ERIC LANDER: And so the issue
became how does DNA replication work. And so I'm about
to go into it. Now, I'm going to note we're
going to be starting this DNA goes to RNA, goes to protein,
and DNA goes to itself. DNA is replicated. It makes RNA. The RNA is used to
make protein. This will be what
we'll be talking about today and tomorrow. So the first step of that
is, how does DNA give rise 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. I got to choose a cell in which
I'm likely to find an enzyme, 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? So 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'll see if one of those fractions
has the ability to copy DNA. If we're going to run
an assay, we have to give it a substrate. What substrate would you
like to give it? What do you think it needs? AUDIENCE: [INAUDIBLE]. ERIC LANDER: It better have
some free nucleotides otherwise, 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 a double helix. So what should we give it? AUDIENCE: [INAUDIBLE]. ERIC 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 little sequence actually, here. Let's say A phosphate, T
phosphate, G phosphate, C phosphate, A phosphate, T
phosphate, T phosphate, A phosphate, G phosphate,
G phosphate. I'm going to not write the
phosphates too much longer, guys, but anyway C phosphate,
C phosphate, T phosphate, like that. Pretty soon, in fact, almost
immediately, I'm going to start dropping the phosphates
in here. But that's the way it goes. All right. That's a template. We need floating around in the
solution some trinucleotides. We have some nucleotides
floating around. And now will this enzyme work? We would try different fractions
and see if it's able to just install the right
letters in the right place. Now, it turned out it needed one
more thing, and the person who 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, phosphate C, phosphate G,
phosphate T, phosphate A, let's say like that. So this is the five
prime end of DNA. Remember the phosphate is
hanging off the five prime carbon, right? What's look at the other end. The other end ends in the
hydroxyl on the three prime end of the ribose. Since this is anti-parallel,
this strand is going five prime 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 five prime and three prime. There you go. If you're handed a primer to get
a head start, and you're handed a template, and you hand
it some nucleotides, you then assay different fractions
exactly as you suggested and we see is one of them capable
of extending this strand by putting in an A, putting in a T,
putting in a C, putting in a C, putting in a C, putting
in a G. That's the assay. And Arthur Kornberg discovered
an enzyme that could do this. And the biochemists went nuts. They thought, wow. This is so cool. Kornberg is able to discover
an enzyme that can accomplish this. The enzyme polymerizes DNA. Coincidentally, what is
the enzyme called? AUDIENCE: DNA polymerase. ERIC LANDER: DNA polymerase. Accidentally, has a nice name. Good. DNA polymerase. Excellent. Now, notice what it does. It takes this triphosphate, puts
it in here, and it joins it 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. What direction is the synthesis
proceeding? Starts here at the five prime
end, and it moves adding on to the three prime end. So it's five prime to three
prime direction. That's the direction it moves. It adds to the three
prime end. It adds to the free
nucleotides to the 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. You see, suppose we were
going the other way. Suppose the primer
was this way. Where would as we added each
base, the triphosphate would be on the strands, right? And we'd be adding to the
three prime end here. That means the energy supplied
by the triphosphate would be on the growing strands rather
than in the free nucleotides. Why would it be a terrible idea
to put your energy source on the growing strand? MIKE: [INAUDIBLE]. ERIC LANDER: Well Mike, you
know, those triphosphate bonds are pretty unstable. They hydrolyzed by themselves
at some frequency. If you're a free nucleotide
and the triphosphate hydrolyzes, big deal. That free nucleotide floating
around loses its triphosphate. But what if I'm the growing
strand, and I lose my triphosphate? AUDIENCE: [LAUGHS] ERIC LANDER: Exactly. AUDIENCE: 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 want your triphosphates, those
hydrolyzable triphosphates to be 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 going and how it is, and it's
kind of interesting. Any way. All right. So Kornberg wins the Nobel
Prize for this. Good 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. So let's suppose I have a double
strand of DNA, and I'm just going to open it up here,
five prime to three prime, five prime to three prime. I need to get like
a primer here. Then the primer can be extended
by polymerase. Well, where's the primer
come from? It turns out there is an enzyme
specially devoted to making those primers. Kornberg didn't know it,
but there's an enzyme. And by coincidence, it
is called primase. Exactly. Primase makes the primer. So you need a primer here, and
the primer is made by primase. Once primase makes a primer,
polymerase can chug along and do it just fine. Let's check out the
other strand. Primer here, polymerase
chugs along. But now as this double
helix opens up, what happens over here? The synthesis going this way. So what do I have to do here? AUDIENCE: [INAUDIBLE]. ERIC LANDER: Another primer. Need another primer. Then as it opens up more,
what do I need? AUDIENCE: Another primer. ERIC LANDER: Another primer. So the two strands are
experiencing very different kind of replication. In one place, one primer in the
five prime to three prime direction is enough
to keep going. In the other strand, as it keeps
opening up, you gotta keep making primers. You have all these little
fragments there. Now, those little fragments were
