Understanding DNA Replication: The Science Behind Cell Division

what's up ninja nerds in this video today we're going to be talking about dna replication but before we get

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account go check that out all right ninja nerds let's get into it all right ninjas when

we talk about dna replication the first thing that we need to talk about is a couple of fundamental points very

important things that we're going to build on throughout the process of this lecture and so the first thing i need

you guys to know is what in the heck do we do dna replication for and the whole point is is that in order

for cells to be able to replicate and make more cells

we need the dna within those cells to replicate because the dna is pretty much the

the genetic portion of the cell it's what makes a cell what it is so in order for us to really

understand dna replication i really want you to understand that the whole purpose of it

is to allow for cell replication okay or sometimes we refer to this as the cell cycle

okay so the cell cycle i know you guys know the cell cycle when in the cell cycle what's kind of

the really quick part of it you start off you go g1 then you go s phase g2 phase and then you go into

the mitosis part and then out of that you get two cells where you take one cell

that cell enters into the g1 s g2 goes through mitosis and makes two cells the big thing i want you to know is that

dna replication primarily occurs within a particular part of the cell cycle when a cell's replicating making more cells

it primarily occurs within the s phase so in the s phase that is where dna replication is occurring so the first

fundamental that you need to know is why do we perform dna replication in order for our cells to replicate make

more cells so whenever they go through their cell cycle the particular point when the

dna is actually replicating is in the s phase of the cell cycle now real quick what in the heck is cell

replication it's really simple i'm taking this cell here which has 23 maternal and 23 paternal chromosomes

and all i'm doing is i'm making two identical cells that look just like this so i have to replicate

the dna within this chromosome and the dna within this chromosome and i'll make two of this

and two of this and that's going to give me these two identical daughter cells and that's the basic process of cell

replication so that's the first thing i need you guys to know the second thing that i

need you guys to know about dna replication is that it occurs in what's called a

semi-conservative model what the heck does that mean zack i got you

so the next thing you need to know is that dna replication is semi-conservative

semi-conservative means let's take that a piece of dna here right so dna has two strands and we're going

to call these we're going to give them two names we're going to call these blue dna

strands parental strands or let's call them old strands what i'm going to do is when i want to

replicate this dna i have to separate them and we'll talk about how we do that when you separate the dna into separate

strands here i have two old parental strands separated

what i'm going to do is i'm going to replicate the dna in a complementary

fashion so what does that mean complementary that means if for example this

nucleotide was adenine or a this would be t if this was t this would be a

this was g this would be c if this was c this would be g you get the point i'm going to make

nucleotides that are complementary on that but do you see how the color is

different and i synthesized a new strand or a a daughter strand if you will that's one aspect of it i'm

going to do the same thing to this other strand so i'm going to use this old parental strand

and make a new strand with complementary nucleotides on the other old parental strand i'm going to again

make dna that is complementary to this old or parental strand so the whole concept here is that i start with

old if you will let's use the term old and i make new mixed with the old

that's really the easiest way of understanding dna replication is i'm taking

two old parental strands separating them and making two new dna strands that are complementary to them

so what i did is i took this dna and made two new double-stranded dna molecules

isn't that cool and when i did it i did it in this semi-conservative

process the next really important thing that you guys need to know is that dna replication occurs in a very

very specific direction dna replication has to occur okay the direction okay replication direction

is very important it's kind of annoying we'll mention it a lot throughout the process of this lecture

but dna direction always has to occur from the five prime end to the three prime

end i can't stress that enough super important it's going to come up a lot what the heck does that mean really

quickly do you guys remember from the dna structure video what was on the five prime end of a

nucleotide the phosphate group what was on the three prime end of the nucleotide the oh group

so when i'm adding nucleotides i'm adding a phosphate group onto a three prime

group of the preceding nucleotide let's show you an example of that so let's say that we took this old dna

strand we're gonna do replication following the semiconservative model here i have two old parental dna strands

separate them i'm going to replicate it via the semiconservative model but i'm also going to follow this

process where i have to replicate five to three so let's say on one end of this old dna

strand i have a three prime end here what does that mean that means there's an o h group here on

the five prime end of this dna strand i have a phosphate group here okay same thing here

oh group and then the phosphate group right here so that's basically what the five prime three prime end is

and you guys remember that dna is antiparallel so if it's three prime on one end five prime on the other

end the other dna strain has to be flipped so it's five prime on the same and then

it's three prime and three prime on the same end that it was five prime that's important

so when dna replication occurs it has to occur five to three so here's the three prime end

when i make a new dna strand it has to be five prime first three prime end here

and then what will i do i'll add another nucleotide and then this connection here will be

between what i'll have a three prime end here and a five prime end of this next nucleotide

i'll have a three prime end here i'm gonna add another nucleotide so when i make nucleotides i make them

