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So in the last lecture I spent quite
a while trying to convey a sense of

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how the structure of DNA was
discovered.  The crystallographic

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data that led to it,
as I said, was collected by Roslyn

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Franklin.  And I saw there was some
confusion about this picture that I

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showed you next.
This is not a photograph of a

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double helix.
This is what happened when she

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bounced the x-ray off the crystal of
DNA.  This is the diffraction

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pattern that she saw.
And then one works backwards from

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that trying to figure out what kind
of structure it was that would have

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caused that diffraction pattern.
And you have to be a pretty good

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x-ray crystallographer to draw any
kind of inferences from that.

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And there people, including Francis
Crick, who saw the implications

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of it right away.
But the point was she collected the

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data and then two people that I told
you about then whose name you know

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so well, Jim Watson and Francis
Crick, were the two individuals that

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came up with the model that
explained the diffraction pattern.

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And therefore we learned the
structure of DNA as a

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double-stranded helix.
I also tried to make the case that

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it wasn't two geniuses who sat down
in the room, took a look at this and

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popped up with the model.
It was a story of real people with

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misadventures and mistakes and
recovery from mistakes and so on

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getting it.  It was also a very
small group.  And I'm going to take

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just a very small minute at the
beginning of the class because I

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have a colleague,
Vernon Ingram who's sitting down

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here in the front,
who was a member of this very small

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group with Jim Watson and Francis
Crick.  So here was there where all

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this happened.
And almost nobody in the world has

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had a chance in your generation to
hear directly from somebody who was

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there when it happened.
So asked Vernon if he would come

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and just talk to you for a little
bit just what it was

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like to be there.

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Well, thanks, Graham.
You seem to be at a very exciting

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state in 7.014.
This structure of the secret of life,

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no less.  And it's interesting that
immediately when Watson and Crick

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put together a model of the DNA
molecule that fit the x-ray data,

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that was the point, how do you know
a model is correct?

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Because there are certain distances
in the model, and those have to

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correlate exactly with the distances
of the x-ray spots in the

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diffraction pattern that you saw.
That's how you know that a model

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that you've built to certain
specifications corresponds to what

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the molecule of itself in the
crystal that you're examining

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actually is composed of.
It was by sheer accident that I

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happened to be working as a
biochemist in the MRC,

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The Medical Research Council lab at
the Cavendish Laboratory where

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Watson and Crick were working.
Sheer accident.  It was a very

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crowded lab, as Graham said.
And that's something that you should

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remember.  When you're choosing a
lab to work in,

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always go to a lab that's
overcrowded.  Never go to a lab

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where there's lots of space because
a really successful lab attracts so

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many coworkers,
visitors that it rapidly gets

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overcrowded.
And that was the case in this

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laboratory.  The director was Max
Perutz.  Co-director John Kendrew

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doing x-ray crystallography of
proteins for almost the first time,

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and solving the protein structure.
Francis Crick was a graduate student

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of Max Perutz's doing his PhD work.
And the first thing I remember about

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Francis was when I went there as a
biochemist to work with Max Perutz,

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when I went there, there was this
tall gangling guy constantly

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circulating between the top floor of
the building, his office in the

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middle and the x-ray machines at the
bottom.  He was constantly going up

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and down.  And in those days the
buildings didn't have an elevators

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or lifts as the English
called them.

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So he was in excellent physical
shape.  Very crowded,

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a very modest lab.  And what's
usually forgotten is a key member of

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that group, an engineer,
Tony Broad, key person because he

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invented what was then the world's
best and most efficient x-ray

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machine, a rotating anode
x-ray machine.

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And because to the x-ray
crystallographers in that group this

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machine was available,
because of that they were the

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preeminent x-ray structure group in
the world.  My job was as a

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biochemist protein biochemistry
putting a heavy atom,

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mercury, very heavy atom into Max
Parutz's hemoglobin crystals

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in specific places.
That has a predictable effect on the

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x-ray pattern and that enables the
Fourier diagram to be constructed

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with real phase values for the x-ray
diffractions, for the physicists

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among you here.
Are there any physicists here?

