this lecture will cover replication as it relates to the DNA strand so let's get started um as you see here on the screen replication is essentially what we're using when we're trying to duplicate or make another copy of our DNA strand so we're going to use that original DNA template as kind of like a guide or a blueprint as we make more copies so you've copied something before you'll have an original copy which will be your DNA strand and then you're going to use that strand to duplicate it or make a brand new strand based off of that original so let's talk about a few points before we get too deep into the process with DNA replication we have a starting point and that starting point is actually called the origin of replication so this is the particular place or opening where replication begins so you have seen a strand of DNA at this point of course course is double stranded and if you look here let's say replication will begin at this point that will be called your origin of replication as we start to unwind or pull apart those two strands we are going to create a space called a replication bubble so you see here as we're opening the strands we're unwinding the strands we're creating this replication bubble on either side of the replication bubble you're going going to see these Forks so just like a fork in the road where the road will split you'll see each end of the bubble has a replication fork um and DNA replication is going to proceed outward so they'll proceed towards the fork so you see right here this is the replication bubble you're going to have replication moving to the left towards one fork or to the right towards the other Fork so it's going to be imagine pull pulling those strands apart if you had a finger here and a finger here pulling them apart the bubble will start getting wider on either side and that's where the replication will occur so go to the left over here and then it'll go to the right over here so for bacteria they usually have a single origin of replication that's because their back um excuse me their DNA is usually circular so it has the ability to be able to you know just literally start in one place and go around the circle but our DNA is very very long it can be up to 3et of DNA per cell so we typically have multiple origins of replication it's not just in one particular area so DNA replication is actually a little complex so I want to talk about it um and before we get too deep into it we got to talk about some of the proteins that help to make it possible so I have a little picture to the right and we're going going to I'm going to give you some definitions and explain it and we'll continue to refer back to this image so the first protein or enzyme because you see the uh ASC at the end is called DNA helicase so DNA helicase is going to bind to one strand and it's going to travel from five Prime to three prime to actually break those bases apart so a few reminders DNA does replicate only in the five Prime to three prime Direction so that's very important we've already talked about it but it only replicates from five Prime to three prime um and the DNA helicase is like a zipper so you understand what a zipper looks like right you have um let's say you have a a hoodie or a jacket that has a zipped up portion think of the DNA healer case is being the zipper that is actually going to unzip those two sides um so you have to ask yourself what is it breaking it's breaking the base pairs apart so remember the a will interact with the T or the G will interact with the c but they're held together by which Bond a hydrogen bond I hope you said that so a hydrogen bond is going to be what holds them together right remember a hydrogen bonds are the weakest bonds but our DNA helicase will help to separate those two strands because we need to open the strands up so that we can actually copy their code and then make new strands but for DNA helicase to work we need another enzyme called DNA Topo isomerase so DNA Topo isomerase moves a little bit ahead of the zipper or the helicase to stop the DNA from coiling amongst itself um so the best example I have is um if any body knows how to braid hair if you're braiding a long braid you actually have to use your fingers to to run through the hair at the bottom of the braid so that it doesn't tangle um if you don't know how to braid hair then I'm just reminding you that as we start to unzip this portion of the DNA the bottom portion of the DNA starts to kind of wind on itself and it will become a big knot if we don't have something to kind of smooth it out out so that's what the DNA Topo isomerase is doing it actually moves a little bit in front of the DNA helicase to try to stop the DNA from coiling or creating some type of knot and that coil that it would create is actually called a super coil so DNA Topo isase is usually going to be first and then you're going to have your DNA helicase after it um which is doing the actual unzipping or unbinding between that DNA molecule and then you have another set of proteins called single stranded excuse me single strand binding protein so ssbp and this has a big job in making sure that the strands that you separated don't stick together again so imagine a zipper going down the DNA once it zips the DNA if this area doesn't have any buffer between it or any type of bumper there it is going to readhere it's going to stick together again so by putting these little single stranded binding proteins on the Strand it's going to make sure that the Strand stays separated and they don't rebond um after the helicase and the Topo isomerase move down the DNA strand so again this is just a really big picture of everything again our direction of replication is going to the left remember every time you see a fork your replic will go in that direction um you have the Topo isomerase that's going to be the first protein to help keep it straight you have DNA helicase which actually does unzip the the strands and then you have single stranded binding proteins which will stick on the Strand and help to keep it apart so there's some more proteins that we need um after we have the DNA strand opened we need to start adding some proteins that are going to make the New Strand so remember DNA is semiconservative we have the original or parent strand here in red and we will be making a brand new daughter strand under that if you're unfamiliar with that make sure you look at the previous lecture