and synthesize them from five to three okay same thing if i'm going to do it off of

this strand here's the five end here's the three end of the parental strand if i want to make the new strand

this is the five prime end so i'm going to be synthesizing dna in which direction

in this direction here right and again doing this according to the complementarity rule

okay so that's the important thing i need you guys to remember is that dna replication occurs in a five to

three direction the last fundamental thing that is really important here

is that dna replication is bi-directional and you're like why the heck do i need

to know that i think oftentimes when we're looking in textbooks we only focus on one end where dna

replication is occurring but what's really important is we're going to talk about this in a second but

what we do is we take the dna and we already have an idea that we're going to separate the two older parental strands

away from one another so that we can create new dna from that when we do that we create these little

ends here these little y-shaped regions called replication forks okay so it's called the replication fork

and you have two of them one on this end one on this end there's going to be enzymes called

helicases which are going to come in and unwind the dna on both sides moving it in this

direction and moving it in this direction and then enzymes called dna polymerases

which we'll talk about they're also going to move into these areas and follow the helicase synthesizing new

dna off of that parental strand in a bi-directional fashion

so the big fundamentals i need you guys to take away is that why do we do it dna replication in order for cells to

replicate and make more cells it occurs in a semi-conservative fashion taking old

making a mixed old and new double two double-stranded dna molecules it occurs in a five to three direction

and it occurs bi-directionally from what's called the origin of replication or where these

replication forks are okay now that we have the fundamentals let's now talk about the steps of dna

replication all right so the first thing that we have to talk about when we're talking

about the stages of dna replication there's three stages of dna replication initiation elongation and termination

okay initiation elongation and termination initiation is really an easy process

it's not too hard to remember so what happens is let's say that here i have my double-stranded dna okay and

let's say there's a particular region in that double-stranded dna which is

really really a nice little area that i want to go and i want to separate the dna so that i can

create two separate dna strands parental strands that i can use as templates to make new dna

that area is going to become our origin of replication all right so this area is really what's

going to be our origin of replication and why do we need to know this okay

so whenever we need we're picking this spot how do we determine what that we're picking that spot like okay i know

that there's a bunch of regions on the dna why is the this point here the

particular origin and there's a really cool reason why there's particular

nucleotides in this region which are really highly concentrated with adenine and thymine so it's an adenine

and thymine rich area now let's talk about this for a quick second

why would i pick adenine and thymine as the area that i really want to target as compared to

guanine and cytosine do you guys know why so adenine and thymine how many hydrogen

bonds are there between them two how many hydrogen bonds are there between guanine and cytosine

three it's going to be easier to break two hydrogen bonds and it is going to be able to break three hydrogen bonds

so in this area there's going to be particular areas which are really concentrated with adenine and thymine

nitrogenous bases or nucleotides and that's going to be better suited as the area we want to

kind of separate why because there's only two hydrogen bonds and that's going to

require less energy okay to break those hydrogen bonds as

compared to guanine and cytosine okay so that's the first thing we have particular areas now

the next thing i need you guys to understand is in eukaryotic cells is there only one

origin that okay there's just one area here where there's a lot of adenine and

thymine i just have enzymes bind to that portion and separate it and the enzymes just work from the

center and go to the ends no what's really important that you guys need to know is that in eukaryotic cells

there is multiple origins sometimes represented or e c origins of replication

so that's really really important so for example i may just be representing this one portion but there may be another

origin of replication right here and another origin of replication here which is really rich in adenine thymine

nucleotides okay so that's one important thing the next thing is what type of structure

is going to bind onto these areas and help to break the bonds between the adenine and thymine

what do we have there's a really interesting protein it's got one heck of a name they always

do don't they and this protein here is called the pre-replication

pro pre-replication protein complex one heck of a name so here we're going to draw a cute

little enzyme here okay this is a cute little enzyme and this enzyme is going to come in and

bind onto these areas and when they bind onto the areas they separate

the adenine and thymine nucleotides in that area to separate the dna what is this protein here called that

separates them it's called a pre replication protein complex

so again it binds to the origin of replication and separates the adenothymine

uh nitrogenous bases now once it does that what's that going to look like well let's take the next step

we had this protein bind on to the origin of replication where there's a lot of adenine and thymine nucleotides

we separated the bonds between them the two hydrogen bonds and we create this little like bubble if

you will you know what we call this it's not hard it's called the replication bubble that's literally what

it's called i know it sounds crazy but now we kind of form this little bubble due to this whole process and

it's called the replication bubble now once we form this replication bubble there's a couple things that i

need you guys to know we have separated the nucleotides so if you imagine here we're not showing them but let's say

that here i show a couple you know here's my nitrogenous bases that are coming off this

sugar phosphate backbone right which is made up of again the deoxyribose sugars the phosphate groups

