downstream the dna starts bunching up
19:42and it creates things these things
19:44called super coils
19:46okay it creates these things called
19:47super coils
19:50and this is caused by the helicase
19:52really unwinding the dna
19:54why are super coils bad if you really
19:56bunch up the dna in front of it it's
19:58going to really impede the helicase
20:00from continuing to unwind the dna it's
20:02going to face a lot of restriction
20:04because it's really bunched up here so
20:06it'll just keep getting bunched up until
20:07you relieve
20:09those super coils so we need enzymes
20:12that can come
20:12in there and fix these supercoils where
20:15the dna is
20:16really really tightly wound so how do we
20:18do that
20:19all right so what little enzymes do we
20:21have or special little things that come
20:23into the play to really alleviate these
20:25super coils
20:26these enzymes are crazy interesting so
20:29these guys are really cool
20:34and they are called topo
20:38high sama races they're shape of the t
20:40right so they're called what
20:42toppo is
20:47now topoisomerases there's actually a
20:49couple different types
20:50okay there's particularly type 1
20:56type 2 and then there's type 4.
21:00for the most part they all do the same
21:02kind of thing and what is that
21:04okay this enzyme has two little
21:08arms if you will let's say here it has
21:09one little arm
21:11and another little arm on one arm
21:14it can go to where this area of the
21:16supracoil is let's say that the
21:17supercoil's right here
21:19it can use a little enzyme a little
21:21domain of it
21:22and cut this dna strand
21:26if it cuts the dna strand what does that
21:28allow for it to do
21:29it kind of allows the dna to kind of
21:30unwind a little bit and
21:32unravel and so there's a particular
21:35name to this domain on that
21:37topoisomerase enzyme
21:38it's called a nuclease
21:41domain and what does it do it creates a
21:44cut
21:45or breaks the phosphodiester bond in the
21:47dna strands one maybe two dna strands
21:49allows it to unwind there's a problem
21:51with that though
21:52if i just cut the dna strand and allow
21:55it to continuously unwind
21:56that may be problematic the dna could
21:58continue to fragment
22:00i don't want that so what i do is
22:03i use this other arm and after the dna
22:07super coils have been alleviated so
22:08let's now kind of draw
22:10new dna after the super coils have been
22:13alleviated
22:14so we alleviated the super coils we got
22:16rid of all of those
22:17overwinding of the dna
22:20look now it's beautiful it's not
22:23overwinded
22:24but we have a break in that dna and
22:26that's a problem so now we need to use
22:28the other
22:28arm of this topoisomerase where we cut
22:31this portion here
22:33and allow it to unwind we need to use
22:35this portion
22:37called the ligase domain
22:40and once it's unwound this portion here
22:43what does it do
22:44it re-stitches this area back together
22:48after it's unwound the super coils
22:50okay so again the topwise sama races
22:52what were their function again
22:54two unwind
22:57the super coils now
23:01why in the heck did i take all this time
23:03to mention the topoisomerases and
23:05there's different types
23:07let me explain why topoisomerases
23:10can be in both cells so we primarily
23:12haven't really discussed
23:13that dna replication can occur in
23:15bacterial cells and it can occur in
23:16eukaryotic cells or we call it bacterial
23:18cells like prokaryotic cells
23:20and eukaryotic cells human cells
23:22primarily
23:23they have type one and two
23:25topoisomerases
23:27and in prokaryotic cells they primarily
23:29have two
23:30and four so again type one and two are
23:33primarily for
23:34eukaryotic cells
23:38and then type 2 and 4 are primarily for
23:41prokaryotic
23:43cells big thing i need you guys to know
23:46is
23:47is that type 1 topoisomerase
23:51does not require any atp to unwind the
23:54supercoils so let's put that next to it
23:56that just for this one just for this one
24:00it no atp is required in order for it to
24:04perform this unwinding of the super
24:05coils
24:06but for type 2 and 4 let's pick a
24:08different color so that we don't confuse
24:10it here
24:11for type 2 and type 4 these do
24:14require atp
24:18in order for them to unwind the super
24:20coils there's also one more thing i
24:22don't want to get too far in depth in it
24:24that type two and four can do that are a
24:25little bit different from type one
24:27and that's that if you really look in
24:29the the textbooks
24:31they can actually take and cut that
24:33little
24:34supercoil area allow for unwind and then
24:36insert in what's called negative
24:38supercoils which also helps to kind of
24:39relax the dna and
24:41prevent that kind of bunching up region
24:43let's not get too far into depth in that
24:45what i really want you guys to know
24:47is why in the heck did i spend so much
24:49time talking about these topoisomerases
24:51in your usmles you have to know
24:54particular drugs that we can target on
24:56these
24:57and eukaryotic cells i want you to think
25:00about a reason just try to think for a
25:01reason
25:02why would i want to target this enzyme
25:06in eukaryotic cells when this is
25:07important for dna replication
25:09if these enzymes aren't really working
25:11dna replication won't occur
25:14when a eukaryotic cell what if i have a
25:16cancer cell
25:17so if i have cancer the cells will
25:20continue to replicate
25:22so they'll replicate and replicate and
25:23replicate well
25:25i could maybe use some drugs that could
25:28target these topoisomerases in my cancer
25:31cells
25:32and prevent them from replicating how do
25:34i do that
25:35well there is a particular name of
25:37there's a couple drugs that you guys
25:38definitely need to know
25:40for topoisomerase one in eukaryotic
25:43cells
25:43the cancer drugs that we can use for
25:46topoisomerase
25:47one is called irenotecan
25:52and what's called topotechin and again
25:55these are
25:56anti like they're chemotherapeutic drugs
