• [categories_slider autoplay="1"]
  • DNA Replication | MIT 7.01SC Fundamentals of Biology

    Copy Help
    • Public/Private: Change the visibility of this video on your My Videos tab
    • Save/Unsave: Save/Unsave this video to/from your Saved Videos tab
    • Copy: Copy this video link to your system clipboard
    • Email: Copy this video link to your default email application
    • Remove: Remove this video from your My Videos or Saved Videos tab
    search-icon
    Watch at: 00:00 / 00:00:20ERIC LANDER: And so the issue became how does DNAreplication work.And so I'm about to go into it.Now, I'm going to note we're going to be starting this DNAWatch at: 00:20 / 00:40goes to RNA, goes to protein, and DNA goes to itself.DNA is replicated.It makes RNA.The RNA is used to make protein.Watch at: 00:40 / 01:00This will be what we'll be talkingabout today and tomorrow.So the first step of that is, how does DNA giverise to more DNA?Well, how do you find an enzyme?How do you do biochemistry?What do you do?AUDIENCE: Assays.ERIC LANDER: Assay.So you've got to grind up the cell.Watch at: 01:00 / 01:20I got to choose a cell in which I'm likely to find anenzyme, grind it up, break it up into different fractions,and test each fraction.That's all biochemists do, right?So what cell might have the enzyme we're looking for?What cells might be able to copy DNA?How about all cells?Watch at: 01:20 / 01:40So let's use a simple cell.What's a simple cell?Let's use bacteria.So we'll take some bacteria, we'll grow it up,we'll grind it up.We'll fractionate it into different fractions, and we'llsee if one of those fractions has the ability to copy DNA.If we're going to run an assay, we haveto give it a substrate.What substrate would you like to give it?Watch at: 01:40 / 02:00What do you think it needs?AUDIENCE: [INAUDIBLE].ERIC LANDER: It better have some free nucleotidesotherwise, how are we going to make DNA.What else?Are you going to ask it to make DNA all by itself?We want something that can copy one of the strands of adouble helix.So what should we give it?AUDIENCE: [INAUDIBLE].Watch at: 02:00 / 02:20ERIC LANDER: Sorry?AUDIENCE: Half a helix.ERIC LANDER: Half a helix.A strand of DNA, the strand to be used as a template.So let's give it a template strand.So we'll take a template strand of DNA.There's my template of DNA.Let's actually give it a littleWatch at: 02:20 / 02:40sequence actually, here.Let's say A phosphate, T phosphate, G phosphate, Cphosphate, A phosphate, T phosphate, T phosphate, AWatch at: 02:40 / 03:00phosphate, G phosphate, G phosphate.I'm going to not write the phosphates too much longer,guys, but anyway C phosphate, C phosphate, Tphosphate, like that.Pretty soon, in fact, almost immediately, I'm going tostart dropping the phosphates in here.But that's the way it goes.Watch at: 03:00 / 03:20All right.That's a template.We need floating around in the solution some trinucleotides.Watch at: 03:20 / 03:40We have some nucleotides floating around.And now will this enzyme work?We would try different fractions and see if it's ableWatch at: 03:40 / 04:00to just install the right letters in the right place.Now, it turned out it needed one more thing, and the personwho discovered this, Arthur Kornberg, thought of it.It needed a head start.It needed a primer.So the primer goes let's say, phosphate T, phosphate A,Watch at: 04:00 / 04:20phosphate C, phosphate G, phosphate T, phosphate A,let's say like that.So this is the five prime end of DNA.Watch at: 04:20 / 04:40Remember the phosphate is hanging off the five primecarbon, right?What's look at the other end.The other end ends in the hydroxyl on the three primeend of the ribose.Since this is anti-parallel, this strand is going fiveWatch at: 04:40 / 05:00prime phosphate to three prime hydroxyl.You're going to need to know five prime and three prime.So I'm doing this so you get used to fiveprime and three prime.There you go.If you're handed a primer to get a head start, and you'rehanded a template, and you hand it some nucleotides, youWatch at: 05:00 / 05:20then assay different fractions exactly as you suggested andwe see is one of them capable of extending this strand byputting in an A, putting in a T, putting in a C, putting ina C, putting in a C, putting in a G. That's the assay.And Arthur Kornberg discovered an enzyme that