all that and these are just my nitrogenous bases that are popping out off of it right

so i'm going to have these exposed now so these were once really connected nicely together like in

these regions i really separated them so they're really kind of like vulnerable right now and you know what's

really important these when you separate them they want so badly

to re-anneal with one another all right so what is this protein that really helps to protect

these vulnerable separated parental dna strands

you know it's really ironic uh you know sometimes science is there's a big old protein

that comes and binds to this end it would do the same thing on this one and this protein you know what it's

called it's called the single stranded binding protein i'm not even joking that's literally the name of it

and it's perfect it's not hard to remember why because it's a protein binding to a single

parental strand so you would have one over here i'll just draw a portion of it but you'd have the same thing over here

another single strand of binding protein binding onto this parental strand and what is the purpose of these single

stranded binding proteins that's what i really want you to remember one of the functions is that it prevents

the parental strands from re-annealing what the heck does that mean what's that term re-annealing from reconnecting to

one another right so that's important so it present prevents the parental strands from

re-annealing to one another because they honestly they really want to click back to one

another the other thing that they do is that when you have

these uh parental strands separated as kind of these single strands if you will they're very vulnerable to very nasty

little enzymes there's some nasty little pac-man-like enzymes that want to come to the area

and break the phosphodiester bonds these are called nucleases so what these single-stranded

binding proteins do is they kind of act as a barrier and protect these single strands from

exonucleases or endonucleases so again it prevents it protects from nucleases

okay so so far free replication complex binds to the origin of replication or the at rich area separates it forms a

replication bubble single strand of binding proteins bind to the single strands prevent them from

kneeling and protect them from nucleases the next thing is once you form a replication bubble you form these two

ends here okay and these two ends we already kind of mentioned it a little bit

this end here where it kind of like makes like a y shape if you will this right here is called your

replication fork okay there's going to be an enzyme that we're going to talk about in just a

second ah frickin we'll talk about them now he hops in here

this little enzyme is like the energizer bunny he's got so much energy as long as you

keep feeding them and he hops in here look at this cute little enzyme look at this guy

this enzyme will come in at this point here and really unwind the dna at

both replication forks what is the name of this enzyme that really works

in these replication forks unwinding the dna in front of them this enzyme is called helicase

and the big thing i really want you to know about the helicase enzyme is that he requires a ton of atp

in order to perform this process okay so step by step again real quick pre-replication

complex binds to the origin of replication at rich area separates it single-stranded binding proteins bind

protect them from exonucleases and endonucleases prevent re-annealing whenever you do

that separating them you create a replication bubble at the ends of them you have

replication forks helicases highly atp dependent hop in there and start unwinding the dna

in front of them after they do that something happens which is really important

that we definitely need to know let's come down here and this is a really important area that i really really need

you guys to understand so let's say that we come back to this point here

again what we have binding right here to these two ends single stranded binding proteins we're

not going to show it on the top but the same thing here and then again what enzyme let's just

focus on this area right here just on this replication fork what enzyme would be in this area here

really working and unwinding the dna in front of it again that's called helicase

what happens is as helicase continues to unwind the dna the whole purpose of that as it unwinds the dna separates the

strands so that enzymes would be able to use those parental strands to make new dna

okay but there's a problem that happens as dna helicases just you know going through those mofos and unwinding the

dna constantly in front of it it bunches up the dna in front of it distal to it okay from that replication

fork so distal to the replication for downstream the dna starts bunching up and it creates things these things

called super coils okay it creates these things called super coils

and this is caused by the helicase really unwinding the dna why are super coils bad if you really

bunch up the dna in front of it it's going to really impede the helicase from continuing to unwind the dna it's

going to face a lot of restriction because it's really bunched up here so it'll just keep getting bunched up until

you relieve those super coils so we need enzymes that can come

in there and fix these supercoils where the dna is really really tightly wound so how do we

do that all right so what little enzymes do we have or special little things that come

into the play to really alleviate these super coils these enzymes are crazy interesting so

these guys are really cool and they are called topo high sama races they're shape of the t

right so they're called what toppo is now topoisomerases there's actually a

couple different types okay there's particularly type 1 type 2 and then there's type 4.