25:59that are going to inhibit the
26:01topoisomerase one in the cancer cells
26:03the type two in eukaryotic cells
26:07we can use drugs called etopocide
26:11and tinopocide and i'm going to explain
26:15how they do that because that's really
26:16important i really want you to
26:17understand how they do this
26:18but that's for the eukaryotic cells
26:21think about prokaryotic cells
26:23if you were infected by a bacteria and a
26:25bacteria infected your lungs
26:27and it just kept replicating and
26:29replicating within your lungs could
26:31i maybe target the topoisomerase two and
26:33four and prevent that replication
26:34process of the bacteria in my lungs
26:36yes so in bacterial infections
26:40let's say because it's a prokaryotic
26:41cell and bacterial infections
26:44these bacteria will continue to keep
26:46replicating
26:48so if i use particular drugs that can
26:50maybe prevent the dna from replicating
26:53in these bacterial cells
26:54that's important and we can do that via
26:57inhibiting the topoisomerase primarily
26:59type 2
27:00and we use the drug called
27:04fluoroquinolones
27:08some of you may have heard of these
27:10ciprofloxacin levofloxacin
27:12oxyfloxacin all of those guys they're
27:14inhibiting
27:15the topoism race too but i really want
27:18to take a second because i never
27:19understood this completely until i
27:21really dug into the mechanism of how
27:22they actually do this i really want to
27:24quickly say how they do it
27:26what these drugs are really doing
27:29is these drugs so let's kind of just put
27:31here drugs
27:33they're exciting in increasing the
27:36activity of the nuclease domain
27:38what the heck is that going to do that's
27:40going to just chop
27:42all those like portions of the dna and
27:45it's going to continue to fragment them
27:47right
27:48remember we had another another domain
27:50of this enzyme that re-annealed them and
27:52kind of stitched it back together to
27:53prevent that fragmentation
27:55that was the ligase binding domain what
27:57if i use drugs
27:59to enhance the nuclease domain
28:02but inhibit the ligase domain so now
28:06i'm going to have this enzyme go in and
28:08kind of cut where the supercoils is
28:10but i'm never going to re-stitch it
28:12together the dna will just
28:14fragment over time and that's important
28:17because then you can't replicate the dna
28:19within what kind of cells eukaryotic
28:21cells like cancer cells
28:22or bacterial cells and that's why those
28:24drugs are important doesn't it make
28:26sense
28:26all right cool it's important to take a
28:28clinical application and tie it
28:30to the the basic foundational science
28:33okay
28:34so we kind of went through talked about
28:36the topoism races
28:38that was the big thing and how they
28:39unwind the super coils
28:41let's get back to the foundational
28:42science and now that we've talked about
28:44that the next thing that we have to go
28:46into
28:46is elongating the dna all right ninja
28:49nerds so we already know we have a
28:51replication bubble
28:52we got a replication forks we have our
28:55protein here called the single strand of
28:56binding protein which is stabilizing
28:58these single strands
29:00we have the helicase enzymes that are in
29:01these replication forks working like a
29:03son of a gun
29:05to unwind the dna we've got those
29:07topoisomerases over here that are
29:09kind of unwinding those coils
29:12okay now we've really separated this
29:16we've stabilized it and we're ready to
29:18begin elongating the dna
29:20okay here's what's really interesting
29:23there's an enzyme that comes into play
29:25here
29:26and it does something really cool
29:29it's called primase so an enzyme called
29:32primase will come into play here so what
29:36primase does
29:37is it's an enzyme that
29:40lays down okay that get makes what's
29:43called
29:43rna primers so this takes a really quick
29:47turn where we've got to understand
29:48zach you just said that we're making dna
29:51why the heck
29:52would i make rna there's a reason why
29:56there's an enzyme that we'll talk about
29:57a little bit later called dna polymerase
29:593 that will
30:00make dna but the only way it can do that
30:04is if it has some type of primer or
30:08three prime ohn to build off of so
30:11what's the purpose of this primase this
30:13enzyme
30:14it lays down rna primers which enable
30:19dna polymerase particularly type
30:22three to
30:26make dna and i'll kind of show you that
30:29in a little bit in a second okay
30:31so primase comes in so imagine here i
30:34just have like this cute little enzyme
30:36here called primase
30:38okay and this cute enzyme comes in here
30:41and let's say here what's this strand up
30:43here this top part remember we're going
30:44to say that this is
30:45three prime end where the o h would be
30:48this is the five prime end where the
30:50phosphate would be
30:51and again the opposite strand here would
30:53have to be antiparallel
30:54so five prime end here three prime end
30:57here right we already know that
31:00the primase is gonna come in and it's
31:02gonna read the nucleotides and it has to
31:04go in a particular fashion
31:05it reads it from the three all the way
31:08to the five end
31:09so what does it do the first thing it
31:11does is it reads
31:14the dna strand from three to five
31:18after it reads it from three to five
31:20what did i tell you that's super
31:22important what does dna replication
31:24occur even though this isn't dna
31:25it's the same concept it synthesizes
31:29rna primers or nucleotides in a
31:33five to three fashion so it's going to
31:35take and make a couple nucleotides
31:37generally it's about 10 nucleotides
31:39we're only going to draw a couple here
31:40but it'll have has to be again five
31:42primes starting here
31:44and i'm just going to make a couple i'll
31:45make like four nucleotides here
31:48okay so here it's going to have five
31:52all the way to the three prime end here
31:54okay that's my five prime end here that
31:56i just
31:57started with and i'm synthesizing it in
31:59the three direction
