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Genome Replication & Central Dogma
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0:15
so we're gonna get started this is this is a complicated lecture to choreograph but I'm gonna do my very very best because I think it's there's some pretty amazing stuff that we have to explain that is carried out in nature and one of those things is how do we replicate the entire genome of organisms in one fell swoop almost perfectly sometimes there are little errors and fast so we're gonna refer to these numbers in a moment but first of all I just want to get you back
0:47
in the picture of where we were at the end of the last section the biochemistry section and in the next few classes we're going to address issues related to the central dogma and the use of nucleic acids for information storage and information transfer and before I do that I just want to highlight a couple of things just in terms that you should recall and recap from the biochemistry section about nucleic acids so nucleic acids form complimentary double strands through base pairing and that base
1:23
pairing involves hydrogen bonding between the nucleobases one purine to one pyrimidine and the a T base pair is worth two hydrogen bonds the GC base pair is worth three hydrogen bonds and those are actually pretty useful facts to remember because they tell us about the stability of double-stranded DNA where it's easier to tease it apart and other characteristics so it's kind of a useful thing to keep in mind so the base pairing between what's the purine pyrimidine pair which sets the exact
1:59
register down the double-stranded DNA that the backbone is the exact distance apart because you always combine a small pyrimidine with a much larger purine so that's something you should feel comfortable about the components are organized in an anti parallel orientation so one end is good runs 5 prime to 3 prime the other end runs 3 prime to 5 prime and I showed you last time an image that allows us to state quite clearly that the anti-parallel orientation is significantly more stable than the
2:37
parallel orientation because you can't make all those great hydrogen bonds well in the parallel organization apart from everything else that becomes complicated another important thing that you're going to have to remember specially in this lecture is that we add new nucleotides to the 3 prime end of a nucleic acid and so I'm just gonna put this little guy in the corner here this is the ribose sugar that would be where the nuclear base is attached and it's a deoxy sugar so there's not no
3:16
substituent at that carbon this is the one position where the base is the 2 position where it's deoxy the 3 position and these are all prime numbers remember the sugars have prime numbers the bases themselves have numbers without primes to distinguish what we're talking about so 3 4 5 and they're all prime and when we grow the the single strand of DNA we always add new basis to the 3 prime end that's a an import enum burring system so we have that piece of chalk so we
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always grow from 5 prime to 3 Prime oops
4:08
and that will be important to you as we go through the discussion today and then finally a feature of DNA quite double-stranded DNA quite unlike proteins which sometimes you'll thermally or with pH melt out and will often have a problem reforming their reliable structure because instead of the structure refolding they aggregate double-stranded DNA doesn't aggregate because it's got all their negative charges that would be quite repulsive it would be very difficult for DNA
4:43
strands to aggregate as such because there's too much of a concentration of negative charges that would be repulsion amongst the same type charges so DNA can be peeled apart with heat and it will really to its partner complementary strand once you get to six seven eight base pairs that is a nice interaction between the strands and it forms faithfully if we have a mistake in the strands the stability of the double strand would be a bit less and we can measure that through physical
5:15
measurements that we carry out in the lab so we can measure the denaturation or the thermal melt temperature so we call this interaction we call this process rather after you separate double-stranded DNA we call it reeling or it's also designated as hybridization to form a hybrid which is the composite of the two structures those terms are used fairly interchangeably all right so the goals in the next four class is to show you how the structures of the nucleic acids really a purpose for the
5:51
sorts of processes that they undergo so the replication of DNA the conversion of a strand of DNA into complimentary messenger RNA once the process of protein synthesis needs to be initiated that second step the first step is called replication the second step when you transcribe DNA into RNA is called transcription and then finally when you translate messenger RNA into proteins so going to a completely new language we call it a translation and that's the basis of these of these lectures there's
6:30
a little bit of stuff in these lectures about more complicated issues in the mammalian cell where we have to process the messenger RNA a bit before we can have it leave the nucleus and I'll explain the time at there when it comes so this is the lineup for the four lectures all right so what I already talked to you how when you add I mentioned it here when you add sighted een triphosphate gtp ATP TTP these are the activated nucleobases so they're the deoxy nucleotide
7:07
nucleoside triphosphates they are nucleotides because they include phosphate but when we describe them we'll call them nucleoside triphosphates if we're gonna mention the phosphate part and I know that probably doesn't make any sense at all right is everyone okay with that if you're just generically describing something with a phosphate in it you call it a nucleotide if you want to be a little bit more specific you say nucleoside and then you say how many phosphates alright and so
