Cell Biology | DNA Replication 🧬

what's up ninja nerds in this video today we're going to be talking about dna replication but before we get started please continue to support us by hitting that like button commenting on the comment section and please subscribe also down in the description box we have links to our facebook instagram patreon 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 called the hayflick limit it's called the hay flick limit and that is basically the maximum amount of times that this this kind of dna can replicate before it starts to involve genes now let's talk about why these telomeres are shortened really quickly let's say here we take and let's say that we use this as our example this is our leading strand and this is our lag strand right again

let's just say here we have our three prime end we're going to use our rna primer here and then from here the rna primer was made by the what enzyme primase dna polymerase type 3 will then add on to that 3-prime end and start making dna continuously all the way down the leading stream remember it was continuous so this will happen all the way down okay on the leading strand the lagging strand is where it becomes a problem remember you have that three prime end right and the five prime end the prime ace will have

to add on to that three prime n so let's say it adds on right here at this three prime end when it does that it gives a little primer and then again dna will build built off of that here's another primer and then dna would be built off of that right so we kind of know that process this is pretty much you know a review of what we just talked about but here's what's different here watch what happens you guys your minds are about to get blown remember dna polymerase one what does he come in

and do comes and plucks off the rna primer and then makes dna right on the leading strand on the lagging strand it'll come off and pluck this portion here okay that rna primer and when it plucks off the rna primer it still has a what remember this is the three prime end what would this be here five prime three prime so remember five prime this is a three prime end right here it still has a three prime n that it can use to build off of and make dna at this point here but watch what

happens down here comes down to this end here plucks off these rna primers uh oh do i have a three prime end that i can add off of somewhere i don't dudes why because look this is my five prime end i have no three prime end here that that dna polymerase can add on nucleotides to so dna replication won't occur at these points here and that is problematic because guess what this was the old dna strand you replicated and made a new dna strand this one's going to get shorter it's shorter than the original one

guess what happens when this one replicates it'll get the new strain will be shorter than it and then the next one and the next one and the dna will continue to get shorter and shorter and shorter and eventually start involving those genes so thank goodness in particular cells where we have a need a lot of replication to occur we have a special enzyme that comes in and says hey i'm going to elongate those telomeres for you so that way whenever dna replication does occur you don't start really taking away too much of the telomeres and

involving these really important genes so what is the name of this special enzyme that we should give great thanks to this wonky looking enzyme here has two arms and this enzyme is called telomerase and telomerase is a really interesting kind of ribonucleoprotein one arm comes here okay so remember we're just looking at this portion here so we're only zooming in on this lagging strand about right here okay and zooming in on it so here we're on this portion where we didn't finish and synthesize the nucleotides because again we didn't have that three prime oh region

here to add nucleotides off of the dna polymerase so what the telomerase does is it takes one arm and brings that arm out and on this arm is something really really cool it expresses nucleotides and a particular type of nucleotides that you guys really need to know okay and so what are these nucleotides that it has well what it expresses is complementary nucleotides that are commonly seen on the telomeres telomeres always have and the easiest way there's a mnemonic to remember them telomeres always have a particular repeat of nucleotides on them on their three prime

end and the easy way you can remember that repeat is the mnemonic tell them all genes gotta go that is the repeat that you constantly see on that three prime end of that parental dna so what telomeres does is it comes in and says hey i have all the complementary rna nucleotides to this sequence that's commonly seen here so let's kind of write down what would the complementary portion be if it was t it would be a t it would be a a it would be u g all the way across it would be c

c c so it expresses that with its one arm the other arm is really cool the other arm will then use this rna strand as a template to make dna that's complementary to it so if it does that it's going to take this rna read it and then what would be the complementary t t a g g g this is really interesting you want to know why i elongated my three prime n okay which is important i elongated it so that way next time this dna replicates i won't really take too much of the dna

and involve those genes because the telomerase but what i did is i used rna and from that i made dna i need you guys to understand what that's called what is that called when you go from rna to dna reverse transcription this is called reverse transcription and so what this kind of telomerase does in a way is it has again it's a protein with it which expresses nucleotides it expresses rna and then it has this other arm this other arm that reads the rna and says oh okay this is a i'm going to make t

on the parental strand that's a i'm going to make t on the parental strand so it can take rna and make dna elongating the telomeres why would we want to elongate the telomeres we obviously know to prevent gene loss and so we don't shorten those telomeres significantly and what cells would you want there to be a lot of telomerase enzymes are a lot high activity highly replicating cells cells that are replicating so much that those telomeres would shorten if we didn't have it so this is important you need lots of telomerase enzyme and what kind

of cells primarily in like stem cells so if you're like if you're a zygote and you're starting with one cell you need those cells to have lots of telomerase activity to replicate and make the whole human body or your hema hematopoietic stem cells which are making red blood cells white blood cells all those different things you need those to be able to replicate and have enough telomerase enzymes so that it can replicate without hitting and losing those genes that's important one last thing clinical point telomerase what if we figured out a way cells certain damaging

and really nasty cells figure out a way to evade uh the cell replication where they can just continue to keep replicating without not being able to stop what are that what is that called cancer neoplasia so cancer cells you know what they can do that we believe that they can do is they upregulate the activity of their telomerase enzymes and if they up regulate the activity of their telomerase enzymes they continue to elongate the ends of the dna on the chromosomes which allows for them to continue to keep replicating and replicating and replicating without shortening

the telomeres enough that it starts to involve genes within that cells that's really interesting so really big thing i need you guys to take away telomeres shorten with every dna replication we can prevent that with telomerase enzymes which perform what kind of process here reverse transcription use one arm which is rna and build dna on the parental strand to elongate that and what types of cells normally would you see lots of telomerase activity normal stem cells highly replicating cells in our body or cancer cells which dysregulate or upregulate the telomerase enzymes all right ninja nerds

that covers everything for dna replication all right ninja nerds so in this video we talk about dna replication i hope it made sense and i hope that you guys did enjoy it alright engineers as always until next time [Music]