32:01now the reason why this is important is
32:03on that three end what do we have here
32:05the o h that's what's on the three prime
32:07end
32:08i need that o h the reason why
32:12is another enzyme called dna polymerase
32:16type 3
32:17comes in so there's an enzyme called dna
32:20polymerase type 3
32:25and he comes in and he needs that three
32:28prime oh from the rna primers in order
32:30for it to continue to build nucleotides
32:32so again a big thing i need you to
32:34remember is it needs
32:36the three prime o h of
32:40rna primer in order to carry out its
32:43activity if it doesn't have it it can't
32:44do it
32:45so now that it has that this dna
32:48polymerase comes in
32:49and it says okay i have my three primo h
32:51region perfect
32:53now what i'm going to do is i'm going to
32:54read my dna and i'm going to do it the
32:56same way that the primase did
32:58i'm going to read the dna from the three
33:00direction
33:01to the five direction so i'll read it
33:03boom boom boom
33:05once i read and figure out what kind of
33:07nucleotide is then i'm just going to
33:08synthesize
33:10those nucleotides in the 5 to 3
33:13direction
33:14and it's the same process here so now
33:16let's make a different color since it's
33:18a different enzyme
33:20we kind of picked red over there so
33:22we're going to start off and we're going
33:24to say okay
33:25i'm going to take that oh and i'm going
33:26to add a phosphate group
33:28onto it of a of another nucleotide so
33:31when i do that i'm going to continue to
33:33keep synthesizing
33:34in a five to three direction moving
33:36towards the replication
33:38fork again when i do that i read it
33:41i say okay let's say that this is
33:43adenine i'll put a thymine
33:45this is guanine i'll put a cytosine this
33:47is cytosine i'll put a guanine
33:48and so on and so forth and i'll just
33:50keep reading the nucleotides three to
33:52five
33:53and making a dna strand in what
33:55direction
33:56five to three okay
34:00and again important to remember it
34:03needed this three prime end of that oh
34:05of the rna primer to build off of it
34:08now here's what's really interesting the
34:10primase will give it kind of a little
34:11leading point and the dna polymerase
34:13will just
34:14go all the way towards the replication
34:15fork
34:17this strand is very continuous where
34:19there's just one rna primer and then dna
34:21the rest of the way
34:22this strand is very important we give it
34:24a particular name
34:25the strand that's very continuous where
34:27the dna polymerase moves towards the
34:29replication fork
34:30is called the leading strand
34:34okay it's called the leading strand on
34:36this other string which we're going to
34:38talk about is called the lagging strand
34:40something different happens where you're
34:42still going to have
34:44the are the primase it'll come to this
34:46area okay on this other strand and again
34:48this is the three end on this part
34:50five end on this part and what it'll do
34:53is
34:53it'll read it from three to five and
34:56then synthesize a couple nucleotides
35:00from five to three so this is the three
35:01end it's going to synthesize from five
35:04to three and again the same thing will
35:07happen we created
35:08a primer of a couple nucleotides with a
35:11three prime
35:12o h end that the dna polymerase type
35:14three can build off of
35:16so now the dna polymerase type 3 will
35:18just pop on and say oh perfect i have my
35:203 prime n to use
35:22i'm going to go ahead and just read the
35:23dna from 3 to 5
35:26and synthesize it from 5
35:29to three okay
35:32so i'm gonna do all that perfectly
35:36now something interesting happens where
35:38it's gonna look it looks perfectly the
35:40same you're like zach i don't get the
35:41difference here
35:42let's say that the gila case continues
35:44to unwind the dna
35:46so it continues to unwind the dna
35:48something interesting happens that we
35:50have to talk about
35:51okay so now let's come down here so
35:52let's say let's pretend
35:54right that for a second here we have
35:57that primer
35:58let's kind of continue off this let's
35:59say that the helicase unwound the dna a
36:01little bit more
36:02and we along we kind of opened up the
36:03dna and created a more
36:05longer length of nucleotides so again
36:08let's say that here we had that primase
36:10came in here read this from one end
36:13again this would be your
36:15three prime end this would be your five
36:17prime end so it'll read from three to
36:18five
36:19and synthesize from five to three
36:21creates a little primer with a three
36:23prime oh end
36:24the dna polymerase 3 says okay perfect
36:27i have everything i need i can continue
36:29to grow and let's say that it just came
36:30up to like this point here
36:32but then you kind of unwind the dna
36:34again that dna polymerase 3 doesn't stop
36:37it just
36:38keeps on going and keeps on moving
36:40reading the dna from three to five and
36:43continuously synthesizing nucleotides
36:45from five to three so again this would
36:47be your five prime end
36:49this would be a three prime end on this
36:52other chain this is on the leading
36:53strand it continues
36:55on the lagging strand here's where it's
36:56a little bit different
36:58let's say that we continue to move on
37:00here and let's say that like
37:01at this point here this was where the
37:04previous rna primer was from above
37:06where it had again reading this portion
37:09of the dna this is the five prime end of
37:11this part
37:12three prime end of this part it read
37:14this sequence of dna from three to five
37:16and let's say that it synthesized a
37:18couple nucleotides to give your rna
37:20primer
37:21from five to three and then what do we
37:23say happen from that part above
37:26we have the dna polymerase three use
37:28that three prime oh end
37:30read the dna from three to five and
37:33synthesize the nucleotides
37:34from five to three here's what happens
37:39the primase lay down a primer here but
37:41the dna polymerase
37:42iii has to use that primer to continue
