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DNA Replication Mechanism
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this video will focus on dna replication and the molecular mechanism by which that occurs in cells before we do that i just want to begin with a very quick refresher on the structure of dna itself because its structure and biochemical makeup is centrally important to the mechanism by which it's copied these two gentlemen are james watson and francis crick and they are famous for having discovered the structure of dna deoxyribonucleic acid not discovering dna but for discovering the structure
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of dna with the title of their paper that they published in april of 1953 a structure for deoxyribose nucleic acid and at the beginning of that paper there's just this very simple sentence a bit humble we wish to suggest a structure for the salt of deoxyribose nucleic acid dna this structure has novel features which are of considerable biological interest and you know they could not have been more right because in solving the structure of dna essentially they solved the puzzle of heredity
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and how genetic information can be passed on down the generations this was a seminal discovery in the history of biology they didn't make this discovery in isolation though they were actually building on the work of a lot of people who had gone before who had discovered some very important things about dna oswald avery and alfred hershey and martha chase definitively showed that dna is the heritable material that gives the cell its characteristics the key insight that dna has a helical
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structure was deduced based on rosalind franklin's x-ray crystallography data that a colleague of hers maurice wilkins shared with watson and crick without her knowledge or consent which is a crazy and compelling story i encourage you all to read erwin chargaff had shown that the amount of adenine that you find in a cell was roughly equivalent to the amount of thymine and the amount of cytosine was roughly equivalent to the amount of guanine
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which watson and crick thought likely meant that a and t were paired and g and c were paired but looking at the chemical structures they had drawn they couldn't figure out how until their colleague jerry donahue corrected their erroneous structures and showed them what the nucleotide bases really looked like without all of this other work they wouldn't have made their great discovery in 1962 watson and crick were awarded the nobel prize along with maurice wilkins unfortunately
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rosalind franklin died before that prize was awarded and the nobel is not awarded posthumously all of this to point out that even though we like to give credit to individuals for huge discoveries like this science is really a collaborative effort and watson and crick relied on many many discoveries that other people had made and were able to put that information together to solve the puzzle of the structure of dna as you've seen before in the course the
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most common form of dna found in cells is the structure that we call bdna which is a right-handed double helix made up of two nucleic acid strands that are hydrogen bonded with one another in anti-parallel orientation there is a phosphodiester backbone illustrated by this ribbon structure that runs along the outside of the helix with the nitrogenous bases of the nucleotides tucked away to the interior hydrogen bonding with one another but also accessible from the outside via the
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major groove double helix is referring to the fact that there are two individual molecules of dna that are hydrogen bonded with one another right handed refers to the direction of coil of the helix imagine pointing the helix away from you if it coils away from you in a clockwise direction that's right-handed if counterclockwise that would be left-handed the term anti-parallel orientation refers to the directionality of the phosphodiester backbone to which those
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bases are bonded remember that each nucleotide is made up of a nitrogenous base that's bonded to a ribose or deoxyribose sugar depending on whether you're looking at rna or dna in this case dna and bonded to the five prime carbon of this sugar we have one or more phosphate groups if it's already incorporated into the chain then it would be just one within each individual nucleic acid strand these nucleotides are bonded to one another by their sugars and
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phosphates in a characteristic directional fashion with the phosphate group of the next nucleotide in the chain being bonded to the three prime carbon position of the sugar of the preceding nucleotide and we can trace the linkages phosphate attached to five prime carbon through the sugar three prime carbon phosphate attached to five prime carbon three prime carbon phosphate attached to five prime carbon three prime carbon phosphate attached to five prime carbon and then here's our three
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prime hydroxyl group that is dangling off of the end now this is sort of implying that there's another nucleotide down here but if there wasn't then this would be a free hydroxyl group that's available for bonding by the next nucleotide to be added in the chain so the directionality of this nucleic acid on the left is five prime to three prime going top to bottom and if we look at the phosphodiester backbone of the partner strand on the right it's running five prime to three prime
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bottom to top here's our five prime phosphate three prime carbon five prime phosphate three prime carbon so the directionality here is going from the five prime end of this backbone to the three prime end and again up here we would have our free hydroxyl group available for bonding to the next nucleotide to be added this antiparallel orientation allows the phosphodiester backbones to pack close enough to one another to allow this hydrogen bonding interaction and we discussed the specific base