for the most part they all do the same kind of thing and what is that okay this enzyme has two little

arms if you will let's say here it has one little arm and another little arm on one arm

it can go to where this area of the supracoil is let's say that the supercoil's right here

it can use a little enzyme a little domain of it and cut this dna strand

if it cuts the dna strand what does that allow for it to do it kind of allows the dna to kind of

unwind a little bit and unravel and so there's a particular name to this domain on that

topoisomerase enzyme it's called a nuclease domain and what does it do it creates a

cut or breaks the phosphodiester bond in the dna strands one maybe two dna strands

allows it to unwind there's a problem with that though if i just cut the dna strand and allow

it to continuously unwind that may be problematic the dna could continue to fragment

i don't want that so what i do is i use this other arm and after the dna super coils have been alleviated so

let's now kind of draw new dna after the super coils have been alleviated

so we alleviated the super coils we got rid of all of those overwinding of the dna

look now it's beautiful it's not overwinded but we have a break in that dna and

that's a problem so now we need to use the other arm of this topoisomerase where we cut

this portion here and allow it to unwind we need to use this portion

called the ligase domain and once it's unwound this portion here what does it do

it re-stitches this area back together after it's unwound the super coils okay so again the topwise sama races

what were their function again two unwind the super coils now

why in the heck did i take all this time to mention the topoisomerases and there's different types

let me explain why topoisomerases can be in both cells so we primarily haven't really discussed

that dna replication can occur in bacterial cells and it can occur in eukaryotic cells or we call it bacterial

cells like prokaryotic cells and eukaryotic cells human cells primarily

they have type one and two topoisomerases and in prokaryotic cells they primarily

have two and four so again type one and two are primarily for

eukaryotic cells and then type 2 and 4 are primarily for prokaryotic

cells big thing i need you guys to know is is that type 1 topoisomerase

does not require any atp to unwind the supercoils so let's put that next to it that just for this one just for this one

it no atp is required in order for it to perform this unwinding of the super coils

but for type 2 and 4 let's pick a different color so that we don't confuse it here

for type 2 and type 4 these do require atp in order for them to unwind the super

coils there's also one more thing i don't want to get too far in depth in it that type two and four can do that are a

little bit different from type one and that's that if you really look in the the textbooks

they can actually take and cut that little supercoil area allow for unwind and then

insert in what's called negative supercoils which also helps to kind of relax the dna and

prevent that kind of bunching up region let's not get too far into depth in that what i really want you guys to know

is why in the heck did i spend so much time talking about these topoisomerases in your usmles you have to know

particular drugs that we can target on these and eukaryotic cells i want you to think

about a reason just try to think for a reason why would i want to target this enzyme

in eukaryotic cells when this is important for dna replication if these enzymes aren't really working

dna replication won't occur when a eukaryotic cell what if i have a cancer cell

so if i have cancer the cells will continue to replicate so they'll replicate and replicate and

replicate well i could maybe use some drugs that could target these topoisomerases in my cancer

cells and prevent them from replicating how do i do that

well there is a particular name of there's a couple drugs that you guys definitely need to know

for topoisomerase one in eukaryotic cells the cancer drugs that we can use for

topoisomerase one is called irenotecan and what's called topotechin and again

these are anti like they're chemotherapeutic drugs that are going to inhibit the

topoisomerase one in the cancer cells the type two in eukaryotic cells we can use drugs called etopocide

and tinopocide and i'm going to explain how they do that because that's really important i really want you to

understand how they do this but that's for the eukaryotic cells think about prokaryotic cells

if you were infected by a bacteria and a bacteria infected your lungs and it just kept replicating and

replicating within your lungs could i maybe target the topoisomerase two and four and prevent that replication

process of the bacteria in my lungs yes so in bacterial infections let's say because it's a prokaryotic

cell and bacterial infections these bacteria will continue to keep replicating

so if i use particular drugs that can maybe prevent the dna from replicating in these bacterial cells

that's important and we can do that via inhibiting the topoisomerase primarily type 2

and we use the drug called fluoroquinolones some of you may have heard of these

ciprofloxacin levofloxacin oxyfloxacin all of those guys they're inhibiting

the topoism race too but i really want to take a second because i never understood this completely until i

really dug into the mechanism of how they actually do this i really want to quickly say how they do it

what these drugs are really doing is these drugs so let's kind of just put here drugs

they're exciting in increasing the activity of the nuclease domain what the heck is that going to do that's

going to just chop all those like portions of the dna and it's going to continue to fragment them

right remember we had another another domain of this enzyme that re-annealed them and

kind of stitched it back together to prevent that fragmentation that was the ligase binding domain what

if i use drugs to enhance the nuclease domain but inhibit the ligase domain so now

i'm going to have this enzyme go in and kind of cut where the supercoils is but i'm never going to re-stitch it

together the dna will just fragment over time and that's important because then you can't replicate the dna

within what kind of cells eukaryotic cells like cancer cells or bacterial cells and that's why those

drugs are important doesn't it make sense all right cool it's important to take a

clinical application and tie it to the the basic foundational science okay

so we kind of went through talked about the topoism races that was the big thing and how they

unwind the super coils let's get back to the foundational science and now that we've talked about

that the next thing that we have to go into is elongating the dna all right ninja

nerds so we already know we have a replication bubble we got a replication forks we have our

protein here called the single strand of binding protein which is stabilizing these single strands