7:40
for the building blocks it's a nucleoside triphosphate and believe me I get that wrong all the time and I my students correct me so I don't have a problem if you if you're not perfect with that nomenclature so these are the building blocks for DNA for polymerization and I just showed you how you grow from the 5 prime end that numbers not shown there but it's shown over here and you add to the 3 prime end and the convention remember when we describe a strand of DNA because we make
8:14
it 5 prime to 3 prime we write it 5 prime to 3 prime just so that we're all consistent and we know what we're talking about now I want to talk about 2 experiments but enlist the use of isotopes because these were quite useful earlier early on to describe some of the characteristics of replications so some of the details of replication and also the fact the DNA was the genetic material okay so isotopes are elements that share the same number of protons and electrons but they differ in the
8:51
number of neutrons okay so the common isotopes of the elements that are in all the covalent structures of the body are hydrogen
9:06
carbon and what I'm putting is the number next to it is the common isotope so it's the number that number designates the sum of the number of neutrons and protons so carbon-12 phosphorus 31 I should have put nitrogen here let's see wait I mean I'm gonna put these in order because it makes more sense phosphorus 31 nitrogen 14 phosphorous 31 and sulfur what's the sulphur isotope 32 so these are the common isotopes that we that comprise the majority of those elements
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within the body but there are lots of experiments done that have different isotopes of these atoms that we can use as tracers or markers kind of the same thing but you'll see when I mentioned the word tracer to me it brings up the radioactive isotopes because we use very little of them so when we talk about these elements there are different isotopes that we use commonly there's the hydrogen that has an extra Neutron that's also called deuterium and then there's the hydrogen that is radioactive
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it's metastable it will decay and emit a radioactive particle so this one we would call it the heavy isotope and this one we would call the radioactive isotope so we could trace certain hydrogen's in biomolecules by either making those hydrogen's the heavier ones or making them the radioactive ones carbon also has useful isotopes carbon 13 is the heavy isotope and carbon 14 is the radioactive isotope so they're used quite commonly and there things get a little bit different for
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nitrogen we use a heavy isotope of nitrogen which is n15 just one extra Neutron but while there is a radioactive isotope of nitrogen it's too short of a half-life to work with in the lab it's not useful to us so we never talk about it but we I will describe to you an experiment with the heavy n15 when we talk about the mechanism of replication and then for phosphorus and sulfur the most important ones are P 32 which is a radioactive phosphorus and s 35 which is
12:00
a radioactive sulfur there's another radioactive phosphorus that's P 33 has a slightly longer half-life it's kind of handy to work with if you have certain experiments the half-life is very a lot but the half-life of tritium c14 a long that's why we do carbon dating the half-life of P 32 a short you know less day time frame and sulphur 35 is a little bit longer but those are nuances that you don't you don't need to worry about now how are these isotopes useful
12:33
as tracers and markers to tell us about biology and details of biology so what I'm going to just show you here is a particular experiment that's carried out with what are known as baculovirus so viruses infect eukaryotic cells baculovirus --is infect bacteria so there's a commonality there and when baculovirus has infect bacteria just as the parallel with eukaryotic cells they deposit material into the bacteria so they can replicate so they can hijack the bacteria to make new viral new
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baculovirus so what an early experiment was done to ask what is the genetic material that transfers the information from the baculovirus to the to the bacterial cell in order to make more baculovirus right so they were able to demonstrate in this Hershey and chase experiment that you could raise you labor you could label protein and DNA with particular isotopes and find out what part of the baculovirus was important for transferring information for the production of new baculovirus so
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they wanted to label the capsid protein so the proteinaceous material that's on that's on the outside of this lunar lander this is a baculovirus it's um it's sort of amazing how it looks so I wanted to label the protein but they also wanted to label the contents of the baculovirus the DNA with tracer Radio isotopes so what would be the best isotopes to use if you wanted to differentiate between protein and nucleic acid and what you want to think about is what does protein have that
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nucleic acids don't have and what the nucleic acids have the proteins don't have which which are the elements that are important for differentiating yeah over there yes phosphorus going do you want to give a little bit more of a with
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okay so the answer is we use phosphorus to label nucleic acids because that phosphodiester backbone it's rich in phosphorus there are a few phosphates in proteins but they're very transient they're part of signaling but the boat you know every nucleotide has phosphorus in every building block and then in proteins they have sulfur in them where nucleic acids do not and the sulfur is in two amino acids cysteine and methionine so you can use those two traces for the building blocks and the