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pairing earlier in the course adenine pairs with thymine and guanine with cytosine the consequence of this highly specific base pairing interaction is that if you know the sequence of one strand of dna then you can automatically predict the sequence of the opposing strand of dna watson and crick immediately recognized this highly specific pairing interaction would very likely be used by the cell as a means of copying the dna which is what we mean when we're talking
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about heredity at the cellular level it's dna being copied and passed on to daughter cells through cell division towards the end of their very short one page paper watson and crick have this sentence it has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material and of course they were right we now know the mechanism by which dna is copied and it's exactly what they predicted the two parent strands would be
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separated and each would serve as the template for synthesis of a new opposing complementary strand by the base pairing rules after replication each double helix consists of one old and one newly synthesized molecule of dna so this is referred to as semi-conservative dna replication here we're looking at one old parental strand being copied the two strands of the parent dna would have been separated here we're looking at just one of those strands in this case the
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strand that runs from five prime to three prime bottom to top it's serving as a template for the enzymes which are called polymerases that will copy this and produce the new opposing strand there's a whole pool of all the possible nucleotides in the cell and the enzyme will look for the nucleotide that matches up correctly by hydrogen bonding opposite this t there will be an a opposite this g there will be a c opposite this c there will be a g
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and so on as we talked about before the way that this incoming nucleotide is going to be added on to this growing chain is by a new covalent bond that's going to be formed between this first phosphate on this incoming nucleotide and the three prime carbon of the sugar of the preceding nucleotide this covalent bond holding these two terminal phosphates on is going to be cleaved and the first or alpha phosphate is now going to be joined to the three prime carbon of the sugar here
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joining it to the growing nucleic acid strand the two terminal phosphates are going to leave as a pyrophosphate group this set of reactions is catalyzed by enzymes collectively called dna polymerases rna polymerases catalyze essentially the same reaction but during transcription of dna to produce rna all known dna and rna polymerases have evolved to only synthesize nucleic acids in the five prime to three prime direction in other words adding nucleotides onto
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the three prime end this imposes some limitations and causes some difficulties when we start thinking about copying dna since dna is double stranded and these two strands of parental dna run in opposite directions which means that the two strands have to be copied in opposite directions we'll come back to that a bit later an important thing to recognize about dna replication is that it's going to begin at specific sequences in the genome called
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origins of replication if we look at a simple prokaryotic cell like an e coli bacterial cell what we would find is that the genome of that organism is maintained in one circular chromosome which consists of approximately 4.6 times 10 to the six base pairs so about four and a half million base pairs or so and all of that dna can be served by one origin of replication which in e coli is referred to as oric we would first see separation of the two parental strands illustrated in dark blue here
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forming a replication bubble and we would then see synthesis of two new daughter strands here in light blue that are complementary to these two parent strands that replication would be bidirectional proceeding in both directions away from that origin with both strands of the dna being copied at the same time this is illustrated by both of these dark blue parental strands being paired with a light blue newly synthesized daughter strand the same thing over here on the other
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side of the replication bubble as this proceeds what happens is this replication bubble gets bigger and bigger as the parental dna is separated here on both sides at what's referred to as a replication fork this is the point where the parental dna strands are being peeled apart so they can be copied so you have two replication forks for each replication bubble and synthesis would be going all the way around this chromosome in this direction all the way around the chromosome in
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this direction and eventually that bubble is going to expand and get to the other side of the dna you would then have two double-stranded dna molecules two chromosomes intertwined and they would be enzymatically separated to yield two completely separate daughter dnas each of which would be segregated to a daughter cell during binary fission if you move to the eukaryotic world we know that eukaryotic cells in general do not have circular chromosomes they have
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multiple linear chromosomes for example a yeast cell like saccharomyces cerevisiae or baker's yeast has a genome consisting of 16 linear chromosomes and 12 times 10 to the sixth base pair so 12 million base pairs of dna replication of those linear chromosomes would be served by 400 replication origins and if we look in human cells we would see an even more complex genome with 6 times 10 to the 9th base pairs of dna distributed across 46 linear chromosomes being served by 40 000 replication