37:44to keep building off
37:45if you unwind the dna a little bit more
37:47now now you have a couple of the
37:49nucleotides
37:50so now let me kind of just so we have
37:52enough room here
37:53let's say i draw a couple more
37:55nucleotides
37:57so now here i have a couple more
37:58nucleotides now that primase
38:01after it just made down this primer for
38:03the dna polymerase three to use
38:05it comes down to the next part of the
38:07replication fork and it says okay here i
38:09got another three prime
38:11end here let me again read from three to
38:13five
38:14and synthesize a couple nucleotides from
38:16five to three so i laid down my rna
38:18primer
38:19dna polymerase says okay cool i got my
38:22three prime oh end here
38:24let me go ahead and use that to make
38:27my dna and i'm going to read the dna
38:29from three to five
38:30and synthesize it from five to three
38:34do you notice something really
38:35interesting here on this strand which we
38:38called again what did we call this
38:39strand
38:40well we had one primer and then dna for
38:42the continuous way towards the
38:43replication fork
38:44we called this the leading strand so the
38:47big thing i want you to know is that you
38:48have one rna primer
38:51and then a continuous dna strand from
38:53that point on
38:54on this strand called the lagging
38:59strand something different happens here
39:02where you have a couple rna primers
39:05okay and then kind of stretches
39:09of dna between those rna primers
39:12this kind of like broken up portion
39:14where there's rna
39:15dna rna dna and if we continue to keep
39:18elongating it we'd have more rna dna rna
39:20dna
39:21this gives a particular name which is
39:23called
39:25oka zaki fragments
39:29okay okazaki fragments and again it's
39:32basically where you have multiple
39:34rna primers
39:37and multiple stretches of dna
39:42stretches okay so multiple dna stretches
39:45and then multiple rna primers it's a mix
39:47of them
39:48and that's a problem okay because you're
39:50going to see now there has to be another
39:52thing that we have to do
39:54we want everything when we replicate dna
39:56it has to be all dna we can't have it be
39:58dna with a little bit of rna so we're
40:01only using these primers as just kind of
40:03a point to build off of after we've
40:05built some stuff
40:06we're just going to go in and cut those
40:08things out because we don't really need
40:09them anymore
40:10so now let's talk about the next part
40:12which is
40:13we've started to kind of create these
40:15primers that we needed to build the dna
40:17off of
40:18now we don't need the primers and we got
40:20to get rid of it how do we do that
40:21the next thing that you guys need to
40:22understand is okay we've used our rna
40:24primers for the dna polymerase 3 to kind
40:26of build off of and
40:27make dna from we don't need those
40:29primers anymore we got to get rid of
40:31them so let's draw the diagram that we
40:32had previously
40:33which again we only had what a little
40:36stretch of rna primer here
40:39and then the rest of the length down
40:41going towards the replication fork which
40:43again what is this strand here called
40:45the leading strand is going to be all
40:48dna okay
40:52so now this is really important we're
40:54going to talk about one more thing
40:55in just a second and again if you guys
40:57remember on this strand the lagging
40:59strand
41:00we had a couple rna primers
41:04that were in between
41:07the stretches of dna creating what's
41:09called okazaki fragments
41:12on the lagging strand right we talked
41:14about that
41:15so now the next goal here is that we
41:17have to remove
41:18those rna primers but before we do that
41:21i got to mention one more thing
41:22so before we talk about how we remove
41:25these rna primers i really want to take
41:27a quick second here
41:28to explain something else that dna
41:30polymerase type 3 can do
41:32so we know that it reads the dna okay
41:35from three to five and then synthesizes
41:36nucleotides off of the rna primer from
41:38five to three
41:39but it also has one other function it's
41:41called a proofreading function which is
41:43very important before we talk about the
41:45rna primers
41:46and this proof reading function is
41:49helpful to prevent
41:50mistakes and what it does is
41:53let's say okay it reads three to five
41:56reads all the nucleotides from a three
41:57to five direction and then synthesizes
41:59nucleotides in a five to three after it
42:00does that it says okay
42:02let me check my work it goes back and it
42:05finds the connection
42:06between this point says okay is this a
42:08good connection yeah that's a good one a
42:09and t are connected together
42:10oh g and c are connected together a and
42:13g oh
42:14this isn't a correct uh complementary
42:18kind of base pair
42:19i need to cut that out so it
42:22reads from three to five and if it finds
42:26any mistakes it uses what's called a
42:29three prime to five prime exonuclease
42:36activity where it says okay let me read
42:40here
42:40i'm gonna read it and i says okay three
42:43to five i'm reading
42:44a t and again it's complementary base it
42:46should be
42:47t should be a g a oh not a correct one
42:51i'm gonna cut that out read it again
42:53make sure i have it okay it was g
42:55that has to be c and synthesize the
42:58correct nucleotide in the five to three
42:59direction
43:00so big thing i need you to remember for
43:02dna polymerase type three
43:04reads dna three to five synthesizes
43:06nucleotides five to three
43:08proof reads back in three to five and if
43:10that's incorrect
43:12uses a three to five prime exit nucleus
43:14to cut it out
43:15and put in the correct complementary
43:17nucleotide very important
43:20okay now we got to get rid of these rna
43:23primers
43:23how do we get rid of the rna primers
43:25well the dna polymerase 3 is not the
43:26answer
43:28the next enzyme as if there isn't enough
43:30enzymes
43:32is called dna polymerase type 1. so dna
43:35polymerase type one
43:40comes to the rescue and what it does is