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way the experiment works is if you've labeled the protein with sulfur you infect a cell the virus replicates and what you're gonna do is then centrifuge the bacterial cells to see what's in the cell and then you can alternatively label the DNA and in this case I've shown it as green but that would be the other isotope let it infect the cell and deposit its contents in the cell centrifuge the cells out and you want to know where the radioactivity is and the radioactivity is strictly associated
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with phosphorus-32 because the genetic material that coded for the production of new baculovirus is the thing that stays associated with the bacterial cell in contrast there's no radioactivity associated when you label the cells with s 35 all right nice experiment easy way to do it quite a rather a nice way to do it the other experiment I want to describe to you very briefly because it relies on centrifugation technology that's very powerful and it's a method that utilizes in 15 very very nicely
16:36
okay so let me tell you about that experiment there are things in the laboratory that we use every day Ultra centrifuges and regular centrifuges that spin at a very fast speed where we can really differentiate things by molecular mass which relates to this the sedimentation coefficients how fast does the particle spin to the bottom of the tube when it's under high centrifugal force the heavier it is the faster it will spin so the question that came up very early on it
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looks like a nonsense question now because it looks so obvious that we replicate DNA the way we do but originally wasn't absolutely certain where the DNA was replicated through a semi conservative mechanism where the strands came apart and you made two copies a copy of each strand to make two identical daughter new double-stranded DNA alternatively could you have a mechanism that was quite conservative you kept your DNA and somehow you made a copy of it a little harder for me to understand
17:39
so your two new strands one would look like the original one but one would be very different and they're all dispersive where you're just copying bits of the DNA and somehow reassembling this gigantic jigsaw puzzle the centrifugation experiment allowed people to absolutely in clearly state that the rep occasion is via a semiconservative process so let's walk through it and what was done is that the nucleotides within the DNA were all labeled with heavy nitrogen so for every nucleic acid
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let's say for example when there's an adenine in the backbone there are five nitrogen's so that would be five atomic mass units heavier the nitrogen 14 in that nuclear base and 15 nuclear base five mass units heavier than n 14 nuclear base makes sense to everybody all right so it's heavier by some amount it's not a massive amount but it's enough to differentiate in a centrifugation experiment so the bacteria will first grown up in heavy nitrogen so all of the DNA sediments at
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a certain rate and place and it's all gone in 15 in it so it's as heavy as it can said iment and then they let the bacteria replicate in the presence of n 14 the lighter isotope of nitrogen and what one would anticipate to happen is whenever you replicate you peel apart the two heavy strands and each one pairs with a light strand okay so you now have two copies two new identical copies of DNA where half of the straf of this one strand has n 15 one strand has N 14 so
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it will sediment less quickly because it is of a different density a lighter density than the all N 15 so you would get an intermediate weight band in this centrifuge tube when you're sedimenting if you peel those two strands apart again you've got a heavy and a light if you're growing in in 14 the light will combine with another light that and there are two of those and then the heavy will combine with the light so you'll get new sedimentation where you still have a band that's the combination
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of the light and heavy but then you start having some material that is all of the light so this would be this cycle so two that are still light plus heavy and then two that Sibley light you keep on replicating and your keep on diluting the heavy does that experiment make sense it's kind of a cool experiment you can hardly believe it's feasible but the centrifugation ability to differentiate with these isotopes is really valuable so I just want to put in a plug for the
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use of isotopes obviously they're used in nuclear physics we use a lot of the radioactive isotopes for different reasons but in biology they are indispensable for some of these experiments and even to this day you can do things with isotopes you can't do with a lot of that you can't do by other experiments either the radioactive are heavy there's a great deal of work done nowadays in proteomics using mass spectrometry and heavy isotopes where you can really track where things go and
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treat cancer cells differently from healthy cells but actually track what's happening by putting heavy isotopes in one of the growing dishes of cells okay so I like both of those experiments a lot even though they're I would put them in the category of oldies but goodies all right now I've got some details over here that we've got some explaining to do as we move forward and I just want to first of all start with a couple of details that I want to highlight on this