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Yeah, I thought so.  That was a big
step forward and that was also a big

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step in figuring out the structure
of the DNA samples semi-crystals

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that Professor Walker
just referred to.

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All dependent on the engineer Tony
Broad who is never mentioned in any

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of these histories,
but without him this would not have

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happened.  So it was an exciting
place to work in,

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very exciting.  We were all young in
those days.  And living the lives of

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young men and young women with all
the complications that arise when

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you put a whole bunch of very
energetic young men,

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very energetic young women together.
And by that I mean the interpersonal

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relationships which when you're in a
crowded, very active situation can

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sometimes interfere.
And always very entertaining,

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I can tell you that.  I could give
you chapter and verse.

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But it isn't really so very
different from people

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your age now, right?
I mean I'm not saying it interferes

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with you, sometimes it might.
But it was an exciting lab, an

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exciting time to be there because we
were not the only group trying to

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figure out the structure of DNA.
A huge competitor was Linus Pauling

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at Caltech who had beaten that same
group once before,

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quite recently, over the alpha helix,
the crucial component of

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protein structure.
He got the right answer first,

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1.5 angstrom reflection, the alpha
helix.  And our group,

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Max Parutz and our group had been
wrong.  So the group was smarting

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under that kind of defeat,
if you like.

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And competition is a wonderful spur,
as long as you don't let it get out

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of hand.  Well,
needless to say we didn't,

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but the competition with the Pauling
lab was certainly so severe that we

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awaited the next letter.
You see, in those days new

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scientific information arrived not
by publications,

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that too much too long,
but by personal letter.  And,

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in fact, the NIH has put together
all these various letters in the

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Francis Crick collection.
And when you have time you should

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look at those.
They're quite interesting because

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they tell you in a way a scientific
paper does not tell you.

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What I feel about my experiment
results.  What she feels about her

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experiment results.
What it means to me as a person,

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to her as a person, to him as a
person.  So we were constantly

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watching the mail and discussing the
news as it came in,

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mostly over a beer at the pub next
door.  It was very conveniently

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located.
But being a small group crowded

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together made communication within
our group very easy indeed.

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And we had fights.  I don't mean
physical fights.

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We had scientific fights.
And as a biochemist I was able to

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settle a crucial fight among the
crystallographers Crick and Watson

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who were building the model.
Because, quite frankly,

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they didn't know much chemistry.
And were trying to build a model

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with the wrong confirmation of the
peptide bond.  They didn't realize

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that the peptide bond has two
possible confirmations.

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And they had at one point a
terrible time trying to fit

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everything together because they
were using the wrong confirmation.

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I'm talking about lactam-lactim for
those of you who are organic

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chemists and it means something,
a confirmation.  And once they got

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the first confirmation then the
model clicked into place.

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So we all helped, that's what I'm
trying to say.

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We all helped with one great aim in
mind.  It was clear.

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And you know from what Professor
Walker said, that the DNA structure,

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in its structure held the clue to
crucial physiological

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behavior of DNA.
And Crick and Watson said this in

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their first paper,
the structure itself because of its

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complimentarity gives you an
immediate clue as to how it

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replicates.  And replication of DNA
structure from generation to

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generation is,
of course, the crucial thing about

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DNA.
The copying, the precise copying

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from generation to generation.
And that fell out the of x-ray

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structure.  That's why the x-ray
structure was so very important,

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because it gave you an immediate
understanding of the role of DNA in

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modern biology.  So that's
what we did.

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And eventually the people in the
group, the group got so overcrowded

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they built a huge lab that was
beautiful, like any new lab is.

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But the thing I remember most of
all was the atmosphere in that place.

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So remember, when you go and choose
a lab, choose one that's overcrowded.

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It will pay off.  [APPLAUSE]

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Thank you so much.
That was really wonderful.