could do this.Watch at: 05:20 / 05:40And the biochemists went nuts.They thought, wow.This is so cool.Kornberg is able to discover an enzyme thatcan accomplish this.The enzyme polymerizes DNA.Watch at: 05:40 / 06:00Coincidentally, what is the enzyme called?AUDIENCE: DNA polymerase.ERIC LANDER: DNA polymerase.Accidentally, has a nice name.Good.DNA polymerase.Excellent.Watch at: 06:00 / 06:20Now, notice what it does.It takes this triphosphate, puts it in here, and it joinsit into a sugar phosphate chain.Where does it get the energy for that synthesis?Hydrolysis of the triphosphates, right?It's the hydrolysis of the triphosphate.That's the energy.Watch at: 06:20 / 06:40What direction is the synthesis proceeding?Starts here at the five prime end, and it moves adding on tothe three prime end.So it's five prime to three prime direction.Watch at: 06:40 / 07:00That's the direction it moves.It adds to the three prime end.It adds to the free nucleotides tothe three prime end.Why not do it the other way?AUDIENCE: [INAUDIBLE].ERIC LANDER: Sorry?AUDIENCE: Phosphates.ERIC LANDER: Can't hear you.Shout loud.AUDIENCE: Phosphates.ERIC LANDER: Phosphates, yes.Watch at: 07:00 / 07:20You see, suppose we were going the other way.Suppose the primer was this way.Where would as we added each base, the triphosphate wouldbe on the strands, right?And we'd be adding to the three prime end here.Watch at: 07:20 / 07:40That means the energy supplied by the triphosphate would beon the growing strands rather than in the free nucleotides.Why would it be a terrible idea to put your energy sourceon the growing strand?MIKE: [INAUDIBLE].Watch at: 07:40 / 08:00ERIC LANDER: Well Mike, you know, those triphosphate bondsare pretty unstable.They hydrolyzed by themselves at some frequency.If you're a free nucleotide and the triphosphatehydrolyzes, big deal.That free nucleotide floating around loses its triphosphate.But what if I'm the growing strand, and I lose mytriphosphate?AUDIENCE: [LAUGHS]ERIC LANDER: Exactly.Watch at: 08:00 / 08:20AUDIENCE: There goes your chain.ERIC LANDER: There goes my chain.So you know, life's not stupid.It doesn't do it that way.It does it this way.No one has ever found a polymerase that goes this way.They find them all going that way for just that reason.Exactly.Bingo.That was why life evolved it that way, because you wantyour triphosphates, those hydrolyzable triphosphates toWatch at: 08:20 / 08:40be floating around freely rather than investing.Now just think about that.It's a kind of cool thing.It doesn't matter.Your book doesn't talk about it.But to me, it helps me remember which way it's goingand how it is, and it's kind of interesting.Any way.All right.So Kornberg wins the Nobel Prize for this.Watch at: 08:40 / 09:00Good stuff.It's very deserved, but you know, there's some questions.Where does the primer come from in life?See, Kornberg gave this test tube a primer.But suppose I'm replicating some DNA.Watch at: 09:00 / 09:20So let's suppose I have a double strand of DNA, and I'mjust going to open it up here, five prime to three prime,five prime to three prime.I need to get like a primer here.Watch at: 09:20 / 09:40Then the primer can be extended by polymerase.Well, where's the primer come from?It turns out there is an enzyme specially devoted tomaking those primers.Kornberg didn't know it, but there's an enzyme.Watch at: 09:40 / 10:00And by coincidence, it is called primase.Exactly.Primase makes the primer.So you need a primer here, and the primer is made by primase.Watch at: 10:00 / 10:20Once primase makes a primer, polymerase can chug along anddo it just fine.Let's check out the other strand.Primer here, polymerase chugs along.Watch at: 10:20 / 10:40But now as this double helix opens up, whathappens over here?The synthesis going this way.So what do I have to do here?AUDIENCE: [INAUDIBLE].ERIC LANDER: Another primer.Need another primer.Watch at: 10:40 / 11:00Then as it opens up more, what do I need?AUDIENCE: Another primer.ERIC LANDER: Another primer.So the two strands are experiencing very differentkind of replication.In one place, one primer in the five prime to three primedirection is enough to keep going.In the other strand, as it keeps opening up, you gottaWatch at: 11:00 / 11:20keep making primers.You have all these little fragments there.Now, those little fragments