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screen on this board rather first of all bacteria and so prokaryotes such as bacteria E coli and eukaryote such as human cells have some differences in their DNA so the size of the bacterial genome is about five million base pairs the size of the human genome is a foul not quite a thousand times bigger but really a good deal bigger the DNA in bacteria is circular whereas the DNA in eukaryotic cells is linear and it's actually in the formula you see all these little images where you see the
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pairs of chromosomes are sort of stuck together at the same and that's linear DNA from one end to the other in bacteria the DNA is circular so it doesn't have an end so they're different all right so replicating circular DNA is a little bit different from replicating linear DNA all right now in both cases the DNA has to be packaged up so it will fit in the cell otherwise it's just too much of a disordered thing in the case of bacteria the circular DNA is wrapped up
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and super coiled with what are known as poly amines compounds that are very positively charged to neutralize all that negative charge in the DNA okay and in man and other eukaryotes the chromosomes are wrapped around very very positively charged proteins known as histones they're similar principles but they're different entities okay so when we start talking about replication of a prokaryotic cell here's the typical circular DNA so you can see the double-stranded DNA and I've put a
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little psy symbol here that I said that I call the Ori
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so that's origin of initiation so that is the place where you start copying your DNA and we're going to talk about how to spot these places in genomes in a moment and so the bacterial genome is copied bi-directionally so you can go in both directions copying you know in the appropriate direction we'll talk about that in a moment to make the entire circular DNA and a perfect copy of the DNA with bi-directional copying and that's not bad for small genomes because you've only got one origin of
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replication you've got to get all the way around the circular DNA all right it's quite a bit smaller than the human one but what you what do you do with gigantic genomes such as the one that's about you know thousand times bigger than the bacterial one how do you manage to still catalyze the replication of the entire genome quickly enough to make the copy of DNA in some reasonable amount of time and that is taken into taking also into consideration that the speed of
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bacterial replication is a thousand base pairs per second pretty impressive thousand of those bonds made every second whereas in eukaryotes it's somewhere down from 50 base pairs per second depending on conditions their numbers these are not very explicit numbers there's a little bit of variability so how would you do it if you had to if you had this massive chunk of gene and you've got a copy it just really pretty quickly in order to turnover replicate an entire cell contents in 8 hours what
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would you do how could you expect things yeah yes so the answer here is starting a lot of different places so there's a lot of collaborative work going on all along that large genome and so when the the replication of the long linear chromosomes that are in eukaryotic cells you end up with just a lot of origins of replication so you're basically starting all over the place so you can get the job done quickly enough for it to make sense does that make sense to everybody
26:09
so starting a lot of places it's a pretty easy thing all right now I mentioned very briefly that when just before you can copy DNA you have to unpack it now we've got a lot of we think a lot about packaging DNA less about unpacking it but that's so I've got these pictures sort of in a reverse direction so it's only this form of DNA stretched out that can be copied whereas the DNA in your cells is bundled up very tightly into chromosomes how are they put together those chromosomes are made
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up of chromatin which is a bunch of balls which are histone protein with DNA wrapped around them chromatin that's compact chromatin oh sorry sorry ice whoops we go back not to go forwards so this is chromatin in a compact form this is chromatin now unraveled they look more like beads on a string DNA wrapped around each histone protein to form those nucleus own structures and then each nucleus own looks like this it's got a protein in the middle and DNA wrapped around it so in order to go
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forward and replicate we have to unpackage DNA there are lots of signals to unpackage the DNA that we will talk about later the nucleosomes look like this so you can see you can trace the DNA and the proteins bundled in the middle and if we have time at the end I will show you the video of the packaging of DNA and the video of the replication of DNA there about a minute and a half each so it's not a whole bunch later on we will talk about what happens when you
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make that there is a determination that a cell needs to replicate its genome a bunch of things have to happen a signals to do the unwrapping and one of those things is actually to alter the charged state of the histone proteins so you neutralize the positive charges so the DNA can unravel for it it makes sense it's still just a plain old electrostatic interaction so the synthesis of DNA is what's known as template driven polymerization