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origins specific sequences distributed along those 46 linear chromosomes each chromosome would have multiple origins of replication each of which would be opened up to form a replication bubble and we have two replication forks at each of those replication bubbles moving in opposite directions with both strands of parental dna being copied at the same time and newly synthesized strands being created opposite them so for example you'd see this
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replication bubble expanding out in both directions and likewise for all of the other replication bubbles along that linear chromosome and they would keep expanding until eventually they'd run into one another and again we would have two completely distinct new dna daughter molecules each of which is composed of an old parental strand and a newly synthesized daughter strand these would be the sister chromatids you learned about earlier in the course
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which would be held together by cohesins until separated in anaphase of mitosis or meiosis now that we've had a general overview of what happens during dna replication we're going to delve into the details what are the enzymes and other proteins required to make this happen and how do they accomplish this work the mechanism by which dna replication occurs in a prokaryotic cell like e coli and a eukaryotic cell like a yeast cell
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or a human cell is virtually identical but what happens in e coli involves a lot fewer specific molecules and enzymes so we're going to be looking at dna replication as it happens in e coli because it's a simpler model of the process everything that we learn about can be applied to eukaryotic cells it's just that the complexity and the number of proteins and enzymes and other players would be much greater let's zoom in on one replication origin and even more specifically to one
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replication fork that's proceeding from that origin in one direction so just to make sure you're understanding what we're looking at here we're talking about a replication fork where the two parental strands of dna are being separated from one another and this fork is opening up the parental dna towards the left and there'd be another fork over here moving to the right after the origin has been opened up we'll see an enzyme called helicase come in
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represented as this green blob and this is the enzyme responsible for unwinding the two parent strands peeling them apart so that each single strand can be copied remember that what's holding these two strands together is hydrogen bonding interactions between base pairs so what would happen if this helicase came through and separated the two strands and then just moved on down the line well those two parental strands would quickly re-anneal those hydrogen bonding interactions
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would reform and to prevent that from happening there's a set of proteins called single-strand binding proteins that bind to single-stranded dna and prevent re-annealing during replication as you can imagine this peeling apart the two strands of dna causes a dramatic strain on the dna helix ahead of that stress being applied this can induce super supercoiling and even tangling of dna ahead of the replication machinery there's a whole set of enzymes
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collectively called topoisomerases that relieve the stress and untangle dna so that the replication machinery can continue copying now the situation is ready for enzymes to come in and start reading the dna sequence and laying down a new strand opposite that parental strand a key limitation of dna polymerases is that they cannot just sit down on dna and start copying and building a new dna from scratch all they can do is add on to an existing strand of dna and so they have to have some sort of
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primer a short piece of nucleic acid to get them going this primer is created by an enzyme called rna primase primase synthesizes a short segment of rna just 10 to 20 nucleotides long and once that primer has been laid down now you can have a dna polymerase which i'll just abbreviate as dna pall bind to the end of that primer and start adding nucleotides onto that three prime end of the primer there are numerous dna polymerases some of which repair dna and
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some of which are involved in dna replication in e coli there are two specific dna polymerases that are involved in replication dna polymerase 3 and dna polymerase 1 and they do different jobs during replication dna polymerase 3 is the main replicative polymerase it's responsible for doing the vast majority of the dna synthesis during dna replication it extends the rna primers with dna pol3 is a very large complex of more than 15 individual proteins or
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polypeptide subunits that come together to form the overall enzyme that we call dna polymerase iii there are four subunits that i want you to know the functions of and those are the alpha subunit the beta subunit the epsilon subunit and the tau subunit each of these subunits has specific functions and or enzymatic activities that contribute to the overall process of dna replication the alpha subunit is what actually synthesizes the new dna
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it's an enzyme with 5 prime to three prime polymerase activity this enzyme moves along the parental template strand and synthesizes a new strand opposite that template incorporating nucleotides using the base pairing rules in the five prime to three prime direction the beta subunit is shaped like a doughnut with a hole in the middle that the dna goes through this subunit clamps the polymerase complex onto the dna that is being copied
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and increases the efficiency or processivity of the replication process if the beta subunit clamp did not hold the rest of the complex onto dna then every 20 to 50 nucleotides this whole complex would fall off and copying would come to a halt and it would have to be reloaded back on the beta subunit prevents that from happening the epsilon subunit is also an enzyme but it has a very different enzymatic activity from alpha it's actually a