were discovered by Okazaki,and they are called Okazaki fragments.Watch at: 11:20 / 11:40Again, I just mention these things.They are known to molecular biologists.But these little guys are Okazaki fragments, and theytell you that you're on the right track here.This is indeed how it's working.You can see those little fragments there.But now, what's the problem with the Okazaki fragments?Watch at: 11:40 / 12:00They're not connected, right?The primase makes a primer.The polymerase copies the DNA, it bumps into the next primer,but you've got to connect them.So that's a problem.That's a real problem.I'll redraw that here.Watch at: 12:00 / 12:20Here was my primer.I got a new primer over here.I got a new primer over here.Right there.Right there.They're not contiguous connected.The word we use for connecting two pieces of DNA, which is aWatch at: 12:20 / 12:40standard English word not used that often is to ligate twothings together.Ligature, for example, in music.You ligate things together.How do you think the cell deals with ligating thesethings together?An enzyme called--AUDIENCE: Ligase.ERIC LANDER: Exactly.Watch at: 12:40 / 13:00So ligase does the ligation.Ligase ligates.It is so lucky that these words turn out to haveaccidentally made sense.It's really cool.So ligase ligates.Now, I'll tell you a factoid, but don't worryWatch at: 13:00 / 13:20about it too much.Primase actually doesn't make DNA.We haven't gotten there yet, but it turns outprimase makes RNA.Turns out to be easier to start an RNAthan a DNA from scratch.Cell doesn't like to start DNA from scratch.Watch at: 13:20 / 13:40It likes to start RNA from scratch as we'll get to amoment with transcription.So as a factoid, I'll mention to you that those littleprimers are actually RNA primers, and what happens isthey get extended into DNA, and they bump into and kind ofdisplace the previous RNA, so it's slightly more complicatedthan I told you.You're welcome to forget that.If you would like to believe that primase is actuallyWatch at: 13:40 / 14:00making little segments of DNA, it'll be just fine.But in fact, it doesn't actually.It's making little segments of RNA so there's a whole othermachinery that has to deal with that.But the basic concept five prime to three prime, littleprimers, getting extended, getting ligated, that's howyou make your DNA.And you can check it out, and it works.All right.Watch at: 14:00 / 14:20Well, it turns out to even be a little more complicated.That was how we got the synthesis going, but we alsoWatch at: 14:20 / 14:40have a little bit of a topological problem.This again, says a lot about how people do science.You gotta just like not worry about certain things.If Kornberg had said, oh my goodness.Watch at: 14:40 / 15:00I can't give my test tube a primer, because I don't knowhow the cell would make a primer, he wouldn't have madeany progress.So he throws in the primer and says, the cellwill figure it out.I'm just giving it a primer, and I'll see what happens.Now, there's another problem, this topological problem thatalso can make your head hurt.Let me try to explain what the topological problem is.Watch at: 15:00 / 15:20Suppose I have DNA like that.Make that a little prettier.So I have some DNA like that.Watch at: 15:20 / 15:40And maybe it goes around for a very long distance like acircle or something like that.I now want to copy that DNA.So I have one strand, and I'm copying it.I have this other strand, and I'm copying it.Watch at: 15:40 / 16:00And remember, these two strands are wrapped around,and around, and around, and around each other.One is going like this.One is going like that, and there's some wrapped around.And as I tug them apart to make a new strand, tosynthesize a new strand, those two new double helices are soWatch at: 16:00 / 16:19totally intertwined with each other.Every turn that there was in the double helix is now atwist and turn connecting the two, sort of entangling thetwo helices.Watch at: 16:19 / 16:40So I have the two new double helicesentangled with each other.Why is that going to be a problem?I'm going to send these to two daughter cells.These are the two genomes for the two daughter cells.In fact in particular, if this thing was a circle, the twonew circles will be totally wrapped around each other withWatch at: 16:40 / 17:00a gazillion wraps.No way