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okay so template driven deer polymerization you get a sense of exactly what that means we know the two strands are complementary and we're gonna use one strand as the code to make a new complementary strand so when we're making DNA we have one strand there that's the what's known as the template strand I want to make a complimentary copy of that strand so what I'm gonna do what the DNA polymerase is gonna do is systematically add bases sorry add nucleotides to the 3-prime Oh
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H of the ribose and then you keep growing but the base that gets put in is the one that's complementary to the base on the template strand so if it's I mean on the template strand its adenine puttin on the new strand and so on all right and just to get these numbers again the new strand is grown from 5 prime to 3 prime the old strand is red 3 prime to 5 prime so you're reading a strand and you're reading it's 3 to 5 and you're making 5 to 3 that's how you end up with complementary antiparallel
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strands all right ok with that everyone alright and when you form the bond you go in with nucleoside triphosphates you form a new phosphate diaster were just one of those phosphorous --is and you kick out phosphate phosphate which we would call pyrophosphate or diphosphate ok so here you could just sort of see just check what you think you know what you're doing I'm gonna just say it's this is really a pretty simple thing we're gonna put in a T then we're gonna put in an A
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then we're gonna put in a see because our template is telling us to put in the appropriate purine or pyrimidine that forms a nice base pair with that so there's a question here but that's I've already given you the answer to it so it should be very straightforward for you to look at a template strand and decide what would be on the complementary strand and also decide on the directionality of the complementary strand all right now origins of replication what do they look like we're
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in that big weird where is the old where do I start problem where do I start what's the best place to start someone not you not you anyone else want to answer here where I've got this whole genome I gotta get going I got to stop me copy of it where was the best place to break into it to start making copies yeah correct it's simple it's the difference between two and three if you go to the area where there's lots of G's and sees every GC has three hydrogen bonds if you go to a different area
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where there happened to be a lot of A's and T's in a row each pair is only worth two hydrogen bonds you're gonna pick the stretch that has the most days and T's because it's the place where the base pairing is weaker and it's a it's actually a very simple concept so it would be the 8e rich region is the place where origins of replication and the predominant okay so what we're gonna do now and this is going to be tricky but I'm gonna make this work is we're gonna
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talk about replicating an entire chunk of gene and what I've done here is I put the pieces that we're gonna discuss over here that's my menu and we're gonna work through what we need to copy a large chunk of DNA all right everybody with me so I redrew this this morning I promised myself I'm gonna be super tidy on this blackboard which is a far stretch from me so replication we know where replication starts a tea rich region we could pick those out in the genome we know that
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we've got to unwind the DNA to make a copy of it so we must have had to unwrap the DNA beforehand these are all known things so let's now take a look at double-stranded DNA and try and figure out how we use all the components such I'm going to bring this board down because I don't see it as well up there how do we make use of all these components that are part of the menu that we need to make the new strand of DNA so we have double stranded DNA
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and there are base pairs across so that's typical double-stranded DNA that's what we're starting from unwrapped I haven't made it helical because my Gila C's will get messy and so we need to start so what the first thing that will happen is that the the important enzymes like the helicase will scan to find an origin of replication so somewhere for the for the process to start cuz that's where they're gonna break in so David one of your TAS works for ste in Steve Bell's lab and they
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study the mammalian origin of replication complex which is this much more complicated situation than what I'm going to describe to you today but they do fabulous single molecule work to assemble to show how those pieces are assembled but I'll tell you the truth as dorky as I am the thing that I find the most cruel about the origin of replication complex is that the the expelled orc and if any of you are Lord of the Rings fans who can't be excited by a complex called the or complex
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alright so there's going to be the the screening of the genome for an Ori and I want you to remember that what is up here is the rest of the chromosome it's not just a fuzzy end it's something that's got a lot of stuff there that still be spared so the first thing that happens is that helicase needs to start unzippering the two of the double-stranded DNA so you get to a new intermediate we're gonna draw like this
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and the helicase is going to intercept to basically start separating those two strands of DNA in order to do the replication don't forget that these are still base paired and these now are single strands so I'm gonna need something from that menu I'm gonna need it quickly what do you think's the next thing we're really gonna need in order to be able to move forward in our job what do we need from over here we're we're already using the double-stranded DNA it's not gonna be too difficult to