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three prime to five prime exonuclease nucleases are enzymes that degrade or break apart the covalent bonds between nucleotides in nucleic acids an exonuclease would chew from the ends exo meaning outside an endonuclease would break covalent bonds within or internal to the nucleic acid endo meaning inside the epsilon exonuclease is a three prime to five prime exonuclease so it specifically recognizes the three prime end and chews way from that end towards the five prime end of the molecule
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this subunit functions as an editor of the sequence it deletes mistakes when incorrect nucleotides get incorporated into the new strand the polymerase can recognize that the shape of the molecule is wrong and remove the recently added nucleotides we'll come back to this in a minute the last subunit you should know something about is the tau subunit and this subunit tethers the two polymerases copying the two parental strands of dna together so that their activity is coordinated
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at each replication fork there are two strands of dna being copied each has its own alpha beta and epsilon subunit and the tau subunit holds these two complexes together for this class we won't worry about the functions of the rest of the subunits some of which aren't even known in any case so that's the structure and properties of dna polymerase iii what about dna polymerase 1 the function of dna polymerase 1 and dna replication is very different remember that each newly synthesized dna
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starts with an rna primer that rna has to be removed and replaced with dna and that's the dna that paul 1 is responsible for making it's only responsible for synthesizing short sequences of dna 10 to 20 nucleotides long once rna primers have been enzymatically removed polymerase 1 is a single polypeptide one protein but interestingly that one protein has three different enzyme activities two of which are used during dna replication
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there are three different active sites that catalyze three different reactions like dna polymerase iii pal1 has a five prime to three prime polymerase active site that allows it to synthesize dna matching up the sequence of the new strand to the parental strand template according to the base pairing rules also like dna polymerase 3 paul 1 has a 3 prime to 5 prime exonuclease activity which is very important for that editing or proofreading function in case of mistakes
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it also has a five prime to three prime exonuclease activity that's not used during dna replication but that's very important for dna repair dna polymerase 1 is a critical enzyme that participates in a lot of the dna repair pathways that are going on in e coli now depending on the text you may still see references to this 5 prime to 3 prime exonuclease being used to remove the rna primer but that's incorrect the primer is removed by a separate protein called
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rnase h an exonuclease that binds to the rna dna hybrid molecule and chews away the rna portion let's talk a little bit more about this three prime to five prime proof reading exonuclease activity and its importance during dna replication why is it needed it turns out that the error rate of the polymerase activity of dna polymerase is unacceptably high and without proofreading the cell would incur thousands more new mutations in every round of replication
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on its own the polymerase activity of prokaryotic dna polymerase has an error rate of 10 to the minus 4th to 10 to the minus 5th meaning the polymerase makes a mistake every 10 000 to 100 000 nucleotides with a genome that's over 4 million base pairs long that works out to over 40 to 400-ish new mutations in the genome per replication the proofreading function of polymerase reduces that error by two to three orders of magnitude and dna repair after replication reduces the mutation
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load another two to three orders of magnitude so how does this proofreading function work this figure is illustrating a polymerase enzyme you can visualize this as a hand wrapping around the dna that's being copied in this case this is our parental template strand being read three prime to five prime in this direction and this is our newly synthesized strand in the lighter blue here's the active site that has the polymerase activity it's reading the sequence of nucleotides
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in the parental strand and synthesizing a partner strand opposing that by the complementary base pair rules so if this is a t it's going to be adding in an a if this is a c it's going to be adding in a g extending this three prime end of the growing strand as it synthesizes but let's say there's an a in the template and the polymerase activity makes a mistake and adds in a c in the new dna that's a mismatch c and a won't hydrogen bond in the correct pattern and this enzyme is going to be able to
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feel that the pair doesn't feel right because the two are not going to match up in the way that they should if they had true complementarity in their hydrogen bond donors and acceptors the structure of this bit of dna sitting in the palm of this enzyme's hand won't feel right and when that occurs the enzyme undergoes a shape change that flips the end of the newly synthesized dna down to the other active site which has the three prime to five prime
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exonuclease activity which will chew away the nucleotides that were just incorporated or to the epsilon subunit if we're talking about dna polymerase 3. how many nucleotides get chewed away is sort of random it's on the order of 5 to 10 maybe even 15 nucleotides it's kind of like the delete button on your computer delete delete delete it removes the mismatch then the three prime end gets flipped back up to the polymerase active site
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and this enzyme gets a do-over the enzyme picks up where it left off and hopefully gets it right this time and doesn't misincorporate nucleotides we'll talk more about replication errors and other mutation sources as well as dna repair in a later video now that we've had an overview and we've looked at the molecular players and we know the enzymes and other proteins that are involved in the process of replication let's revisit the problem that i alluded to earlier