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Thank you.

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I don't know if some of you realized
quite how rare that was,

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this discovery of the structure of
DNA.  As I said,

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probably one of the big discoveries
of mankind.  Because,

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as Vernon said, you could see so
many of the secrets of life as soon

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as you saw that structure.
Very few people have ever heard

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from someone who was there at the
time.  Maybe you'll forget a bunch

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of stuff down the line,
but I hope you'll remember you heard

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somebody who was there when Wesson
and Crick were there and maybe his

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extra piece of advice about
choosing a lab.

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To say one thing quickly,
some of you I think understood what

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I've been trying to do.
I spent quite a bit of time talking

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about science being done by real
people doing real experiments.

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Thanks for your comments.  A few of
you have gone out of your way to say

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that this was a total waste of time
and you didn't understand why I

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didn't teach you something instead
of doing something on the test.

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Well, I'm making up the test.  And
if you don't think there'll be

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something on scientific process on
the second exam you'll

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be surprised.
So I'm spending a lot of time on

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this, and the reason is because you
are MIT student.

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You know, you can go many places in
the country to many high school

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biology courses and you can memorize,
someone will tell you to memorize

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everything that's in the book,
and you'll get tested whether you

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can memorize it.
You guys are at MIT because you

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have the potential to be leaders in
whatever you do.

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I've made the transition from being
an undergrad sort of trying to

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memorize stuff in a textbook to
working on a cutting-edge.

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I've made some reasonably
significant discoveries in science,

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as have my other colleagues in the
department, some of them making

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greater than I.
But nevertheless if you're on the

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cutting-edge then you're dealing
with all the stuff I'm trying to

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tell you about in this thing.
You're working as a part of a group.

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There's competition.
There are interpersonal

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relationships.
You make mistakes.

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You recover from them.
You're making inferences.

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You're testing models.  This is a
very complex, very real,

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very dynamic, very human interaction.
I hope you got a little bit of

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whiff of that from Vernon.
And I wouldn't be,

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I'm quite capable of reproducing
diagrams from the textbook without

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trying to give you a deeper
understanding,

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and that's what I'm trying to do
here.  And I hope if it hasn't made

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sense to you by the end that at
least a few more of you will get it.

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And those of you who I think saw
what I was doing I appreciate your

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telling me that in the things.
These are anonymous so I don't know,

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but a couple of you are certainly
trying to make it clear that you

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didn't think it was worth your time
coming to lecture.

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I'm trying to tell you why I'm
trying to do it.

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I'm trying to teach you in a deeper
way.  And this is a required course.

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It's important for your life.  I
hope some of you will see that or if

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you don't see it now you'll see it
later in your career.

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OK.  Now, we're going to talk about
DNA replication.

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I'm going to start to drive into
some of the details that maybe are

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more the kind of things you're
expecting.  I just want to make one

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quick point here.
I've talked about cell division and

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we saw this, how cells come from
other cells going to make more cells.

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I showed you this little movie
you've seen a few times of a yeast

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cell dividing,
but all cells divide.

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Here's a cancer cell dividing.
If you get a cancer it's a cell

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that's forgotten how to stop
dividing and is growing to make a

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tumor.  There's this cancer cell
dividing.  It looks not unlike a

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yeast on a molecular level,
very, very similar.  But there's

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another point.
I told you how the structure of DNA

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with the complimentary strands with
G pairing with C and A pairing with

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T immediately gave rise to an
insight as to how the genetic

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material could be replicated.
And you guys know that it's held

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together by hydrogen bonds between
base pairs which are about

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one-twentieth the strength of the
covalent bonds.

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So you're able to peel the strands
apart without breaking the covalent

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bonds.  And then by pairing A with T
and G with C and doing that on both

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strands then you can end up with two
identical copies.