discovered by Okazaki, and they are called
Okazaki fragments. Again, I just mention
these things. They are known to molecular
biologists. But these little guys are
Okazaki fragments, and they tell 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? They'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. Here 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 a standard English word not used
that often is to ligate two things together. Ligature, for example,
in music. You ligate things together. How do you think the cell deals
with ligating these things together? An enzyme called-- AUDIENCE: Ligase. ERIC LANDER: Exactly. So ligase does the ligation. Ligase ligates. It is so lucky that these
words turn out to have accidentally made sense. It's really cool. So ligase ligates. Now, I'll tell you a factoid,
but don't worry about it too much. Primase actually doesn't
make DNA. We haven't gotten there
yet, but it turns out primase makes RNA. Turns out to be easier
to start an RNA than a DNA from scratch. Cell doesn't like to start
DNA from scratch. It likes to start RNA from
scratch as we'll get to a moment with transcription. So as a factoid, I'll mention
to you that those little primers are actually RNA
primers, and what happens is they get extended into DNA, and
they bump into and kind of displace the previous RNA, so
it's slightly more complicated than I told you. You're welcome to forget that. If you would like to believe
that primase is actually making 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 other machinery that has to
deal with that. But the basic concept five prime
to three prime, little primers, getting extended,
getting ligated, that's how you make your DNA. And you can check it
out, and it works. All right. Well, it turns out to even be
a little more complicated. That was how we got the
synthesis going, but we also have 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. I can't give my test tube a
primer, because I don't know how the cell would make a
primer, he wouldn't have made any progress. So he throws in the primer
and says, the cell will figure it out. I'm just giving it a primer,
and I'll see what happens. Now, there's another problem,
this topological problem that also can make your head hurt. Let me try to explain what the
topological problem is. Suppose I have DNA like that. Make that a little prettier. So I have some DNA like that. And maybe it goes around for
a very long distance like a circle 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. And 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, to synthesize a new strand, those
two new double helices are so totally intertwined
with each other. Every turn that there was in
the double helix is now a twist and turn connecting the
two, sort of entangling the two helices. So I have the two new
double helices entangled 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 two new circles will be totally
wrapped around each other with a gazillion wraps. No way they're going to
two daughter cells. Now, here is where
mathematicians are very useful, because it is a theorem
that if I take two circles wrapped around each
other like that, there is no topological deformation
possible that can separate them. It's like these puzzles, you
get some strings wrapped around each other
separate them. It's a theorem that two circles
wrapped around each other 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 to separate 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. Now, what it does, is it takes
those double helices. I'll represent the double
helix as a thicker kind of thing now. That was my double helix,
this other double helix wrapped around it. It's got to cut it. Now, when I take two DNAs that
are wrapped around each other or two DNAs that are separate,
have I done any chemistry 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 from each other. So they're still the same
chemical bonds, the same molecules, but when I separate
these two double helices now, the difference between these
is that they are what are called topoisomers. They are isomers because they're
exactly the same chemical formula. But they're topoisomers
because they have different topology. They're not wrapped around
each other anymore. So it turns out there is an
enzyme that just gets in there and makes a double stranded
cut in one of the double helices, grabs the two ends,
passes it around the other side, and ligates them back
together, and keeps doing that until they're disentangled. Pretty clever. Cut, paste, cut, paste till it
can separate those two double helices from each other. Remarkably, this enzyme is
called topoisomerase. This job is done by
topoisomerase, actually, by topoisomerase II. There's a couple of different
topoisomerases, and it's topoisomerase II that does this
particular job, cuts and seals up that double-stranded
break. All right. It 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. I 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 opposite direction there. I now add on. If 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. ERIC 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 the wrong base to go in? It's not impossible, it's just
disfavored, because it's energetically 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 for putting in the wrong base, and
that is K equilibrium is about 10 to the third for the right
base, 10 to the minus third for the wrong base. Thank goodness. So 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, it means 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 you are satisfied with? AUDIENCE: No. ERIC LANDER: No. How big is a typical gene? Typical gene is, in terms
of its protein coding information, you guys already
know about DNA goes to RNA goes to protein. It's about 2,000 bases of
protein coding information. That guarantees two mistakes