43:44it starts here and it finds this okay so
43:46let's say here we had our three prime
43:48end here
43:49five prime end here the new strand would
43:52be synthesized from five
43:54to three what this enzyme will do is
43:57it'll come
43:57in and it'll cut out these primers
44:01going from the five to three direction
44:04so it removes primers or it plucks those
44:07little
44:07primers out in a five to three
44:12exonuclease
44:15activity right particularly for what to
44:18pluck out
44:19the rna primers so this guy will come in
44:22and it'll say okay
44:23pluck remove that one pluck remove that
44:26one
44:27and then what it'll do is once it plucks
44:29those out it then says okay
44:32i'm gonna read this strand from three to
44:34five
44:35so it reads the one that it plucked out
44:36and says okay that's a adenine
44:38what do i need to add here i need to add
44:41in
44:41thymine oh this is thymine i need to add
44:45in here
44:46adenine so it plucks off the primers
44:49then what does it do it reads the dna
44:55from three to five and then it
44:58synthesizes
45:02from five to three
45:06you know what else it can do one more
45:09function
45:10let's say okay it plucked off the rna
45:12primer reads it as adenine
45:14puts a thymine reads it as guanine
45:16accidentally puts an adenine
45:18has to go back and proof read it though
45:20because that's always the thing that
45:21they have to do
45:22proof reads it and says not a good
45:24connection
45:25i don't want that what do i need to do i
45:27need to pluck that thing out of there
45:29and put the correct nucleotide
45:32so the last thing dna polymerase type
45:34one can do
45:36is again it has that proof reading type
45:38of activity
45:40where it can do what it can read from
45:43three to five it
45:44finds an incorrect base pair connection
45:47it cuts it out and when it cuts it out
45:50it cuts that at a three
45:52to five exonuclease type of fashion
45:56okay so the big difference that if you
45:59ever get asked between
46:01what in the heck is the difference
46:02between dna polymerase type one
46:04and dna polymerase type three really the
46:07big difference
46:08is that this guy can do everything type
46:10three can do
46:11it's just it has that five to three
46:13prime exit nucleus activity where it
46:14plucks out the rna primers
46:16everything else is the same though now
46:17the next thing is dna polymerase type
46:19one we talked about on the on this
46:20leading strand on the lacking strain
46:21it's a little interesting
46:23it'll come in and again it'll use its
46:25five to three prime exonuclease activity
46:27so again let's use our combination here
46:29of what we know
46:30this was three this was five so
46:32antiparallel this has to be
46:34five to three for the old strand the new
46:37strand would then be
46:38what read three to five synthesize
46:425 to 3. so this dna polymerase will come
46:45in and it'll start moving down
46:47and at this point it'll pluck off an rna
46:50primer and then what will it do
46:52read 3 to 5 and synthesize
46:56five to three come to the and then
46:58proofread it is it correct
46:59oh it is okay if it's not use my three
47:02to five primax nucleus to cut it out
47:04and then put in a new one it goes to the
47:06next one plucks off the rna primer
47:09and does the same thing reads three to
47:11five synthesizes five to three proof
47:13reads three to five
47:15then it just keeps doing that and
47:16plucking these things off here's the
47:18difference though in the lagging strand
47:19it creates like a couple gaps these
47:20actually don't
47:21completely kind of fuse together so
47:23let's kind of draw where we had
47:25this here and we'll create a little
47:27space between these points here where
47:29the rna primers were
47:31so there's kind of a little space here
47:33let's draw it here in orange
47:34so on the lagging strand it creates like
47:35a little space where it can't like
47:37really fuse these ends
47:39where the primers were to the original
47:41dna
47:42okay so it plucked the rna primers off
47:44and put nucleotides but it wasn't able
47:45to perfectly fuse these ends on the
47:47lagging strand
47:48one more enzyme you're like dude i can't
47:51do no more
47:52i promise one more this enzyme
47:56is called ligase so it's called ligase
48:00now ligase
48:02will come in on that lagging strand
48:06and fuse the dna
48:10ends together okay
48:14those basically where those okazaki
48:17fragments were
48:18it'll come and it'll say okay here's
48:20these ends here
48:21i'm going to fuse these points together
48:24so that it's
48:25perfectly connected and continuous
48:28and now we have a parental dna
48:32with a new daughter dna strand again
48:35a parental dna strand with a whole new
48:37daughter dna strand that is all
48:39continuous and all in sequence no rna
48:41primers no nothing
48:43no breaks it's perfectly set now the
48:45last thing that i want to talk about
48:47here before we go on to termination is
48:49we've elongated our dna we now took the
48:50old parental dna and made new dna
48:53the reason why i want you to remember
48:54this is that there's it's very important
48:56for us emilies to connect foundational
48:58sciences with clinical significance
49:00and so in people who have hiv
49:04okay their t cells okay their t cells
49:08have are infected with the particular
49:10virus called a retrovirus
49:12and it's causing this virus to get
49:13incorporated into the dna and then from
49:15every point that on that these t
49:17cells replicate they continue to
49:19replicate more of the hiv genome
49:21so there's drugs that we use to target
49:25this hiv virus and particularly the t
49:27cell replication process and these drugs
49:30are called
49:31nucleoside reverse transcriptase
49:33inhibitors you're like holy crap
49:35what the heck does that mean i just want
49:37you to remember that they're drugs that
49:38are used for hiv
49:40and they inhibit the replication process
49:43and t cells that have been affected with
49:44hiv let me explain how this works this
49:46is really cool