219
00:19:02,000 --> 00:19:05,000
And so if you do two identical
copies and you do it again you get

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00:19:05,000 --> 00:19:09,000
eight.  One of the things we've
realized over the last two or three

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00:19:09,000 --> 00:19:13,000
years in looking through the exams
is somehow, at least some of the

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00:19:13,000 --> 00:19:17,000
class, didn't connect the business
about cells coming from other cells

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00:19:17,000 --> 00:19:21,000
and DNA duplicating to give daughter
DNA.  And I'm just trying to hammer

224
00:19:21,000 --> 00:19:25,000
home the point that these are
related.  Every time a cell divides

225
00:19:25,000 --> 00:19:29,000
it has to duplicate its
genetic information.

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00:19:29,000 --> 00:19:33,000
That's why I'm going to be telling
you about DNA replication.

227
00:19:33,000 --> 00:19:37,000
Here's a picture of that same
cancer cell, but watch over here.

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00:19:37,000 --> 00:19:41,000
This is the DNA.  And you see it's
doubled.  And see how the DNA,

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00:19:41,000 --> 00:19:46,000
which is the chromosomes, has pulled
apart so that at the end you now

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00:19:46,000 --> 00:19:50,000
have two cells and you've got
identical copies of DNA.

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00:19:50,000 --> 00:19:54,000
So if you're studying cancer,
for example, this sort of thing is

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relevant to you.  OK.  So
the issue of how --

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00:19:59,000 --> 00:20:03,000
Well, before I do that,
I'm sorry.  Just a couple of things

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00:20:03,000 --> 00:20:08,000
about DNA replication before I dive
into this.  So we all started out as

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00:20:08,000 --> 00:20:13,000
a single cell.
I've got a lot more obviously

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00:20:13,000 --> 00:20:18,000
because I'm made up of a lot of
cells.  If I took all the DNA in my

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00:20:18,000 --> 00:20:22,000
body and I wind up all the molecules
in it, do you guys have any idea how

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00:20:22,000 --> 00:20:27,000
long that would be?
Who thinks it would reach let's say

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00:20:27,000 --> 00:20:33,000
across the room?
OK.  Across campus?

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00:20:33,000 --> 00:20:39,000
Across Cambridge?  Around the world?
To the moon?  Anybody left?

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00:20:39,000 --> 00:20:46,000
To the sun?  I've got ten to the
fourteenth cells.

242
00:20:46,000 --> 00:20:52,000
There's about a meter or two in
each cell.  10 to 20 billion miles

243
00:20:52,000 --> 00:20:59,000
of DNA in each of our
bodies, human DNA.

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00:20:59,000 --> 00:21:03,000
They would go back and forth to the
sun multiple times.

245
00:21:03,000 --> 00:21:07,000
So that much DNA had to get
replicated in order for the

246
00:21:07,000 --> 00:21:12,000
fertilized egg we all started out as
to become you.

247
00:21:12,000 --> 00:21:16,000
Another thing,
the accuracy of replication is about

248
00:21:16,000 --> 00:21:21,000
ten to the minus tenth.
Most people, including myself,

249
00:21:21,000 --> 00:21:25,000
don't have a very good feel for
exponents.  So that's one

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00:21:25,000 --> 00:21:30,000
mistake in 10 billion.
You know, it could be one mistake in

251
00:21:30,000 --> 00:21:34,000
10 to the ninety-ninth.
Well, what is one mistake in 10

252
00:21:34,000 --> 00:21:39,000
billion mean?  So let's relate it to
something we know.

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00:21:39,000 --> 00:21:43,000
If I was typing let's say an eight
letter word, 60 words a minute,

254
00:21:43,000 --> 00:21:48,000
24 hours a day, 7 days a week, and I
was as good as DNA replication,

255
00:21:48,000 --> 00:21:52,000
how often would I make a mistake?
So you can each think of how long

256
00:21:52,000 --> 00:21:57,000
you think that is.
But if I was good on average,

257
00:21:57,000 --> 00:22:02,000
I would make a mistake once every 38
years.