per cell division. Not good. Two 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 a lot of cell divisions, you're
in a lot of trouble. So it turns out something
more is needed. Quality control is needed. So later, it was discovered
that the enzyme DNA polymerase, which has a five
prime to three prime polymerization activity also
does a second thing. That same enzyme, DNA
polymerase, is also a three prime to five prime
exonuclease. What do you think an
exonuclease is? AUDIENCE: [INAUDIBLE]. ERIC LANDER: Take stuff out. So it adds bases in the forward
direction, but it also goes backwards and
takes bases out. Isn't that dumb? I thought we were trying to
synthesize, but we're also unsynthesizing. With some probability, it goes
backwards and takes out bases. Turns out that the probability
of taking out a base backwards is higher if it's
the wrong base. It's proofreading as it goes
as I hope you are. It's proofreading. It goes backwards and takes
bases out more often. Sometimes it takes out the
right bases, but it is proofreading its work. And more often when it's the
wrong base, it goes backwards, and so you get the benefit of
a K equilibrium from the original base. And then there's a separate
K equilibrium for the proofreading, and
that helps you. And when you combine the
proofreading with the original accuracy, now, we're down to
something like 10 to the minus five or 10 to the minus
six errors per base, per cell division. It's only making on the order
of one error per million. Now are we satisfied? No. You guys pretty hard nosed. Not good enough, because you
have 50 cell divisions to make more and some cells go
through many, many, many more cell divisions. Not acceptable. But it's a start. So proofreading helps. So we have the fidelity
of replication. Replication makes an error
at a rate of 10 to the minus third. Proofreading brings you down
to 10 to the minus six, and there's another process. There are a set of enzymes that
go around and feel the DNA double helix after it's
finished, and if you put in the 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. Mismatch repair comes along,
and if there was an error right here, the helix bulges
out too much let's say. Mismatch repair cuts, removes
some DNA, and gives the cell another chance to do it again. Mismatch repair gets you down
to something in the neighborhood of 10 to
the minus eighth, 10 to the minus ninth. Let's say for the sake
of argument, 10 to the minus ninth. You're genome is about three
times 10 to the ninth. Now 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 heterozygous for 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, the good copy of the mismatch
repair enzyme? And now that cell in your body
had no copies of mismatch repair enzyme. What do you think is going
to happen to your DNA replication? Instead of being one in a
billion, it would be one in a million accuracy. Turns out you have
an extremely high risk of colon cancer. There are hereditary colon
cancer syndromes that are due to inherited defects in the
mismatch repair system. It is not at all trivial. Hereditary polyposis
coli is due to a defect in this enzyme. It matters. You've got to get it down to
that level because otherwise, you're getting mutations that
cause cancer, that is, when you lose both copies, if
you lost both copies. Most of your cells would be
fine, but if you'd lose the other good copy, by chance,
that cell can go on to cause cancer. So 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 2,000 nucleotides 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% of the time, proofreading as it
goes, and gets the whole thing done 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, except for one thing. Kornberg was a biochemist. Biochemists purify things
in test tubes. He discovered an enzyme,
Kornberg's polymerase. How do we know it's the enzyme
the cell actually uses to copy its DNA? See, 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 of polymerizing DNA. How do I know that's the enzyme
that's actually doing it from the cell copies
its whole genome? What 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 because what else could they do. And 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. What does that tell us? Kornberg actually had
the wrong enzyme. He still deserves a Nobel Prize
for it because he got an enzyme 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 it can still copy the DNA, it
can't be the main enzyme. Turns out what Kornberg found
was a minor polymerase that was used in those mismatch
repair situations that would come along and do the tidying
and clean up. The main enzyme turned out to
be another enzyme, a more complicated enzyme. So my point about biochemistry
and genetics both having to talk to each other, you only
really know something when you have it from a biochemical point
of view and the genetic point 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 only know 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 on the organism. They're all called
polymerases. They're all DNA polymerases. They just get different
names and numbers. Turns out most cells have
multiple polymerases and Kornberg found the kind
of simpler polymerase. The main replication polymerase
also called polymerase but with a different
number, is a different 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? AUDIENCE: [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. They mark their DNA with
methyl groups. There is an enzyme that comes
along and put methyl groups at certain 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 does the new strand lack? AUDIENCE: Little
methyl groups. ERIC LANDER: Little
methyl groups. It'll get them eventually
because that slow enzyme will come 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 that say, this was the old strand,
and this guy is the new stand. It's thought of everything. It's really smart. Very, very smart.
