nm0238: okay good morning everyb-, good af-, it's Friday i don't even know whether it's morning or afternoon good afternoon everyone er i hope you're all comfortable and have switched your mobiles off so that we have a nice quiet time apart from me er first of all a reminder about the practical sessions this afternoon and of course i'll put this up again later er group D are doing practical three in A-M-S-G-eight group A are over in Plant Sciences lab B doing the er mitosis practical i think the miosis practical and group C are doing the wash up practical one with me in G-four we're also er we also welcome to this session a member of the er Centre for Applied Language Studies staff who thinks er that this lecture may be worth recording well there we go my topic for the first session this afternoon is D-N-A and information we've spent a long time now er talking about genetics about m-, genetics at the level of the individual at the level of the phenotype at the level of the population last week and at the level of the gene undefined idealistic gene Mendel's gene which we've identified so far with a locus on a chromosome but always at the back of our minds has been the fact that we all know that we live in the post-D-N-A age and there is another way of explaing genetic behaviour and that is to explain it at the molecular level to explain it in terms of this molecule deoxyribonucleic acid D-N-A and at about the same time that we discovered that D-N-A was the genetic informa-, the genetic er material we also were powerfully influenced by a theory that said genetic material must consist of information this was a theory which was based on early computer theory er but was much supported by the famous physicist Erwin Shrödinger who during the last war lived understandably away from Germany in Dublin and he gave a very powerful series of lectures in Dublin called What is Life in which he propounded the view that at the bottom of life was genetics and at the bottom of genetics had to be some sort of information so we'll look at that information paradigm or model of er genetics alongside er information about D-N-A now i know that there are some of you who will have been talking about D-N-A for many years now and will be if an-, if y-, if it's possible to be and since i'm a D-N-A biochemist i don't think it's possible to be er may actually be bored with it well we'll try and er liven it up as much as we can when we talk the substance of this lecture has three parts er we're going to remind ourselves what the evidence is for D-N-A as the genetic material then we're going to look at nucleic acids as molecules and finally we're going to look at the functionality of D-N-A how this model of D-N-A as information maps onto biochemical mechanisms and in my second talk this afternoon we'll move on to talk a bit more about that so the evidence first of all then for D-N-A as the genetic material and there are two two canonical experiments er which are described in chapter sixteen of Campbell er and one of which we have already talked about so i will talk about the second today the first one is the transformation experiment which we talked about when we were talking about transformation in our consideration of bacterial genetics remember transformation is when an external D-N-A source er is added to a bacterium the bacterium takes up the D-N-A and changes its er phenotype in response to that new genetic information and you'll remember that this was originally studied by the English bacteriologist Griffith when he looked at rough and smooth pneumococci bacteria that caused lung disease and killed mice if they were smooth but not if they were rough and then he showed that an extract of D-N-A from the smooth bacteria could transform the rough bacteria to make those equally pathogenic now of course at that point you remember he didn't know anything about what it was that was the transforming agent as he called it er but subsequently three researchers in Baltimore Avery MacLeod and McCarty went through a series of experiments where they digested that er preparation of transforming agent with different enzymes so they digested it with enzymes which er broke down protein which broke down R-N-A which broke down sugars which broke down D-N-A and they discovered that the only set of enzymes which killed the ability of the extract to transform were the D-N-A breaking down enzymea D-N-A-ases nucleases and so they came to the conclusion that D-N-A must be the transforming agent and hence what it was that was in the smooth bacteria which when donated to the rough bacteria made them pathogenic the second experiment is known throughout biochemistry as the blender experiment because er a material part in the experiment is taken by an ordinary kitchen blender and that is described on this overhead which you have a copy of in your handout it's more properly known at the Hershey-Chase experiment not for those of you who come from the other side of the water because it has anything to do with chocolate bars but because there was a man called Hershey and a man called Chase in fact a woman called Chase er who did this experiment and this brings us back again to our consideration of bacterial genetics because it involves one of those organisms we talked about in bacterial genetics that is the bacteriophage and you'll remember this picture of a bacteriophage looking rather like a lunar module here attaching to a host bacterium well what Hershey and Chase tried to do was to show what it was that a phage injected into the host bacterium which enabled that host bacterium to become then a machine for constructing new phage and they did that using