we have the helicase enzymes that are in these replication forks working like a son of a gun

to unwind the dna we've got those topoisomerases over here that are kind of unwinding those coils

okay now we've really separated this we've stabilized it and we're ready to begin elongating the dna

okay here's what's really interesting there's an enzyme that comes into play here

and it does something really cool it's called primase so an enzyme called primase will come into play here so what

primase does is it's an enzyme that lays down okay that get makes what's

called rna primers so this takes a really quick turn where we've got to understand

zach you just said that we're making dna why the heck would i make rna there's a reason why

there's an enzyme that we'll talk about a little bit later called dna polymerase 3 that will

make dna but the only way it can do that is if it has some type of primer or three prime ohn to build off of so

what's the purpose of this primase this enzyme it lays down rna primers which enable

dna polymerase particularly type three to make dna and i'll kind of show you that

in a little bit in a second okay so primase comes in so imagine here i just have like this cute little enzyme

here called primase okay and this cute enzyme comes in here and let's say here what's this strand up

here this top part remember we're going to say that this is three prime end where the o h would be

this is the five prime end where the phosphate would be and again the opposite strand here would

have to be antiparallel so five prime end here three prime end here right we already know that

the primase is gonna come in and it's gonna read the nucleotides and it has to go in a particular fashion

it reads it from the three all the way to the five end so what does it do the first thing it

does is it reads the dna strand from three to five after it reads it from three to five

what did i tell you that's super important what does dna replication occur even though this isn't dna

it's the same concept it synthesizes rna primers or nucleotides in a five to three fashion so it's going to

take and make a couple nucleotides generally it's about 10 nucleotides we're only going to draw a couple here

but it'll have has to be again five primes starting here and i'm just going to make a couple i'll

make like four nucleotides here okay so here it's going to have five all the way to the three prime end here

okay that's my five prime end here that i just started with and i'm synthesizing it in

the three direction now the reason why this is important is on that three end what do we have here

the o h that's what's on the three prime end i need that o h the reason why

is another enzyme called dna polymerase type 3 comes in so there's an enzyme called dna

polymerase type 3 and he comes in and he needs that three prime oh from the rna primers in order

for it to continue to build nucleotides so again a big thing i need you to remember is it needs

the three prime o h of rna primer in order to carry out its activity if it doesn't have it it can't

do it so now that it has that this dna polymerase comes in

and it says okay i have my three primo h region perfect now what i'm going to do is i'm going to

read my dna and i'm going to do it the same way that the primase did i'm going to read the dna from the three

direction to the five direction so i'll read it boom boom boom

once i read and figure out what kind of nucleotide is then i'm just going to synthesize

those nucleotides in the 5 to 3 direction and it's the same process here so now

let's make a different color since it's a different enzyme we kind of picked red over there so

we're going to start off and we're going to say okay i'm going to take that oh and i'm going

to add a phosphate group onto it of a of another nucleotide so when i do that i'm going to continue to

keep synthesizing in a five to three direction moving towards the replication

fork again when i do that i read it i say okay let's say that this is adenine i'll put a thymine

this is guanine i'll put a cytosine this is cytosine i'll put a guanine and so on and so forth and i'll just

keep reading the nucleotides three to five and making a dna strand in what

direction five to three okay and again important to remember it

needed this three prime end of that oh of the rna primer to build off of it now here's what's really interesting the

primase will give it kind of a little leading point and the dna polymerase will just

go all the way towards the replication fork this strand is very continuous where

there's just one rna primer and then dna the rest of the way this strand is very important we give it

a particular name the strand that's very continuous where the dna polymerase moves towards the

replication fork is called the leading strand okay it's called the leading strand on

this other string which we're going to talk about is called the lagging strand something different happens where you're

still going to have the are the primase it'll come to this area okay on this other strand and again

this is the three end on this part five end on this part and what it'll do is

it'll read it from three to five and then synthesize a couple nucleotides from five to three so this is the three

end it's going to synthesize from five to three and again the same thing will happen we created

a primer of a couple nucleotides with a three prime o h end that the dna polymerase type

three can build off of so now the dna polymerase type 3 will just pop on and say oh perfect i have my