they're going to two daughter cells.Now, here is where mathematicians are veryuseful, because it is a theorem that if I take twocircles wrapped around each other like that, there is notopological deformation possible thatWatch at: 17:00 / 17:20can separate them.It's like these puzzles, you get some strings wrappedaround each other separate them.It's a theorem that two circles wrapped around eachother like that cannot be separated unless,of course, you cheat.What's cheating?AUDIENCE: You cut it.ERIC LANDER: You cut it, obviously.If you cut it, then you can separate it.But otherwise, it's mathematically impossible toWatch at: 17:20 / 17:40separate them.So this could concern people.How could a cell do this?So what does the cell do?AUDIENCE: It cuts it.ERIC LANDER: It cuts it.It's got no choice, right?It's a theorem, right?Even cells can't violate theorems.So it cuts it.The only way to get these things apart is to cut it.Watch at: 17:40 / 18:00Now, what it does, is it takes those double helices.I'll represent the double helix as a thickerkind of thing now.That was my double helix, this other double helixwrapped around it.It's got to cut it.Now, when I take two DNAs that are wrapped around each otherWatch at: 18:00 / 18:20or two DNAs that are separate, have I done anychemistry on them?I'm sorry.Are they chemically different?They're chemically the same molecules.But they're topologically different.Topologically means wrapped around.In one case, they were topologically entangled.In the other case, they're topologically separated fromeach other.Watch at: 18:20 / 18:40So they're still the same chemical bonds, the samemolecules, but when I separate these two double helices now,the difference between these is that they are what arecalled topoisomers.They are isomers because they're exactly the samechemical formula.But they're topoisomers because theyWatch at: 18:40 / 19:00have different topology.They're not wrapped around each other anymore.So it turns out there is an enzyme that just gets in thereand makes a double stranded cut in one of the doublehelices, grabs the two ends, passes it around the otherside, and ligates them back together, and keeps doing thatuntil they're disentangled.Watch at: 19:00 / 19:20Pretty clever.Cut, paste, cut, paste till it can separate those two doublehelices from each other.Remarkably, this enzyme is called topoisomerase.This job is done by topoisomerase, actually, byWatch at: 19:20 / 19:40topoisomerase II.There's a couple of different topoisomerases, and it'stopoisomerase II that does this particular job, cuts andseals up that double-stranded break.All right.Watch at: 19:40 / 20:00It is amazing how this works.Let's take another problem in how we do DNA replication.So let's deal with fidelity.The fidelity, accuracy of replication.Watch at: 20:00 / 20:20I have my strand.Which direction do we go?We go, for this template, five prime to three prime.This way goes five prime to three prime, the oppositedirection there.I now add on.Watch at: 20:20 / 20:40If this is a T, what do I add in?AUDIENCE: [INAUDIBLE].ERIC LANDER: If it's a GCGTAAT, et cetera.Why does the right base go in?Why does the right base go in?Yeah?AUDIENCE: Hydrogen bonding.Watch at: 20:40 / 21:00ERIC LANDER: Hydrogen bonding.It's got that these hydrogen bonds.AT makes two hydrogen bonds.GC makes three hydrogen bonds.The wrong base could never go in.Sorry.In biochemistry, do you ever say never?No, we say K equilibrium.We say how much more unfavored is it for thewrong base to go in?Watch at: 21:00 / 21:20It's not impossible, it's just disfavored, because it'senergetically less good.How much energetically less good is it?What is the delta G for putting in the wrong base?It's not infinity.It turns out that there is an equilibrium constant forWatch at: 21:20 / 21:40putting in the wrong base, and that is K equilibrium is about10 to the third for the right base, 10 to the minus thirdfor the wrong base.Thank goodness.Watch at: 21:40 / 22:00So only one time in 1,000 does it put in the wrong base.That's what that has to mean, right?If it's 1,000 times less favored energetically, itmeans you only make a mistake one letter in 1,000.How do you feel about that for your own genomes?Is that a level of quality control youare satisfied with?Watch at: 22:00 / 22:20AUDIENCE: No.ERIC LANDER: No.How big