what was at that time very new technology they er radioactively labelled their phage and they used two radioactive labels thirty-five-sulphur and thirty-two- phosphorus so the the upper line of this describes what happens er with thirty-five-sulphur and the bottom line describes what happens with thirty-two-phosphorus the reason they used the two labels is that there is quite a lot of sulphur in proteins coming from the amino acids methionine and cysteine and very little phosphate whereas there is a great deal of phosphate in nucleic acids and no sulphur so using these two labels the thirty-five-S showing what the protein is doing and the thirty-two-P showing what the nucleic acid is doing they hoped to be able to show what it was that got into the bacterium and caused the er caused the er genetic changes in the bacterium so in both experiments what you do is to take your radioactively labelled phages in a a flask you infect some E-coli bacteria with those radioactively labelled phages and you allow them to attach and even after they've injected their genetic information into the host cell they will remain attached so we have to detach them and the way we do that is to use the sheer forces generated by an ordinary kitchen blender so you put your mixture into the blender and you whirl it around a bit and then you use a centrifuge to separate the supernatant which contains the phage particles that have broken off from the outside of the bacteria and the pellet which contains the bacteria and when you do that and you do it with thirty-five-sulphur then all the radioactivity remains in the supernatant but when you do it with radioactive phosphorus all the radioactivity is in the pellet so you know that it's the phosphorus from the bacteria from the phage that's entered the bacteria and not the thirty-five- S and so we conclude that it's the nucleic acids in the phage that are carrying the genetic information and not the protein okay another very simple experiment it looks t-, almost trivially simple to us now er but it was very instrumental in showing that D-N-A was the genetic information so now we were able to show that in bacterial transformation and in phage infection the genetic information that was being transferred was a nucleic acid and in particular D-N-A so we now know that D-N- A is the genetic material what sort of stuff is D-N-A and in general what sort of stuff are nucleic acids well nucleic acids as you all know are polymers and in the case of D-N-A strikingly long polymers polymers being chemicals which are constructed from repeating simpler units the base unit of a nucleic acid is what we call a polynucleotide okay and so this is an appropriate point to remind ourselves what a nucleotide is and basically a nucleotide is a combination of three things a base a sugar and a phosphate and for those of you who like like like their chemistry really simple and i'm sure that applies to a lot of you we can think of a nucleotide as being constructed in a very simple way with a base here bonded by a single bond to a sugar and that sugar being again bonded by a single bond to a phosphorus okay now this bond here is an oxygen phos-, er carbon-oxygen- phosphorus bond this bond here is a carbon-nitrogen bond but er that's for the chemists to worry about most of us not too worried about that but those of us who w-, and all of you should have at least some familiarity with what those chemicals look like whether you like it or not we'll look at a slightly more complicated version of what those chemicals look like okay so our bases right can be any of in the case of D-N-A four different bases adenine guanine cytosine and thymine and in the case of R-N-A any of the four bases adenine guanine cytosine or uracil the difference between those different bases what we have there is a picture of thymine and uracil but adenine is a typical base of the other kind so let's just draw the two kinds of D-N-A bases you've seen thymine which looks like this okay that's thymine and thymine is what we call a pur-, a pyrimidine base and it just has a single aromatic ring okay and that aromatic ring contains two nitrogens the difference between thymine cytosine and uracil which are the three pyrimide bases lies in what substituents we have in the case of thymine and uracil it's two oxygens in the case of er cytosine it's an oxygen and a nitrogen if we look at the pyrimidine bases they're slightly different and slightly more complicated just try and draw the bonds right okay that's adenine which is the simplest of these more complex bases which are called purines okay so there are two types of bases purines and pyrimidines one is a single ring and the slightly longer name smaller structure longer name and the other one has a double ring and a shorter name okay as well as thymine in this class we have cytosine and uracil and as well as adenine in this class we have guanine and the differences as i said are in the number of substituents in the ring if you want to know more about that chapter sixteen of Campbell is where you look so those are the bases those bases are linked to sugars deoxyribose in the case of D-N-A ribose in the case of R-N-A both of them sugars containing five carbons one two three four five one two three four five arranged in a five-membered ring with one of their oxygens and then this is linked to a phosphate group and that phosphate group is linked through this carbon here the so-called five-prime carbon and as you see these nucleotides are linked together in a strand up to a polynucleotide by bonds between the phosphate which is on the five-prime on the five-prime carbon of this sugar is linked to