videos so as we put together those nucleotides as we start to build our strand a t GC we're going to need an enzyme that will form a CO bond to link together those nucleotides that enzyme is DNA polymerase so as we build our new D Strand and do a t g c c all of those things together that is going to require a DNA poase the nucleotides are added to the three prime n because remember DNA goes from five Prime to three prime so we have to add it on the three prime end because it's starting at five and it's going down to three so we're adding those nucleotides to the three prime end always there is another enzyme called DNA primase and if anybody has um either painted before so if you paint you might have heard of something called a primer or if you use makeup you may have heard of something called a primer both of these have a role in putting either a CO of coat of paint down like white paint if you're painting a wall or you'll put the primer down before you add your found foundation for makeup all of these have a role in putting something down first before you add what you want whether it's paint or foundation so DNA primase does the same thing it adds a short set of RNA nucleotides or a primer on the Strand so that DNA polymerase can start so DNA polymerase will start to add the Strand AB b c um sorry at GC all of those nucleotides but before it does it needs to have some type of primer on that strand that's because DNA polymerase cannot start on a bare strand it needs to have something down before it starts coly linking those nucleotides together so again I'm just showing you a picture here let's say the red is the parent strand um in this the red is the parent strand you need to put a primer that's yellow you need to put a primer down so that DNA polymerase can start adding some blue nucleotides right so you put your primer down using DNA primase and then from there you can start adding nucleotides to that three prime end so again DNA primase puts the primer down that's made up of RNA nucleotid and then DNA polymerase can start adding those nucleotides in the five Prime to three prime Direction you see the arrow is going from five Prime to three prime that's what's happening here okay so let's talk a little bit about how we actually make that strand so I told you about all the proteins you need to be be able to break apart the Strand and get the Strand started but we're we're going to have a little bit of a challenge because of two rules one of those rules is that this DNA polymerase cannot begin its synthesis on a bare strand so we need to add a primer every time we start synthesizing new nucleotides from DNA polymerase and the other issue is that DNA polymerase only works in a five Prime to three prime Direction so it will only make new or it will only add new nucleotides as it moves from five Prime to three prime so this is going to give us two types of strands that we have to make every time we replicate our DNA one is going to be called the leading strand this one is going to be very simple simple the other one is called the lagging strand lagging means it's a little slow it's behind so we're going to talk about how to make those two strands okay so let's talk about that leading versus lagging strand again I want to remind you that our DNA can only duplicate in the five Prime to three prime Direction um I'm going to try to draw just a little bit it's going to be very challenging with um my lack of software right now but I'm going to try to draw just a few bits of information that I think may help you um understand this one is the direction of replication I'm drawing this with The Mouse and the other is reminding you about the direction of the door strand so that's a five that I drew here and then that's a three that I drew here so let me switch back to my pointer and hopefully this will make some sense okay so for our leading strand let's first just look at our DNA molecule that's right here our parent strand is in Pink So if the top side of the parent strand is five Prime that means this side of the parent strand is three prime and because DNA runs anti-parallel like how 95 North runs right next to 95 South is flipped then then the bottom strand is five Prime here and then on this end it's three prime here so that's the parent strand the pink strand you see the five and the three are opposite from one another same thing with this five and with this three so our new daughter strand has to match those same rules so our new daughter strand for the bottom parent strand it has to be five down here and then this will be three here here that's for the bottom parent strand for the top parent strand if this one is a five Prime anti parallel says my new daughter strand will be three prime here and then if it's three prime here my new daughter strand will have a five Prime you know here or on at least this side of the Strand it would be five Prime so keep those things in mind make sure you're always very aware of how that anti-parallel directionality works so like I mentioned this is our replication fork so every time we replicate we need to be replicating towards the replication fork so the DNA replication is actually going to the right that's why I tried to draw this a little bit if you're looking at this you want to identify the leading strand the leading strand is going to be the Strand that has these following rules if it is a strand that is synthesized in one long molecule it runs continuously meaning it doesn't stop and it also has the ability to attach to the uh thre Prime end of the molecule because if this end is three prime the new door strand will be five Prime so you see here in this picture how the parent strand is thre Prime and the door strand up top would be five Prime that's a good clue to let you know that this is the leading strand so you can tell it's a leading strand you'll put the primer down on the five Prime in and then you will just have your DNA primase I'm sorry DNA polymerase just move on along and continue to synthesize one long continuous molecule so you see as it continues to replicate it's going to make one long molecule so what's on the bottom here you look at uh this second diagram it's on the bottom no problem the third diagram it's on the bottom no problem so this is going to be your easiest strand is the leading strand always on the bottom no it depends on how the parent strand has its five Prime and three prime ends if the parent strand