is a typical gene?Typical gene is, in terms of its protein codinginformation, you guys already know about DNA goes to RNAgoes to protein.It's about 2,000 bases of protein coding information.That guarantees two mistakes per cell division.Not good.Watch at: 22:20 / 22:40Two mistakes per cell division.That's not OK.That's two mistakes per cell division.That would be two errors per cell division, and you have alot of cell divisions, you're in a lot of trouble.So it turns out something more is needed.Quality control is needed.Watch at: 22:40 / 23:00So later, it was discovered that the enzyme DNApolymerase, which has a five prime to three primeWatch at: 23:00 / 23:20polymerization activity also does a second thing.That same enzyme, DNA polymerase, is also a threeprime to five prime exonuclease.Watch at: 23:20 / 23:40What do you think an exonuclease is?AUDIENCE: [INAUDIBLE].ERIC LANDER: Take stuff out.So it adds bases in the forward direction, but it alsogoes backwards and takes bases out.Isn't that dumb?I thought we were trying to synthesize, but we're alsounsynthesizing.Watch at: 23:40 / 24:00With some probability, it goes backwards and takes out bases.Turns out that the probability of taking out a base backwardsis higher if it's the wrong base.It's proofreading as it goes as I hope you are.Watch at: 24:00 / 24:20It's proofreading.It goes backwards and takes bases out more often.Sometimes it takes out the right bases, but it isproofreading its work.Watch at: 24:20 / 24:40And more often when it's the wrong base, it goes backwards,and so you get the benefit of a K equilibrium from theoriginal base.And then there's a separate K equilibrium for theproofreading, and that helps you.And when you combine the proofreading with the originalaccuracy, now, we're down to something like 10 to the minusWatch at: 24:40 / 25:00five or 10 to the minus six errors perbase, per cell division.It's only making on the order of one error per million.Watch at: 25:00 / 25:20Now are we satisfied?No.You guys pretty hard nosed.Not good enough, because you have 50 cell divisions to makemore and some cells go through many, many,many more cell divisions.Not acceptable.But it's a start.Watch at: 25:20 / 25:40So proofreading helps.So we have the fidelity of replication.Replication makes an error at a rate of 10to the minus third.Proofreading brings you down to 10 to the minus six, andthere's another process.Watch at: 25:40 / 26:00There are a set of enzymes that go around and feel theDNA double helix after it's finished, and if you put inthe wrong base, the width of the helix is not right.The shape is wrong.It feels for mismatches.So there is a mismatch repair system.Watch at: 26:00 / 26:20Mismatch repair comes along, and if there was an errorright here, the helix bulges out too much let's say.Mismatch repair cuts, removes some DNA, and gives the cellWatch at: 26:20 / 26:40another chance to do it again.Mismatch repair gets you down to something in theneighborhood of 10 to the minus eighth, 10to the minus ninth.Let's say for the sake of argument, 10to the minus ninth.You're genome is about three times 10 to the ninth.Watch at: 26:40 / 27:00Now making that's one or two errors per genome,that's not so bad.Why do we care?Why am I bothering you with this?Who cares between 10 to minus sixth, 10 to the minus ninth?Big deal.Well, a few percent of you in this class are heterozygousWatch at: 27:00 / 27:20for a mutation in the mismatch repair enzymes.Don't worry.Your cells have the other copy that's good.But suppose one of your cells were to lose, by mutation, theWatch at: 27:20 / 27:40good copy of the mismatch repair enzyme?And now that cell in your body had no copies of mismatchrepair enzyme.What do you think is going to happen to your DNAreplication?Instead of being one in a billion, it would be one in amillion accuracy.Watch at: 27:40 / 28:00Turns out you have an extremely highrisk of colon cancer.There are hereditary colon cancer syndromes that are dueto inherited defects in the mismatch repair system.It is not at all trivial.Hereditary polyposis coli is due to adefect in this enzyme.Watch at: 28:00 / 28:20It matters.You've got to get it down to that level because otherwise,you're getting mutations that cause cancer, that is, whenyou lose both copies, if you lost both copies.Most of your cells would be