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involving the anti-parallel orientation of the two strands of parental dna and the fact that dna and rna can only be synthesized in the five prime to three prime direction let's say we have an origin of replication with the parental strands of dna illustrated as the dark blue and we add some directionality onto this so let's say this is the five prime end and this is the three prime end we know that because of the antiparallel nature of our dna double helix
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that this must be the three prime end of this strand and this must be the five prime end of this strand a replication bubble opens up at the origin and primase binds and synthesizes those short rna primers to start the process off which we'll represent in red with the arrowhead showing the direction of synthesis 5 prime to three prime the rna primer synthesized on the top strand will have to be built five prime to three prime from right to left to be anti-parallel
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to the template this rna primer on the bottom strand will have to be built five prime to three prime going left to right to be anti-parallel to its template so synthesis has to go in opposite directions hopefully you can see where this is going we know what happens next is dna polymerase 3 is going to bind to the end of the primer and extend it with dna so on both of these primers we'll see that happening a helicase enzyme gets loaded on at each
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replication fork one going left and one going right and they open up the dna expanding that bubble this dna polymerase iii copying the top template strand continues tracking right along behind the helicase synthesizing new dna the pol3 on the bottom template strand tracks right along behind the helicase moving in the other direction these two strands that are copied continuously because they're being synthesized in the same direction the helicase is moving
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are referred to as the leading strands but can you see the problem we have with the other strands as this helicase on the left has opened up this replication fork a bit more now we've got another segment of parental template dna that needs to be copied but synthesis can only happen from 5 prime to 3 prime which is left to right for this one so how does this get accomplished as soon as this single stranded template is exposed primase will bind and lay down another
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primer again synthesizing from five prime to three prime left to right dna pol3 will again bind to the end of the primer and extend it with dna until it runs into the previously synthesized dna ahead of it and we just keep repeating that process as helicase continues to separate the parent strands new primers get laid down and extended as these discontinuously synthesized fragments called okazaki fragments after regi and suniko okazaki who demonstrated that discontinuous
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synthesis occurs this strand that has to be copied discontinuously is called the lagging strand the leading strand can be copied continuously because it's being copied in the same direction the helicase is moving the lagging strand must be copied in segments because it's being copied in the opposite direction the helicase is moving and of course at this other replication fork that's moving to the right the bottom strand is the leading strand and so this top strand must be copied in segments
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so it's the lagging strand this process will continue with both parental strands being copied until the entire dna has been replicated but where does dna polymerase 1 come in remember that pal1 is the enzyme that's responsible for replacing the rna primer with dna all of these bits of rna that are currently part of the newly synthesized nucleic acid have to be removed and dna incorporated to replace it first rnach nuclease chews away the primer leaving a gap now remember that all dna polymerases
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require a primer whereas pol3 used the rna primer to begin synthesis dna polymerase 1 uses the 3-prime end of the adjacent dna fragment as its primer it adds nucleotides onto the three prime end of that dna extending it to fill in the sequence until it hits the five prime end of the downstream dna fragment notice that once it fills in the missing nucleotides there will still be a gap between these two bits of dna between this three prime end with its hydroxyl group
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and this five prime end which was previously linked to the primer at this point another enzyme comes into play dna ligase to create a covalent bond between these two nucleotides sealing up this nick in the phosphodiester backbone creating this new covalent bond is an endergonic reaction ligase drives this uphill reaction by coupling it to atp hydrolysis which as we've talked about before is highly exergonic once all the gaps in the backbone have been connected
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dna replication is complete and the two intertwined dna double helices can now be disentangled and segregated to different daughter cells cool as it is we won't go into that process in this class and there you have it replication is now complete again we've looked at the process as it occurs in e coli which is a bit simpler than what happens in eukaryotic cells but the basic mechanism of replication is the same in both in this video i've reviewed the structure of dna the molecular mechanism
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of nucleic acid synthesis and the major protein players and process of dna replication as it occurs in prokaryotic cells in the next video i'll talk about some replication related issues that are unique to eukaryotic cells related to their more complex chromatin structure and the fact that eukaryotes have linear not circular chromosomes
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