the three-prime carbon of the next sugar so what we have is a chain going sugar phosphate sugar phosphate sugar phosphate sugar phosphate sugar phosphate and off it we have the bases so it's like a ladder structure sugars and phosphates but with bases intervening so if we were to put that back on my simple minded diagram okay here's base sugar phosphate what i would have then is this sugar here joining on to another phosphate here and then another sugar here and another base here and then this sugar again joining on to another phosphate and so on and so on phosphate sugar phosphate sugar phosphate sugar so that in very simple terms is how you make a nucleotide something about the nomenclature well we found that there are five bases adenine guanine thymine cytosine and er uracil in fact as nucleotides they all change their names just to be very awkward and adenine changes to adenosine cytosine to cytidine guanine to guanosine and uracil and thymine well there is no no uracil changes to uridine and thymine to thymidine again this is something you don't have to take in now perha-, er any book will tell you those names and we've looked at how to build a polynucleotide we build a polynucleotide by joining these units through base er through sugar phosphate linkages we've also in talking about nucleotides discovered some of the differences between D-N-A and R-N-A we've discovered that D-N-A has thymine as one of its bases whereas r-, R-N-A has uracil that D- N-A has deoxyribose ra-, where er uracil ha-, er where R-N-A has ribose but there are one or two other structural differences that perhaps we might like to look at and one of those while we're looking at it is that D-N-A in general forms a double helix of two chains of polynucleotide and is in general a very long polymer which has a fibrous structure whereas R-N-A is normally consist of one polynucleotide chain it is single stranded whereas D-N-A is double stranded and in general R-N-A chains tend to be short and therefore fold upon themselves to make globular molecules a bit like proteins okay so D-N-A is a double helix long and fibrous R-N-A a single stranded molecule short and globular don't know whether you've ever thought how big D-N-A might be it's a long molecule er my my favourite model for it is a a piece of ordinary sewing cotton which is a roughly one in a hundred-thousand scale model okay it's one in it's a hundred-thousand times larger than a piece of D-N-A er and on this model er the amount of D-N-A in a bacterium would be a couple of hundred metres of this okay couple of hundred metres is actually more or less the contents of this er this rather large bit of cotton if you can imagine me unravelling all this of course you can see what a problem D-N-A is to keep anywhere okay such a long molecule clearly although er one draws it conventionally as a nice little tiny circle is er somewhat complex and rather inclined to get muddled and twisted and that's a problem for a bacterium as i said about two-hundred metres of this stuff er in each cell in your body right using this model you have a hundred-and-six kilometres okay of this stuff okay it's it's actually if the real stuff okay so we scale this down a hundred-thousand times is around one- point-six metres okay one-point-six metres so it's roughly the height of a less than average man that's me er okay er roughly my height in D-N-A if you can imagine an a D-N-A molecule my height and ten-thousand hundred-thousand times thinner than that er that's how much D-N-A you've got inside every one of your cells i said a hundred-and-six kilometres didn't i it's a hundred-and-six miles to well it makes it more familiar a hundred-and-six so a hundred-and-sixty kilometres er roughly from here to Bristol so i would have to stretch this stuff from here to Bristol to model the D-N-A inside each one of your cells so it's very long and even given that inside every one of your cells there are actually forty-some chromosomes so th-, it's not one molecule a hundred-and-six miles long it's er forty molecules adding up to a hundred-and-six miles er forty-eight molecules of course er but er that does give you some idea of scale that contains three- thousand- million nucleotides okay roughly we believe that each each D-N-A D-N-A in one haploid amount of D-N- A in one of your cells is three- thousand- million nucleotides whereas for a our poor old friend er the er E-coli bacterium it's only some four-million a trifling amount just to go back to the molecules for a minute looking at D-N-A you know that D-N-A is a double helix okay there are some pictures of the double helix there two things about the double helix i think we need to er well at least two things we need to talk about one of them is how the double helix is bonded together there are two sorts of bonds which hold the double helix together one of those are so-called er base pairs which are relatively weak bonds what we called hydrogen bonds which bond across the centre of the molecule always between adenine and thymine and between guanine and cytodi-, cytosine with two bonds between adenine and thymine and three bonds between guanine and cytosine that means that a G-C base pair is roughly fifty per cent stronger than a A-T base pair however that's not the only thing that holds the D-N-A molecule together as you see from this picture those D-N-A bases are s-, on top of each other stacked on top of each other in fact this bit of the picture shows you that better in this space filling model here we see the bases stacked on top of each other okay those bases being stacked on top of each other they actually interact with each other like this and that gives