3 prime n to use i'm going to go ahead and just read the dna from 3 to 5

and synthesize it from 5 to three okay so i'm gonna do all that perfectly

now something interesting happens where it's gonna look it looks perfectly the same you're like zach i don't get the

difference here let's say that the gila case continues to unwind the dna

so it continues to unwind the dna something interesting happens that we have to talk about

okay so now let's come down here so let's say let's pretend right that for a second here we have

that primer let's kind of continue off this let's say that the helicase unwound the dna a

little bit more and we along we kind of opened up the dna and created a more

longer length of nucleotides so again let's say that here we had that primase came in here read this from one end

again this would be your three prime end this would be your five prime end so it'll read from three to

five and synthesize from five to three creates a little primer with a three

prime oh end the dna polymerase 3 says okay perfect i have everything i need i can continue

to grow and let's say that it just came up to like this point here but then you kind of unwind the dna

again that dna polymerase 3 doesn't stop it just keeps on going and keeps on moving

reading the dna from three to five and continuously synthesizing nucleotides from five to three so again this would

be your five prime end this would be a three prime end on this other chain this is on the leading

strand it continues on the lagging strand here's where it's a little bit different

let's say that we continue to move on here and let's say that like at this point here this was where the

previous rna primer was from above where it had again reading this portion of the dna this is the five prime end of

this part three prime end of this part it read this sequence of dna from three to five

and let's say that it synthesized a couple nucleotides to give your rna primer

from five to three and then what do we say happen from that part above we have the dna polymerase three use

that three prime oh end read the dna from three to five and synthesize the nucleotides

from five to three here's what happens the primase lay down a primer here but the dna polymerase

iii has to use that primer to continue to keep building off if you unwind the dna a little bit more

now now you have a couple of the nucleotides so now let me kind of just so we have

enough room here let's say i draw a couple more nucleotides

so now here i have a couple more nucleotides now that primase after it just made down this primer for

the dna polymerase three to use it comes down to the next part of the replication fork and it says okay here i

got another three prime end here let me again read from three to five

and synthesize a couple nucleotides from five to three so i laid down my rna primer

dna polymerase says okay cool i got my three prime oh end here let me go ahead and use that to make

my dna and i'm going to read the dna from three to five and synthesize it from five to three

do you notice something really interesting here on this strand which we called again what did we call this

strand well we had one primer and then dna for the continuous way towards the

replication fork we called this the leading strand so the big thing i want you to know is that you

have one rna primer and then a continuous dna strand from that point on

on this strand called the lagging strand something different happens here where you have a couple rna primers

okay and then kind of stretches of dna between those rna primers this kind of like broken up portion

where there's rna dna rna dna and if we continue to keep elongating it we'd have more rna dna rna

dna this gives a particular name which is called

oka zaki fragments okay okazaki fragments and again it's basically where you have multiple

rna primers and multiple stretches of dna stretches okay so multiple dna stretches

and then multiple rna primers it's a mix of them and that's a problem okay because you're

going to see now there has to be another thing that we have to do we want everything when we replicate dna

it has to be all dna we can't have it be dna with a little bit of rna so we're only using these primers as just kind of

a point to build off of after we've built some stuff we're just going to go in and cut those

things out because we don't really need them anymore so now let's talk about the next part

which is we've started to kind of create these primers that we needed to build the dna

off of now we don't need the primers and we got to get rid of it how do we do that

the next thing that you guys need to understand is okay we've used our rna primers for the dna polymerase 3 to kind

of build off of and make dna from we don't need those primers anymore we got to get rid of

them so let's draw the diagram that we had previously which again we only had what a little

stretch of rna primer here and then the rest of the length down going towards the replication fork which

again what is this strand here called the leading strand is going to be all dna okay

so now this is really important we're going to talk about one more thing in just a second and again if you guys

remember on this strand the lagging strand we had a couple rna primers

that were in between the stretches of dna creating what's called okazaki fragments

on the lagging strand right we talked about that so now the next goal here is that we

have to remove those rna primers but before we do that i got to mention one more thing

so before we talk about how we remove these rna primers i really want to take a quick second here

to explain something else that dna polymerase type 3 can do so we know that it reads the dna okay

from three to five and then synthesizes nucleotides off of the rna primer from five to three

but it also has one other function it's called a proofreading function which is very important before we talk about the

rna primers and this proof reading function is helpful to prevent

mistakes and what it does is let's say okay it reads three to five reads all the nucleotides from a three

to five direction and then synthesizes nucleotides in a five to three after it does that it says okay

let me check my work it goes back and it finds the connection between this point says okay is this a

good connection yeah that's a good one a and t are connected together oh g and c are connected together a and

g oh this isn't a correct uh complementary kind of base pair

i need to cut that out so it reads from three to five and if it finds any mistakes it uses what's called a

three prime to five prime exonuclease activity where it says okay let me read here

i'm gonna read it and i says okay three to five i'm reading a t and again it's complementary base it

should be t should be a g a oh not a correct one i'm gonna cut that out read it again

make sure i have it okay it was g that has to be c and synthesize the correct nucleotide in the five to three

direction so big thing i need you to remember for dna polymerase type three

reads dna three to five synthesizes nucleotides five to three proof reads back in three to five and if

that's incorrect uses a three to five prime exit nucleus to cut it out

and put in the correct complementary nucleotide very important okay now we got to get rid of these rna