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figure out that NT piece we're already using the unzip arrays the helicase what are we gonna need yeah primase ah primase shortly but we've really got to do something to stabilize our unzip and stuff anyone else so those two single strands are right near each other and they're complementary what's gonna stop them going straight back together again and not allowing replication so there are proteins that basically sit on the single-stranded parts of DNA that are
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known a single strand binding proteins that stabilize this transiently made complex long enough for it to be copied all right so we've so far we've used the helicase we've used the single strand binding proteins and i'm gonna draw them there all right still remembering that I have an entire chromosome up here now the heavy lifting has to start fairly soon so we need the enzyme that's going to start making a copy of one of those single strands that's going to be a DNA polymerase
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which is right here but DNA polymerase is kind of a finicky enzyme because it doesn't like to just start cold on the genome on the single strand and start making a copy DNA polymerase needs a primer of some kind all right so I'm gonna just put that as a note up here DNA polymerase needs what's called a primer and what is a primer a primer is a sequence I think I've got something up here a primer is a sequence that's complementary to a little bit of DNA so that when DNA polymerase comes along
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it's actually grabbing a double strand and then filling up the rest of the single strand so this would be a typical depiction of a prime a here's a strand we want to copy but the blue one is a primer strand giving you something double-stranded to hold on to and then in this situation DNA polymerase would be happy to fill in the rest of the sequence so for the purpose of this discussion to start we're just going to provide a primer that is complementary to the DNA just to give us a go we have
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an alternative in the cell where is to actually use a primase and RNA building blocks to build a primer but let's keep things simple for a minute and get on with a lot of the major work and then we'll come back to that in a second so this part is a primer and that primer if this is the 3 prime end that's five prime three prime so the primer is complementary to the DNA in that direction is anti parallel and then DNA polymerase can go along and fill in the Strand and I'm gonna draw it dashed and
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it's gonna keep on growing and what I want you to notice his DNA polymerase grows from five prime two three pride that is a cardinal rule the directionality that we grow the new DNA in so basically looking at this picture it's going in this direction alright because the 3-prime o H is free and you're building onto it so you use a primer first and then you use DNA polymerase plus deoxy nucleoside try phosphates so all those 80 CG building blocks so we've been using these we've used the
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single stranded binding proteins we've used a primer but there are an alternative I'll get to that in just a second but we now have a complex where I'm gonna briefly divert you to tell you about in the cell what else you could use as a primer and then we're gonna have to deal with copying this other strand but this first strand that is made is called the leading strand it's the one that's easy I peel apart the DNA I have a primer there and I can just build goes
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really smoothly I'm building in the right direction if you look over on the other side of the fence you've got a problem because I can't build on this side from here right why not yeah I'd be doing it the wrong direction I would be the total mess frankly there would be a crisis in the cell so we're gonna have to deal with that so let me just get you out of the primer just deal with this primer issue in the cell it's not like we can micro inject all these little
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primers to get our DNA synthesized so RNA polymerase believe it or not
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doesn't need a primer so you can build little bits of primer with RNA you don't build very much just build short segments and then once you've got sort of enough there DNA polymerase can start polymerizing so you you use RNA polymerize a nucleotide nucleoside triphosphates not the deoxy ones so later on we've got this piece here that might be made of RNA which is why coming over here to my menu do I have it yes we need an RNA RNA primase and we need RNAs to chop them out so that DNA polymerase
42:43
can come back because now there is double-stranded material and fill in the gap and then we need one more enzyme to stitch together the gaps and that's the ligase the logic of this stuff is great I think it's just really amazing because all these moving parts have evolved to make all these functions fall into place now we need to talk about this pesky other strand okay so we have a problem here because we've got the wrong directionality so what happens in nature
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is a prime as soon as there's enough of a stretch a primer is put in place growing the DNA in the right direction that's from where the origin of replication zipping so we put in another primer and then DNA polymerase that's a white one can build its complementary strand building in the appropriate direction to be consistent so you basically fill in a chunk and then you have to wait awhile til more is on zippered put in another primer and then grow the DNA so in that other piece