49:47let's say here i just i quickly put down
49:49an rna primer
49:51okay and i have my dna polymerase three
49:55it comes in here and it starts making
49:58some dna
49:59right uses that three prime end of the
50:01rna primer and starts making dna
50:03i give them a drug let's kind of put
50:05here an nrti
50:07and again there's many different names
50:08of these like didenosine zydovidine
50:12there's a whole bunch of these but what
50:14i want you to remember is
50:15imagine these as what's called
50:19nucleosides
50:21okay and what they do is imagine here's
50:24my
50:24my basically my my ribose sugar and then
50:27here i'm going to have my phosphate
50:29group
50:29and then here i have like adenine okay
50:33what they do which is really interesting
50:35is usually on your
50:36your deoxyribose sugar you should have
50:38an oh right and you need that oh
50:41on that three prime end in order for the
50:43dna polymerase to continue to keep
50:44adding
50:45what they do is is they remove the three
50:48prime oh region so now
50:52the dna polymerase 3 will get another
50:54nucleotide
50:55it'll read this and say okay this is
50:57adenine i'm going to put a thymine or
50:58something like that
51:00and i'm going to add in this drug this
51:02drug kind of floats around kind of
51:04interestingly
51:05and the dna polymerase iii will then say
51:07okay here this is a
51:09another nucleotide just like the ones
51:10i've been adding let me add this one on
51:13the only problem is is it doesn't have a
51:15three prime o h region
51:17so you know what happens here since
51:19there's no three prime oh
51:22you dna polymerase can't build off of
51:25that
51:26remember what i told you dna polymerase
51:28type three needs a three prime oh region
51:30to build
51:31if you give a drug that doesn't have
51:33that can you continue to build off of it
51:35no so all the dna replication from this
51:37point
51:38is inhibited are you able to replicate
51:41all the dna within these t cells that
51:43have been affected with hiv
51:44no so that's how it does it they're
51:47what's called
51:48kind of like analogs nucleoside analogs
51:50where you're kind of like dna polymerase
51:523 doesn't know the difference it's just
51:53taking nucleotides and adding on
51:55and all of a sudden just by kind of
51:56chance you have this drug that it
51:58attaches on it doesn't know the
51:59difference
52:00it goes to add another nucleotide on
52:02it's like hey that didn't add on what
52:04the heck i can't add this nucleotide on
52:06and the dna never gets completely
52:07replicated so it's a good it's a good
52:09clinical point to understand
52:11that covers our elongation let's hit it
52:13home with termination
52:14and take a quick second to talk about
52:15telomeres all right this is actually the
52:17easiest one of all you're probably like
52:19oh
52:19please zach i can't do any more i know
52:22this this is a lot but let's say that we
52:25have another enzyme okay that helicase
52:26enzyme we're getting to the point where
52:27dna replication has been completed
52:29we got that enzyme what was that enzyme
52:31that was working at these replication
52:32forks and just continuing to unwind the
52:34dna
52:35you know in front of it what are they
52:37called the helicases
52:39and then you had those enzymes the dna
52:41polymerases type 3 and type 1 and all
52:43those guys that were coming and
52:45basically reading the dna three to five
52:46and synthesizing it five to three proof
52:48reading in the three to five all that
52:50good stuff
52:51and you've synthesized the dna and the
52:54helicases are
52:55meeting each other at kind of
52:57replication forks that are about to abut
53:00one another when this happens
53:03when the gila cases meet and you kind of
53:06unwind this portion of the dna
53:08what's going to happen the helicases are
53:10just going to kind of say oh
53:12well hey buddy i guess i don't need to
53:14keep unwinding anymore
53:15and what will happen is you're just
53:17going to kind of have this point here
53:20where the dna polymerase is will just
53:22hop off of the dna
53:24because at this point there's nothing
53:25else for it to read
53:28and so usually once it gets to that
53:29point they'll just say hey
53:31i guess the helicases are done there's
53:33no more unwinding for me
53:35and then after that the dna polymerases
53:37will say hey i've already kind of
53:39hit all the nucleotide regions here
53:42i'm done and i've replicated all of my
53:44dna
53:45so that's important so it's basically
53:47again where there's multiple origins of
53:49replication and they're constantly
53:50moving towards one another whenever they
53:52moved and hit one another
53:53the dna replication at that point stops
53:56now there's something else that you have
53:57to remember though
53:59dna replication will you know start at a
54:01point and then work bi-directionally
54:03it's eventually going to go to the ends
54:06of the dna or the chromosomes which we
54:07call the telomeres
54:09there's a particular nucleotide sequence
54:11at that end where the dna
54:13polymerases have a really hard time
54:15being able to replicate
54:17and that's very important we'll talk
54:18about that next okay but again
54:20termination of dna replication it's
54:21really simple it's when the dna
54:23polymerases
54:24are moving towards one another at a
54:25replication fork and they've just
54:27stopped at that point
54:28they hop off and they no longer perform
54:30their function there's one other part of
54:32it which is with the telomeres which
54:33we're going to finish off with
54:35all right so the next thing that we're
54:36going to talk about is telomeres right
54:37so
54:39dna replication there's a little
54:40interesting issue that happens at the
54:42telomeres so one thing i need you guys
54:43to know is that
54:44telomeres they really shorten over time
54:46so let's say that here
54:48we look at some chromosomes which again
54:50made up of dna and proteins well let's
54:52primarily think of it as dna
54:53and let's say that this goes through
54:55goes through a replication cycle