fine, but if you'd lose theother good copy, by chance, that cell cango on to cause cancer.Watch at: 28:20 / 28:40So this stuff actually matters.Finally, finally, speed.Kind of fun to talk about speed.How fast does polymerase work?It turns out that polymerase is able to polymerize 2,000Watch at: 28:40 / 29:00nucleotides per second.That's very impressive to me.It zips along at 2,000 nucleotides per second,installing the right base, getting it right only 99.9% ofthe time, proofreading as it goes, and gets the whole thingWatch at: 29:00 / 29:20done 2,000 letters in a second.That is impressive engineering.That is really impressive engineering.So that's kind of how DNA replication works well, exceptfor one thing.Watch at: 29:20 / 29:40Kornberg was a biochemist.Biochemists purify things in test tubes.Watch at: 29:40 / 30:00He discovered an enzyme, Kornberg's polymerase.How do we know it's the enzyme the cell actuallyuses to copy its DNA?Watch at: 30:00 / 30:20See, I'm a geneticist.I look at Kornberg and I say, nice job.You showed me an enzyme that in a test tube is capable ofpolymerizing DNA.How do I know that's the enzyme that's actually doingit from the cell copies its whole genome?Watch at: 30:20 / 30:40What does a geneticist want to see?AUDIENCE: A mutant.ERIC LANDER: A mutant.Show me a mutant then I'll believe.So someone went along and took E. colis one at a time becausewhat else could they do.Watch at: 30:40 / 31:00And for every single E. coli they grew up from a plate,they purified Kornberg's enzyme.And you know what they found?They found a mutant E. coli that lacked Kornberg's enzyme,and it could replicate its DNA just fine.Watch at: 31:00 / 31:20What does that tell us?Kornberg actually had the wrong enzyme.He still deserves a Nobel Prize for it because he got anenzyme that could copy DNA.It's actually not the main enzyme that does the job.Because we can make a mutant that lacks that enzyme and itWatch at: 31:20 / 31:40can still copy the DNA, it can't be the main enzyme.Turns out what Kornberg found was a minor polymerase thatwas used in those mismatch repair situations that wouldcome along and do the tidying and clean up.The main enzyme turned out to be another enzyme, a morecomplicated enzyme.Watch at: 31:40 / 32:00So my point about biochemistry and genetics both having totalk to each other, you only really know something when youhave it from a biochemical point of view and the geneticpoint of view.The two have to go together.Kornberg's enzyme is a great enzyme,it's a fantastic enzyme.It just happens not to be the main enzyme, and you can onlyWatch at: 32:00 / 32:20know that by genetics.Of course, you can only purify it by biochemistry.All right.So that's DNA replication.Any questions about DNA replication before I go on?Yes?AUDIENCE: [INAUDIBLE].ERIC LANDER: Polymerase III or polymerase II, depending onthe organism.They're all called polymerases.They're all DNA polymerases.Watch at: 32:20 / 32:39They just get different names and numbers.Turns out most cells have multiple polymerases andKornberg found the kind of simpler polymerase.The main replication polymerase also calledpolymerase but with a different number, is adifferent more complicated enzyme.Yes?AUDIENCE: How does the enzyme know which one is the right..?ERIC LANDER: how does it know which one is right?Watch at: 32:39 / 33:00AUDIENCE: [INAUDIBLE].ERIC LANDER: Because 50% of the time you get it wrong.Do you know what bacteria do?What a great question.How would it know which one to get right?Know what bacteria do?They're very tricky.They mark their DNA, don't worry about this.Watch at: 33:00 / 33:20They mark their DNA with methyl groups.There is an enzyme that comes along and put methyl groups atcertain positions, but that enzyme is kind of slow.So I have a methyl-marked DNA double helix.When I replicate it, the new strand is made, and what doesthe new strand lack?AUDIENCE: Little methyl groups.Watch at: 33:20 / 33:39ERIC LANDER: Little methyl groups.It'll get them eventually because that slow enzyme willcome along and put them on, but mismatch repair is fast.So what is mismatch repair looking for?The little methyl groups that are kind of breadcrumbs thatsay, this was the old strand, and this guy is the new stand.It's thought of everything.It's really smart.Watch at: 33:39 / 34:00Very, very smart.