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you're making little segments of DNA intervened by RNA primers later on those small primers are going to be chewed up by RNAs DNA polymerase is gonna fill them in and ligase is going to join them that makes sense it's a lot to grasp and this other side is called what's known as the lagging strand and there are pieces of short DNA that have transiently made that have had their own name you can remember it or not I just feel I'm not being complete if I don't describe mention their name they're
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called Okazaki fragments after the guy who discovered them and they're longer in bacteria than in humans so Okazaki fragments are the transient short stretches that are made of DNA that are made in the lagging strand and then they get zipped together so that's sort of the story for replicating the DNA I mean make sure I've described everything to you leading strand lagging strand oh I believe I almost got this all right now we got a problem so we've come all this way we've used all the pieces we
45:19
haven't used one of the pieces all right so we're in a small bit of trouble because what's gonna happen when you're for example we're trying to peel apart your DNA what's what trouble are you gonna run into who wants to come up here and get involved in my demo and you've got to be aware that you're gonna be on screen you and you up there you got your hand up first okay I need someone who's going to hold on to this as if their life depends on it you can't let the
45:54
yellow one slip out so you and the rest of the chromosome so make sure that you're really hold and you don't let things come apart now you are helicase okay okay so come on start HeLa casing and so I can get and really pull as hard as you can he's gonna hold it no I don't want you to unwind it because we're in the middle of a chromosome so you you've literally got to do what helicase does yeah keep on pulling come on you can do it can you go much further I mean a your
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arms lightly low but it's almost impossible right we get to a stage where we need hell the help we have coming just from topoisomerase topoisomerase does something so let's walk just one step this way so those yeah topoisomerase comes along and it cuts the DNA it holds the pieces in its hands if you will let's the supercoiling relax and then it joins them back again so topoisomerase thank you very much guys that's it chromosome helicase thank you we really have some tightly wound DNA here so
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topoisomerase is the get-out-of-jail-free card because it allows you to deal with all of this tension you got in place that you cannot unravel so you need someone who's literally gonna cut holes let the thing relax rejoin it and there also there are some topoisomerases called DNA gyrase is but the really cool thing about topoisomerase is that it's a wonderful drug target in both mammalian and bacterial cells because they're quite different in the two types of
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organisms so in bacteria the antibiotic ciprofloxacin is a topoisomerase inhibitor that actually stops your bacteria dividing because if topo doesn't work you can't go on and in human biology and cancer biology there are also topoisomerases there's one called contra Thiessen that does the same thing in the eukaryotic topoisomerase to stop cancer cells dividing rapidly so that those cannot be cannot go on and make larger tumors so I want to show you something and I am
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actually certain that I have time come on now I'm gonna show you in this animation we'll see the remarkable way our DNA is tightly packed up so that six feet of this long molecule fits into the microscopic nucleus of every cell
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the process starts when DNA is wrapped around special protein molecules called histones the combined loop of DNA and protein is called a nucleosome
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next the nucleosomes are packaged into a thread the end result is a fiber known as chromatin [Music] this fiber is then looped and coiled yet again
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[Music]
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leading finally to the familiar shapes known as chromosomes which can be seen in the nucleus of dividing cells I'm going to take you forward using computer animation based on molecular research we are now able to see how DNA is actually copied in living cells you are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand the DNA to be copied enters the production line from
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bottom-left the whirling blue molecular machine is called helicase it spins the DNA as fast as a jet engine as it unwinds the double helix into two strands [Music] one strand is copied continuously and can be seen spooling off to the run things are not so simple for the other strand because it must be copied backwards it is drawn out repeatedly in loops and copied one section at a time the end result is two new DNA molecules [Music] all right okay that's what you saw today
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replication a lot of the mechanics a lot of the moving parts there's this tricky stuff with primers you'll get used to that but this is really the quintessential set of pieces to replicate DNA it is an amazing process look at the speed thousand base pairs a second can you even believe it a chromis entire cyclic circular chromosome copied in 20 minutes so these are really things to think about because they're so impressive that it's a delight to actually be able to teach
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them to you okay I'm done for today
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you
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