had these numbers flipped and you had three prime five Prime on the top and five Prime and three Prime on the bottom then your leading strand would be on the top part of that molecule it's going to follow these rules it's going to be attached to the parent uh three prime to five Prime n because it will make an opposite five Prime to three prime n for the door strand um and it's going to be in one long molecule and run continuously that is your leading strand if you need to rewind that part if you need to restart it please do okay the lagging strand runs discontinuously I'm going to explain why as it runs discontinuously instead of having one long molecule it is made up of many many small molecules called okazaki fragments and this was the scientist who discovered it um Dr okazaki so these okazaki fragments consist of a DNA primer and new DNA once you make these okazaki fragments you have to to glue them or connect them together using a new enzyme called DNA liase so lagging strand runs discontinuously which is opposite than leading it's made up of small fragments which we call okazaki fragments much different than a long molecule and then all these tiny fragments are glued together by DNA liase so let's explain or look why we have to run it backwards remember our direction of replication is to the right but DNA can only move from five Prime to three prime so in this leading strand it has no problem going from five Prime you see five running to three prime it's going five to three five to three all the way from the top or at the bottom it's five 2 three this lagging strand because the parent strand is five here our new daughter strand has to be three on this end which would mean that the right side or basically the replication area the direction we're supposed to be going is actually five Prime so the only way this molecule can move is from here to here it has to still go from five to three 5 to three but obviously that's not the direction of replication so what it does is it goes from 5 to 3 it stops and then it jumps way behind it and then it lands here and then it goes from five to three and it jumps way behind it in five to three so that's was I'll jump down to the very bottom one again we're keeping the same numbers even though they're not on the screen we're starting with the five it goes a little bit to three it jumps five to three it stops falls off starts again over here five to three 5 to three and again once you have these little tiny fragments you would then have the DNA liase come together to glue them together so again please make sure that you take the notes please make sure you listen to the words that I'm saying along with the text that's WR on the slides as well um so again this is just trying to you know show you another picture of how this occurs just for other examples again be really familiar with how these um five Prime and three prime numbers occur see if you can find where the leading Strand and lagging strand would be because we're not um you know in person I can't show you a video but there are a lot of great videos on YouTube all I did was type in Le leading and lagging strand DNA replication and this was just a few of them that is showing you kind of what's happening so you can see some of the terms we talked about primase uh liase you can see here somebody's drawing it you'll be able to see um the primer with the lagging strand versus the primer with the leading strand you see parent is five I'm sorry parent is five and three so that means the daughter is 5 to three which goes in the direction of our replication um you know all these things so please take time to be familiar with these Concepts so that if you ever see them on an exam you'll be prepared again I'm just showing you another example we're going to open up our DNA strand we have these replication forks that are in place as we identify what the numbering of our parent strand are we can then identify okay if the parent strand is three prime here that means my daughter strand is five Prime here the direction of replication is to the left so that makes sense for this to be the leading strand five Prime to three prime and then if you jump down you see on the lagging strand it has to go the opposite way but that's why we're going to start to make multiple okazaki fragments so you'll start here then you'll jump back here and then as this opens up you'll be able to go further back um and then eventually we can link them together through the DNA ligase so DNA replication is very accurate we only have a mistakes maybe one every 100 million nucleotide so that's obviously very very good um and these are three mechanisms that your body can use to make sure you maintain accuracy so one is hydrogen bonding so the bonds that form between the A and the T are very very stable the bonds that form between G and C are also stable however if you try to bond a c with a t or a g with an a it is not stable at all so that mismatch is going to help your body realize that you have had some type of issue with accuracy the second thing is the active site for DNA polymerase is not going to form bonds if they're not paired correctly so we talked about enzymes and how specific enzymes s are and they have very high specificity so if that DNA polymerase is supposed to interact with a particular nucleotide based off of those instructions it will not form um or be very unlikely to form if that is not the correct pair and finally DNA polymerase has a little bit of a backspace button so it can proofread if there are any mismatch pairs so as DNA polymerase is going along just like if you're typing a paper and you misspell something it can back up make that change and then continue to move along um we're only talking about DNA polymerase in this course but there are other DNA repair enzymes in your body that you can use to be able to correct the DNA if you've made a mistake at some point okay so this concludes the lecture that we've had on this material however if there was anything that was confusing please rewatch this lecture this is why I'm recording it so you can watch it pause it rewind it make sure that you understand and process this information if you have any questions please make sure to write them down and then during class time or office hours I will be able to help you to understand it um there is an additional lecture but please review the notes on blackboard to get directions as to where that is thank you