an extra layer of stability to the molecule that's called base stacking so base pairing added to base stacking stablilizes the double helix looking at this diagrammatic model of the double helix here some of the details we can count that there are one two three four five six seven eight nine ten bases per turn off the double helix and that amounts to a distance of some three-point-four nanometres and the width of the double helix is about two nanometres a nanometre remember is one times ten-to-the-minus-nine of a metre and the other thing i need to tell you about is if we go back one thing i need to stress and it's one of the mysteries of nucleic acid structure that always mystifies people if we look at that structure as we've draw it here in very simple terms with our sugar phosphate sugar phosphate sugar phosphate backbone okay we have at one end of the molecule here a something with no phosphate on it we conventionally call that the three-prime end because there's a free three-prime carbon stuck here and at the top here we have a free phosphate and that's attached to a five-prime carbon here so we call that end of the molecule the five-prime end okay so just as proteins have two different ends we call them the amino terminus and the carboxy terminus of a protein we talk about the five- prime and the three-prime end of a nucleic acid okay and if we draw a double helix whereas one of the strands is moving from five-prime to three-prime in this direction the other strand is moving from five-prime to three-prime in the opposite direction that is a double helix with its bonds in between we call it an anti parallel double helix okay because we have two parallel chains one running in one direction and the other one running in the other and half of the problems that people get into thinking about D-N-A has got to do with forgetting that they two run in different directions that was the thing that most people got wrong for instance in the diagnostic test where you were able to to write me the complement of a D- N-A molecule you normally wrote it the wrong way round because the convention is that we always write D-N-A sequences starting at the five-prime and ending at the three-prime so the five-prime is always on the left my right in this case okay and we put out the sequence running from left to right with the five- prime there and the three-prime there now that's a molecule of D-N-A what about a molecule of R-N-A what i just wanted to show you as i said they're short and er generally globular and this picture which you haven't got a copy of but you can look up in figure if you see it's in chapter seventeen of Campbell it's a picture of a T-R-N-A which we'll be talking about again er in the next er part of in the next talk but a T-R-N-A is a short molecule if you could count it's about seventy-five nucleotides long it's trivial compared with the length that we've been talking about in terms of D-N-A okay and as you see it although it's single stranded there's only a single molecule there it coils back on itself making double helical er making base pairs across here and making little bits of double helix as you can see here in this molecule making it in fact almost a a little blob shape like a protein okay so rather than those enormous great molecules three-thousand- million nucleotides long for a piece of D-N-A er we've got something seventy- five base pairs long well R-N-A molecules get larger than that but not a lot larger perhaps a few thousand at most okay so that's looking at the differences between D-N-A and R-N-A and while we were looking at those differences we er talked a little about these forces that bound the D-N-A together okay and you've got that on one of your overheads base pairing with hydrogen bonds two for an A-T base pair three for a G-C base pair and the base stacking bonds which hold the structure together at this point we've talked almost as much as anyone can bear i think about structure so we'll take a two minute break there okay and then we'll go on to talk a bit more about the function of D-N-A nm0238: so D-N-A has to function as the genetic material in most cells and most organisms there are a few organisms very simple organisms who use R-N-A as genetic material most obviously some viruses and some rather odd things that plants have called but for most organisms D-N-A is the genetic material and that means that he has to do two things it has to act as a store we have to be able to store information and we have to be tra-, be able to transmit information and there are basically two modes of transmitting information there's transmitting information from one cell to another and there's what we call expressing information that is moving from genetic information stored to phenotype information expressed which as you remember in the case of molecules is talking about moving from D-N-A to proteins so D-N-A how does it function as an information store we have an incredibly long molecule a copolymer right which links together four different nucleotides adenine the bases you paired your bases adenine guanine cytosine and thymine A G C and T as we call them okay what we have and people er very soon realized this is basically a piece of linear coded information and the best analogy that i can think of is magnetic tape okay now you all vaguely know that what we have in magnetic tape is a series of iron atoms okay which are laid out on this plastic tape in some sort of order okay and the m-, the magnetics the magnetization of those iron atoms is some indication of information it can be read somehow by a machine and interpreted as sound okay we have a linear er a linear succession of