primers how do we get rid of the rna primers well the dna polymerase 3 is not the

answer the next enzyme as if there isn't enough enzymes

is called dna polymerase type 1. so dna polymerase type one comes to the rescue and what it does is

it starts here and it finds this okay so let's say here we had our three prime end here

five prime end here the new strand would be synthesized from five to three what this enzyme will do is

it'll come in and it'll cut out these primers going from the five to three direction

so it removes primers or it plucks those little primers out in a five to three

exonuclease activity right particularly for what to pluck out

the rna primers so this guy will come in and it'll say okay pluck remove that one pluck remove that

one and then what it'll do is once it plucks those out it then says okay

i'm gonna read this strand from three to five so it reads the one that it plucked out

and says okay that's a adenine what do i need to add here i need to add in

thymine oh this is thymine i need to add in here adenine so it plucks off the primers

then what does it do it reads the dna from three to five and then it synthesizes

from five to three you know what else it can do one more function

let's say okay it plucked off the rna primer reads it as adenine puts a thymine reads it as guanine

accidentally puts an adenine has to go back and proof read it though because that's always the thing that

they have to do proof reads it and says not a good connection

i don't want that what do i need to do i need to pluck that thing out of there and put the correct nucleotide

so the last thing dna polymerase type one can do is again it has that proof reading type

of activity where it can do what it can read from three to five it

finds an incorrect base pair connection it cuts it out and when it cuts it out it cuts that at a three

to five exonuclease type of fashion okay so the big difference that if you ever get asked between

what in the heck is the difference between dna polymerase type one and dna polymerase type three really the

big difference is that this guy can do everything type three can do

it's just it has that five to three prime exit nucleus activity where it plucks out the rna primers

everything else is the same though now the next thing is dna polymerase type one we talked about on the on this

leading strand on the lacking strain it's a little interesting it'll come in and again it'll use its

five to three prime exonuclease activity so again let's use our combination here of what we know

this was three this was five so antiparallel this has to be five to three for the old strand the new

strand would then be what read three to five synthesize 5 to 3. so this dna polymerase will come

in and it'll start moving down and at this point it'll pluck off an rna primer and then what will it do

read 3 to 5 and synthesize five to three come to the and then proofread it is it correct

oh it is okay if it's not use my three to five primax nucleus to cut it out and then put in a new one it goes to the

next one plucks off the rna primer and does the same thing reads three to five synthesizes five to three proof

reads three to five then it just keeps doing that and plucking these things off here's the

difference though in the lagging strand it creates like a couple gaps these actually don't

completely kind of fuse together so let's kind of draw where we had this here and we'll create a little

space between these points here where the rna primers were so there's kind of a little space here

let's draw it here in orange so on the lagging strand it creates like a little space where it can't like

really fuse these ends where the primers were to the original dna

okay so it plucked the rna primers off and put nucleotides but it wasn't able to perfectly fuse these ends on the

lagging strand one more enzyme you're like dude i can't do no more

i promise one more this enzyme is called ligase so it's called ligase now ligase

will come in on that lagging strand and fuse the dna ends together okay

those basically where those okazaki fragments were it'll come and it'll say okay here's

these ends here i'm going to fuse these points together so that it's

perfectly connected and continuous and now we have a parental dna with a new daughter dna strand again

a parental dna strand with a whole new daughter dna strand that is all continuous and all in sequence no rna

primers no nothing no breaks it's perfectly set now the last thing that i want to talk about

here before we go on to termination is we've elongated our dna we now took the old parental dna and made new dna

the reason why i want you to remember this is that there's it's very important for us emilies to connect foundational

sciences with clinical significance and so in people who have hiv okay their t cells okay their t cells

have are infected with the particular virus called a retrovirus and it's causing this virus to get

incorporated into the dna and then from every point that on that these t cells replicate they continue to

replicate more of the hiv genome so there's drugs that we use to target this hiv virus and particularly the t

cell replication process and these drugs are called nucleoside reverse transcriptase

inhibitors you're like holy crap what the heck does that mean i just want you to remember that they're drugs that

are used for hiv and they inhibit the replication process and t cells that have been affected with

hiv let me explain how this works this is really cool let's say here i just i quickly put down

an rna primer okay and i have my dna polymerase three it comes in here and it starts making

some dna right uses that three prime end of the rna primer and starts making dna

i give them a drug let's kind of put here an nrti and again there's many different names

of these like didenosine zydovidine there's a whole bunch of these but what i want you to remember is

imagine these as what's called nucleosides okay and what they do is imagine here's

my my basically my my ribose sugar and then here i'm going to have my phosphate

group and then here i have like adenine okay what they do which is really interesting

is usually on your your deoxyribose sugar you should have an oh right and you need that oh

on that three prime end in order for the dna polymerase to continue to keep adding