54:57a couple times i want you to notice what
55:00happens to the ends right so when you
55:01look at a chromosome there's
55:03two primary kind of like structural
55:05points
55:06the point in the center right which is
55:08called your centromere
55:12and then the ends okay these points here
55:16and these are called your telomeres
55:20now what happens is over time as your
55:22cells continue to keep replicating the
55:24dna replicates watch what happens to the
55:26telomeres
55:27they get shorter and shorter
55:31and shorter as that continues
55:34to happen there's a worry with this and
55:37let me explain what that worry is
55:40obviously your dna has particular areas
55:43which
55:43code for rna rna can then get translated
55:46to proteins what are those called
55:48they're called genes so let's say that i
55:50have a gene right here
55:52the telomeres will be there and their
55:54primary function
55:57is that they will it's common for them
55:59this to happen
56:01where the telomeres were short and short
56:02and shortened but the whole purpose of
56:04them
56:04is that telomeres don't code for
56:06anything that's very important let me
56:08write that down
56:09telomeres
56:12do not code for any rna
56:16so do not code
56:19for rna in other words you can't take
56:21the dna from a telomere make rna make
56:23protein that's important
56:25but let's say here there is a gene there
56:27that can make rna
56:28the telomeres will kind of sacrifice
56:31themselves
56:32because dna replication doesn't occur at
56:33this point for a particular reason we'll
56:35explain why
56:36and so because of that they prevent
56:40gene loss so they kind of like take the
56:43hit for us if you think about it
56:44they're like don't worry i don't code
56:46for any rna so you don't have to
56:47continue to replicate me
56:49so with each replication cycle the
56:51telomeres will shorten and shorten and
56:53shorten
56:53but that's okay and for a particular
56:56reason because they do not code for rna
56:58and they help to prevent gene loss but
56:59here's the problem eventually you're
57:01going to get to a point
57:02where the telomeres will shorten so much
57:04that it can interfere
57:06with the genes once that happens where
57:09the cell has reached the replication
57:10limit where it can't replicate
57:12anymore it's reached its maximum number
57:14there's a particular term
57:15that you guys need to know and it's
57:17called the hayflick limit
57:20it's called the hay flick
57:23limit and that is basically the maximum
57:25amount of times that this
57:26this kind of dna can replicate before it
57:28starts to involve
57:29genes now let's talk about
57:33why these telomeres are shortened really
57:35quickly
57:36let's say here we take and let's say
57:38that we use this as our example
57:39this is our leading strand and this is
57:42our lag
57:42strand right again let's just say here
57:45we have our three prime end
57:47we're going to use our rna primer here
57:50and then from here
57:51the rna primer was made by the what
57:53enzyme primase dna polymerase type 3
57:56will then add on to that 3-prime end and
57:58start making dna continuously
58:01all the way down the leading stream
58:02remember it was continuous
58:04so this will happen all the way down
58:07okay
58:08on the leading strand the lagging strand
58:11is where it becomes a problem
58:13remember you have that three prime end
58:16right
58:17and the five prime end the prime ace
58:19will have to add on to that three prime
58:20n so let's say it adds on
58:22right here at this three prime end when
58:24it does that
58:26it gives a little primer and then again
58:29dna will build built off of that here's
58:31another primer
58:33and then dna would be built off of that
58:34right so we kind of know that process
58:36this is pretty much
58:37you know a review of what we just talked
58:40about
58:41but here's what's different here watch
58:44what happens you guys your minds are
58:46about to get blown
58:48remember dna polymerase one what does he
58:50come in and do
58:51comes and plucks off the rna primer
58:55and then makes dna right
58:59on the leading strand on the lagging
59:01strand it'll come off and pluck this
59:02portion here
59:04okay that rna primer and when it plucks
59:07off the rna primer it still has a what
59:10remember this is the three prime end
59:12what would this be here
59:13five prime three prime so
59:17remember five prime this is a three
59:19prime end right here it still has a
59:21three prime
59:21n that it can use to build off of and
59:24make dna at this point here
59:29but watch what happens down here comes
59:31down to this end here
59:33plucks off these rna primers
59:37uh oh do i have a three prime end that i
59:40can add off of somewhere
59:42i don't dudes why because look this is
59:45my five prime
59:46end i have no three prime end here that
59:49that dna polymerase can add
59:51on nucleotides to so dna replication
59:54won't occur at these points here
59:57and that is problematic because guess
59:59what
1:00:00this was the old dna strand you
1:00:03replicated and made a new dna strand
1:00:05this one's going to get shorter it's
1:00:06shorter than the original one
1:00:08guess what happens when this one
1:00:09replicates it'll get the new strain will
1:00:12be shorter than
1:00:13it and then the next one and the next
1:00:15one and the dna will continue to get
1:00:16shorter and shorter and shorter
1:00:18and eventually start involving those
1:00:20genes
1:00:22so thank goodness in particular cells
1:00:24where we have a
1:00:26need a lot of replication to occur we
1:00:29have a special enzyme that comes in and
1:00:31says hey
1:00:32i'm going to elongate those telomeres
1:00:33for you so that way whenever dna
1:00:35replication does occur
1:00:36you don't start really taking away too
1:00:39much of the telomeres and involving
1:00:40these really important genes
1:00:42so what is the name of this special
1:00:44enzyme that we should give great thanks
1:00:45to
1:00:48this wonky looking enzyme here has two
1:00:50arms