these things which we can pull so we can have these sounds in a linear er arrangement as we like music to be okay well you can imagine D-N-A like this so that the linear sequence of the bases along the strand okay is is the information that is the information the sequence of bases on a D-N-A strand okay and exactly like this cassette tape you neither you nor i can tell whether this is Cerys Matthews or Dame tiri game Dame Kiri Te Kanawa whether it's Beethoven or er Catatonia er because just like this the information is redundant useless okay this information is no good unless interpreted by some sort of interpreter which in the case of a cassette tape of course is a cassette player but in the case of D-N-A genetic information is the cell okay so that information only has sense in the context of the cell in which it finds itself okay and if you want to think mechanistically you could think of a cell as a cassette player but it's not really helpful i don't think mechanistic analogies really work okay but something needs to interpret that genetic information that clearly is the cell so the information store consists of the sequence of the bases in the chain the transmission of information as i see it happens in two ways first of all i'm going to talk about transmission by expression and then we'll talk later about what i've called on this slide maintenance of information but it's also transmission to the next generation okay and its replication in order to look at transmission of information i'm going to go to what i've shown you i think once already which is what er Francis Crick immodestly called the central dogma of molecular biology this little diagram which shows you the information flow inside a cell from the D-N-A store of genetic information through a temporary resting place in R-N-A particularly messenger R-N-A to the protein which is equivalent to the phenotype so from genotype to phenotype within a cell when Crick wrote this out first of all he wrote that diagram with simple arrows on it showing that the information moved from D-N-A to R-N-A to protein okay and that's equivalent to the move from genotype to phenotype now that's although Crick would deny it heartily that's actually a very simplified form of the central dogma and we'll look at a more complex form in the next lecture okay but that's basically how the information was transmitted inside the cell to interpret the ph-, genotype as a phenotype what i want to concentrate on in this lecture is the next stage which is replication what the cell has to do because that cell is going to divide and have daughter cells what that cell needs to do in order to pass on that information to maintain a decent store of information in its daughter cells one of the big differences between these two processes transmission and maintenance is that it is much more important for this one to be accurate than it is for this one to be accurate okay when we're trying to maintain the information every time we copy the information if there are mistakes introduced then we've got mutations and we've got a change in our genetic information okay so we want to make absolutely sure if we can that replication is as accurate as be made with transmission of information moving information within a cell from our D-N-A to R-N-A to protein we're not so worried because we're going to throw that R-N-A and that protein away at the end of the day we're going to make new copies and those new copies may be more accurate so although accuracy is the important consideration for both transmission and maintenance it's really the prime problem for maintenance so the process whereby we take a D-N-A double helix and make two D-N-A double helices is the process we refer to as replication and that's what i want to spend the rest of this lecture talking about and D-N-A replication in very simple terms has four important characteristics the first is that it is semiconservative what's that mean one usually makes a political joke here but i won't make a political joke er you can work out a political joke for yourself semiconservative means that if you take a D-N-A double helix and here i'm going to turn to my other favourite model of a D-N-A double helix which is a piece of Velcro okay the important thing about a piece of Velcro is as you know is that it has two sides which better bind to each themselves but which bind to each other this entirely mimics the fact that one chain of a D-N-A double helix absolutely specifies and will only bind to er the other chain okay and we say that the chains of a D-N-A double helix are complementary to each other that's complementary with a E C-O-M-P-L- E- M-E-N-T-R-Y-T-A-R-Y okay they're complementary to each other right we can describe that in very crude terms and say they stick to each other and some people and we do in molecular biology refer to these strands as sticky okay but we've got here's our double helix it's not helical but you could imagine it what happens do we part this double helix make a new chain on here and make a new chain on here and join it up again which would be a conservative model because this helix would remain exactly the same okay or do we throw away both strands of this helix and make a new one well that would be wasteful and clearly we don't do that what we do do is semiconservative replication that is we take these two strands and on each of these two strands we build a new one so for each strand half of it is old there's half of it new and hence semiconservative and that's very clearly shown in the diagram which i think you've got a version of on your handout okay this as i said will be a conservative model where we started