what they do is is they remove the three prime oh region so now the dna polymerase 3 will get another

nucleotide it'll read this and say okay this is adenine i'm going to put a thymine or

something like that and i'm going to add in this drug this drug kind of floats around kind of

interestingly and the dna polymerase iii will then say okay here this is a

another nucleotide just like the ones i've been adding let me add this one on the only problem is is it doesn't have a

three prime o h region so you know what happens here since there's no three prime oh

you dna polymerase can't build off of that remember what i told you dna polymerase

type three needs a three prime oh region to build if you give a drug that doesn't have

that can you continue to build off of it no so all the dna replication from this point

is inhibited are you able to replicate all the dna within these t cells that have been affected with hiv

no so that's how it does it they're what's called kind of like analogs nucleoside analogs

where you're kind of like dna polymerase 3 doesn't know the difference it's just taking nucleotides and adding on

and all of a sudden just by kind of chance you have this drug that it attaches on it doesn't know the

difference it goes to add another nucleotide on it's like hey that didn't add on what

the heck i can't add this nucleotide on and the dna never gets completely replicated so it's a good it's a good

clinical point to understand that covers our elongation let's hit it home with termination

and take a quick second to talk about telomeres all right this is actually the easiest one of all you're probably like

oh please zach i can't do any more i know this this is a lot but let's say that we

have another enzyme okay that helicase enzyme we're getting to the point where dna replication has been completed

we got that enzyme what was that enzyme that was working at these replication forks and just continuing to unwind the

dna you know in front of it what are they called the helicases

and then you had those enzymes the dna polymerases type 3 and type 1 and all those guys that were coming and

basically reading the dna three to five and synthesizing it five to three proof reading in the three to five all that

good stuff and you've synthesized the dna and the helicases are

meeting each other at kind of replication forks that are about to abut one another when this happens

when the gila cases meet and you kind of unwind this portion of the dna what's going to happen the helicases are

just going to kind of say oh well hey buddy i guess i don't need to keep unwinding anymore

and what will happen is you're just going to kind of have this point here where the dna polymerase is will just

hop off of the dna because at this point there's nothing else for it to read

and so usually once it gets to that point they'll just say hey i guess the helicases are done there's

no more unwinding for me and then after that the dna polymerases will say hey i've already kind of

hit all the nucleotide regions here i'm done and i've replicated all of my dna

so that's important so it's basically again where there's multiple origins of replication and they're constantly

moving towards one another whenever they moved and hit one another the dna replication at that point stops

now there's something else that you have to remember though dna replication will you know start at a

point and then work bi-directionally it's eventually going to go to the ends of the dna or the chromosomes which we

call the telomeres there's a particular nucleotide sequence at that end where the dna

polymerases have a really hard time being able to replicate and that's very important we'll talk

about that next okay but again termination of dna replication it's really simple it's when the dna

polymerases are moving towards one another at a replication fork and they've just

stopped at that point they hop off and they no longer perform their function there's one other part of

it which is with the telomeres which we're going to finish off with all right so the next thing that we're

going to talk about is telomeres right so dna replication there's a little

interesting issue that happens at the telomeres so one thing i need you guys to know is that

telomeres they really shorten over time so let's say that here we look at some chromosomes which again

made up of dna and proteins well let's primarily think of it as dna and let's say that this goes through

goes through a replication cycle a couple times i want you to notice what happens to the ends right so when you

look at a chromosome there's two primary kind of like structural points

the point in the center right which is called your centromere and then the ends okay these points here

and these are called your telomeres now what happens is over time as your cells continue to keep replicating the

dna replicates watch what happens to the telomeres they get shorter and shorter

and shorter as that continues to happen there's a worry with this and let me explain what that worry is

obviously your dna has particular areas which code for rna rna can then get translated

to proteins what are those called they're called genes so let's say that i have a gene right here

the telomeres will be there and their primary function is that they will it's common for them

this to happen where the telomeres were short and short and shortened but the whole purpose of

them is that telomeres don't code for anything that's very important let me

write that down telomeres do not code for any rna

so do not code for rna in other words you can't take the dna from a telomere make rna make

protein that's important but let's say here there is a gene there that can make rna

the telomeres will kind of sacrifice themselves because dna replication doesn't occur at

this point for a particular reason we'll explain why and so because of that they prevent

gene loss so they kind of like take the hit for us if you think about it they're like don't worry i don't code

for any rna so you don't have to continue to replicate me so with each replication cycle the

telomeres will shorten and shorten and shorten but that's okay and for a particular

reason because they do not code for rna and they help to prevent gene loss but here's the problem eventually you're

going to get to a point where the telomeres will shorten so much that it can interfere

with the genes once that happens where the cell has reached the replication limit where it can't replicate

anymore it's reached its maximum number there's a particular term that you guys need to know and it's