1:00:51and this enzyme is called telomerase
1:00:57and telomerase is a really interesting
1:01:00kind of ribonucleoprotein
1:01:04one arm comes here okay so remember
1:01:06we're just looking at this portion here
1:01:08so we're only zooming in
1:01:10on this lagging strand about right here
1:01:12okay and zooming in on it
1:01:14so here we're on this portion where we
1:01:16didn't finish and synthesize the
1:01:18nucleotides because again we didn't have
1:01:19that three prime oh region here to add
1:01:22nucleotides off of the dna polymerase so
1:01:24what the telomerase does is it takes one
1:01:26arm
1:01:27and brings that arm out and on this arm
1:01:30is something really
1:01:31really cool it expresses nucleotides
1:01:37and a particular type of nucleotides
1:01:40that you guys really need to know okay
1:01:44and so what are these nucleotides that
1:01:46it has
1:01:48well what it expresses is
1:01:51complementary nucleotides that are
1:01:54commonly seen on the telomeres telomeres
1:01:56always have
1:01:57and the easiest way there's a mnemonic
1:01:58to remember them telomeres always have a
1:02:00particular repeat of nucleotides on them
1:02:03on their three prime end and the easy
1:02:05way you can remember that repeat
1:02:08is the mnemonic tell them all
1:02:12genes gotta go that is the repeat that
1:02:16you
1:02:16constantly see on that three prime end
1:02:19of that parental dna
1:02:21so what telomeres does is it comes in
1:02:23and says hey i have
1:02:24all the complementary rna nucleotides
1:02:28to this sequence that's commonly seen
1:02:30here so let's kind of write down what
1:02:32would the complementary portion be
1:02:34if it was t it would be a
1:02:38t it would be a a it would be
1:02:41u g all the way across
1:02:45it would be c c c so it expresses that
1:02:48with its one arm
1:02:50the other arm is really cool
1:02:54the other arm will then use
1:02:57this rna strand as a
1:03:00template to make dna that's
1:03:02complementary to it
1:03:04so if it does that it's going to take
1:03:05this rna read it and then what would be
1:03:07the complementary
1:03:08t t a
1:03:12g g g
1:03:17this is really interesting you want to
1:03:19know why
1:03:20i elongated my three prime n okay which
1:03:23is important i elongated it so that way
1:03:25next time this dna replicates i won't
1:03:27really
1:03:28take too much of the dna and involve
1:03:29those genes because the telomerase
1:03:32but what i did is i used rna
1:03:36and from that i made dna
1:03:41i need you guys to understand what
1:03:42that's called what is that called when
1:03:45you go from rna
1:03:47to dna reverse transcription
1:03:52this is called reverse
1:03:55transcription and so what this kind of
1:03:58telomerase does
1:03:59in a way is it has again it's a protein
1:04:03with
1:04:03it which expresses nucleotides it
1:04:05expresses rna
1:04:07and then it has this other arm this
1:04:08other arm that reads the rna
1:04:10and says oh okay this is a i'm going to
1:04:13make t
1:04:14on the parental strand that's a i'm
1:04:15going to make t on the parental strand
1:04:17so it can take rna and make dna
1:04:19elongating the telomeres
1:04:21why would we want to elongate the
1:04:24telomeres we obviously know to prevent
1:04:25gene loss and so we don't
1:04:27shorten those telomeres significantly
1:04:29and what cells would you want there to
1:04:31be a lot of telomerase
1:04:32enzymes are a lot high activity highly
1:04:35replicating cells
1:04:36cells that are replicating so much that
1:04:38those telomeres would shorten if we
1:04:40didn't have it
1:04:41so this is important you need lots of
1:04:44telomerase enzyme
1:04:46and what kind of cells
1:04:50primarily in like stem cells so if
1:04:54you're
1:04:54like if you're a zygote and you're
1:04:55starting with one cell you need those
1:04:57cells to have lots of telomerase
1:04:58activity to replicate
1:05:00and make the whole human body or your
1:05:03hema hematopoietic stem cells which are
1:05:05making red blood cells white blood cells
1:05:07all those different things you need
1:05:08those to be able to replicate and have
1:05:10enough telomerase enzymes so that it can
1:05:11replicate without hitting and losing
1:05:13those genes
1:05:14that's important one last thing clinical
1:05:16point
1:05:17telomerase what if we figured out a way
1:05:22cells certain damaging and really nasty
1:05:25cells
1:05:26figure out a way to evade
1:05:29uh the cell replication where they can
1:05:31just continue to keep replicating
1:05:33without
1:05:34not being able to stop what are that
1:05:35what is that called
1:05:37cancer neoplasia so cancer cells you
1:05:40know what they can do
1:05:41that we believe that they can do is they
1:05:44upregulate the activity
1:05:47of their telomerase enzymes and if they
1:05:50up regulate the activity of their
1:05:51telomerase enzymes they continue to
1:05:53elongate the ends of the dna
1:05:55on the chromosomes which allows for them
1:05:57to continue to keep replicating and
1:05:59replicating and replicating
1:06:00without shortening the telomeres enough
1:06:02that it starts to involve genes within
1:06:04that cells
1:06:05that's really interesting so
1:06:09really big thing i need you guys to take
1:06:10away telomeres
1:06:12shorten with every dna replication we
1:06:16can prevent that with telomerase enzymes
1:06:18which perform what kind of process here
1:06:21reverse transcription use one arm which
1:06:22is rna and build
1:06:24dna on the parental strand to elongate
1:06:27that
1:06:28and what types of cells normally would
1:06:30you see lots of telomerase activity
1:06:32normal stem cells highly replicating
1:06:34cells in our body
1:06:35or cancer cells which dysregulate or
1:06:37upregulate the telomerase enzymes
1:06:40all right ninja nerds that covers
1:06:42everything for dna replication
1:06:44all right ninja nerds so in this video
1:06:45we talk about dna replication i hope it
1:06:47made sense and i hope that you guys did
1:06:49enjoy it alright engineers as always
1:06:51until next time
1:07:01[Music]

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