with a double helix when we made a new double helix that was entirely new and the old one was kept right that's a conservative model the semiconservative model is where we start with a double helix we part the two strands and make a new one from each and again when we make that one we part the two strands and and er make a new one on each don't worry about the dispersive model i don't think it's worth thinking about at the moment okay so we say that D-N-A synthesis is semiconservative what evidence do we have for that the evidence that we have for that is the evidence of the Meselson-Stahl experiment which again you have on your handout okay this was an experiment which was done in er the nineteen-fifties er in which what the er the two authors Meselson and Stahl did was to label the strands of D-N-A this time in the Hershey-Chase experiment remember we s-, labelled D-N-A and labelled protein in the men-, Meselson-Stahl experiment what we do what we did was to label the old strands of D-N-A with one isotype not a radioactive one in this case of nitrogen okay and the new strands with the new isotope okay and we then look at the D-N-A molecules that were formed in fact what what they did was to start with nitrogen fifteen and then they substituted it with nitrogen fourteen nitrogen fourteen is lighter than nitrogen fifteen so the density of the molecule went decreased and they showed that density by running the D-N-A molecules in an ultracentrifuge so here's what they started with in the ultracentrifuge this is heavy D-N-A okay and what you see happening is that as we run through one cell cycle the D-N-A moves from this position to all be at a new position here okay and as we run through a second cell cycle it moves to being at two positions if we go back to our model we'll see that that's exactly what we expect from our semiconservative model we start with dark blue heavy and we move through in one cell cycle to everything being a mixture of light an-, and dark blue that is the same density whereas in the conservative model we would move from blue to dark blue and light blue two different densities so at the end of the first generation you have two different densities whereas at the end of the first generation here we just have one density at the end of the second generation however in both cases we will have two densities okay ar-, in conservative three of them will be light one of them heavy in the case of semiconservative two of them will be mixtures and one of er two of them light and if you again look at the Meselson-Stahl experiment you'll see that that's what happens in the second generation we move from this position here to a mixture of things of that density and some lighter things okay so by measuring the density of D-N-A during the experiment in which we substituted a light nitrogen isotope for a heavy nitrogen isotope we were able to prove or Meselson-Stahl were able to prove that semiconservative was the correct model for D-N-A replication what other characteristics do we need to know about D-N-A replication well first of all does it go all over the place no it doesn't it always starts in one place starts at what we call an origin and in the case of er a bacterial chromosome there is only one origin okay there's only one place where we start and replicate bacterial chromosome in the case of your and our my chromosomes which are infant as we saw a thousand times or so larger er i'm afraid we don't start from one origin but we have many origins otherwise it would take us forever to replicate our D-N-A however from that origin we can very easily show that the D-N-A r-, is replicated in both directions okay that replication is bidirectional so if we imagine that we're going to start making a piece of D-N-A so here i have a piece of D-N-A here's a double strand of D-N-A and here is the origin okay obviously the first thing that must happen at the origin is w-, me must dissociate the strands like that and you can in fact see that happening in an E-M photograph a little bubble will appear in the D-N-A okay and then we start making D-N-A in both directions that is we make it on this strand okay and we make it on that strand and D-N-A is always made in the same direction it's always made starting with the five-prime of the new strand and moving towards the three-prime of the new strand which since those two strands as i said are antiparallel this strand has a three-prime here and a five-prime there this strand has a three-prime there and a five-prime there okay those two directions will be the opposite directions here we have the snag of D-N-A replication because unless when i replicate bidirectionally i'm going to be satisfied with going this way all the way round my circular D-N-A if i'm a bacterium okay and come back to meet myself here which will take me a long time and do the same in the opposite direction for the other strand i'm i'm if i'm satisifed with that it will be a very long process and also i will have a single stranded piece of D-N-A hanging around for an awfully long time which you can imagine what it looks like like this you can imagine the problems you'd get into er we need a way of replicating the other strand at the same time and as i've told you we can only replicate D-N-A in one direction and the solution that D-N-A has is that although it replicates D-N-A continuously on one strand it reputat-, replicates the other strand discontinuously okay and that means we make the other strand in fragments so if you imagine the other strand being made what i do is to start here and make a little bit of D-N-A and then i jump back and i start here and make another little bit of D-N-A and then i jump back and start here and make another bit of D-N-A and so on a motion i've described and it will only mean something to the girls among you i guess as being something like blanket stitch okay in out round the back in out round the back okay those of you who think i've got the nomenclature of the stitch wrong can come up and tell me later but i assure you there is a stitch okay but you see we're going forwards the general direction of synthesis is going in this direction okay but i'm always actually making the molecule in this direction that is the five- prime to three-prime direction i clearly i'm going to make a lot of fragments and those fragments have a special name they're named after the Japanese scientist who first discovered them they're named Okazaki fragments so D-N-A replication is semiconservative we retain one old strand in each new double helix it starts from a single place of origin of replication it moves in both directions from that origin moving continuously on one strand and discontinuously on the other of course what's happening on that strand is something very simple okay and the basic mechanism of D-N-A replication is very simple we have a double stranded helix we part it and then we simply match new bases to replace the new strand so where there's an A we put a T where there's a C we put a G and so on until we get two new strands we're matching bases how do we do that how do we do the whole thing what is the mechanism of D-N-A replication and that's where where i want to stop with thinking us through the mechanism of D-N-A replication and we'll then look at the diagram that you've got lastly on your handout which illustrates it so firstly obviously we need to unwind that double helix we need to make our replication bubble as we sometimes call it nature uses two mechanisms to underwi-, unwinding the double helix it uses an enzyme which actually physically unwinds it it's called a helicase okay and it actually does that by untwisting the Watson-Crick double helix and it uses the device of having a protein in the cells which binds strongly to single stranded D-N-A but not to double stranded D-N-A at all a single stranded binding protein okay so as soon as we part the strands this protein binds hard and the the strands are disinclined to come back together then we use two mechanisms for keeping the strands apart then we need to start making some D-N-A and we have a problem we have a problem that problem is clearly we need to get our mechanism right so that we get the right base pairs and the best way to get the right base pairs matching is to have an enzyme which knows roughly how wide a D-N-A double helix is going to be and can fit it okay fit to the helix we've got fit to the distance because an A-T base pair and a C-G base pair are the same size as each other okay and the enzyme can't do that at the same time as starting a strand on its own the enzyme which makes D-N-A will not start a strand on its own so it needs to be helped and it needs to build a primer first okay and that primer's always made of R-N-A okay so we have to first in order to make D-N-A make some R-N-A and what this means basically is going to this going over here let's imagine we have our piece of D-N-A okay imagine this is the five-prime of it and this is the three- prime of it and we're going to make er some D-N-A here we're at the origin we start with a piece of R-N-A which bonds like that okay made from five-prime to three-prime and then the enzyme which makes D-N-A takes over from this point and makes D-N-A in the right direction okay so at the beginning of our D-N-A we have a combination of a primer made of R-N-A and an old strand of D-N-A which we're using to read the correct sequence which we call a template okay so at the beginning we have a template and a primer so we start the chain with R-N-A we make a primer and then we continue D-N-A synthesis on the continuous strand that we call the leading strand okay because it's always slightly ahead of the other strand in making D-N-A okay and on the other strand the lagging strand as we call it 'cause it is slightly behind okay we make those fragments those Okazaki fragments and we need one more process to go on and that is we need to join the fragments together okay so we need a ligase a joining together enzyme to join the fragments together so we can incorporate all that information about the mechanism of replication on the diagram here that i've given you a copy of okay which gives you a brief summary of D-N-A replication here's the parental D-N-A which has started it's being unwound at this point and there are single stranded binding proteins these are the pink fingers binding to the single stranded D-N-A all right then we we have D-N-A being made continuously in this direction okay by the enzyme which makes D-N-A the D-N-A polymerase and on this strand we're making bits of D-N-A but starting with R-N-A primers to make our Okazaki fragments we're then extending our Okazaki fragments and in the end here's the D-N-A ligase enzyme joining two Okazaki fragments together so we unwind continuously make D-N-A on this strand five-prime to three-prime direction we discontinuously make it on this strand starting with an R-N-A primer and then making our D-N-A until it bumps into the next one basically it just falls off and it bumps in and then we have to join up our two Okazaki fragments using a ligase so that is the basic mechanism of D-N-A replication and that's where we shall end this first er talk for this afternoon okay i'll be putting out some more handouts for you for the second talk in just a second we'll take a five minute break