258
00:22:02,000 --> 00:22:06,000
So I'm about to tell you about a
process that's absolutely

259
00:22:06,000 --> 00:22:11,000
astonishing in terms of how fast and
how much you can do and with an

260
00:22:11,000 --> 00:22:15,000
accuracy that goes beyond what we're
used to in our ordinary life.

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00:22:15,000 --> 00:22:20,000
So how does it do this?  It has to
be more than just pulling the

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00:22:20,000 --> 00:22:24,000
strands apart.
And there's been some confusion as

263
00:22:24,000 --> 00:22:29,000
to why I'm emphasizing
5 prime and 3 prime.

264
00:22:29,000 --> 00:22:34,000
Well, each of these subunits,
each nucleotide, this is a 3 prime

265
00:22:34,000 --> 00:22:39,000
hydroxyl and this is the 5 prime
position.  If we were joining

266
00:22:39,000 --> 00:22:44,000
together subunits that had a hook
and an eye it would make a

267
00:22:44,000 --> 00:22:49,000
difference because it's not the same
on both ends.  If we're going to

268
00:22:49,000 --> 00:22:54,000
start hooking together it's exactly
the same thing when we get to a

269
00:22:54,000 --> 00:22:59,000
biochemical level,
the 5 prime end is not the same as

270
00:22:59,000 --> 00:23:04,000
hydroxyl at the 3 prime end because
the whole thing is asymmetric.

271
00:23:04,000 --> 00:23:12,000
So the enzymes that copy DNA are
known as DNA polymerases.

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00:23:12,000 --> 00:23:20,000
And it was a very difficult
challenge to figure out how they

273
00:23:20,000 --> 00:23:28,000
operated, but Arthur Kornberg was
the first person to solve

274
00:23:28,000 --> 00:23:36,000
this problem.
He was an extraordinarily gifted

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00:23:36,000 --> 00:23:43,000
biochemist.  He's still at Stanford.
And what he found was if we have a

276
00:23:43,000 --> 00:23:49,000
5 prime end this would then be the 3
prime end, and there's a 3 prime

277
00:23:49,000 --> 00:23:56,000
hydroxyl which is this one right
here.  And this was paired,

278
00:23:56,000 --> 00:24:02,000
say, with a C and A paired with a T.
And let's say a G paired with a C

279
00:24:02,000 --> 00:24:07,000
here.  And let's say the next
template base was,

280
00:24:07,000 --> 00:24:12,000
let's make it a T.  What Arthur
Kornberg was able to find was an

281
00:24:12,000 --> 00:24:18,000
enzyme activity that catalyzed a
template-dependent replication of

282
00:24:18,000 --> 00:24:23,000
DNA.  That was critical because he
had to find, if you broke the cells

283
00:24:23,000 --> 00:24:28,000
open, somewhere in that gamish of
enzymes and things from

284
00:24:28,000 --> 00:24:33,000
inside a cell.
There had to be something that was

285
00:24:33,000 --> 00:24:37,000
able to copy DNA.
So in order to do that he had to

286
00:24:37,000 --> 00:24:41,000
work out an assay.
And he also had to have some kind

287
00:24:41,000 --> 00:24:45,000
of guess as to what the cell would
be using in order to carry out the

288
00:24:45,000 --> 00:24:49,000
synthesis.  But one thing that was
sort of obvious was a DNA template

289
00:24:49,000 --> 00:24:53,000
because that was being copies.
But the other part was you had to

290
00:24:53,000 --> 00:24:58,000
have energy to form
a covalent bond.

291
00:24:58,000 --> 00:25:02,000
So somehow there had to be something
that was sort of activated with the

292
00:25:02,000 --> 00:25:07,000
energy built into the molecule so
that thermodynamically the whole

293
00:25:07,000 --> 00:25:12,000
thing would slide downhill when you
made a bond.  And he knew that the

294
00:25:12,000 --> 00:25:17,000
cell had triphosphates,
just the same type that we talked

295
00:25:17,000 --> 00:25:36,000
about when we talked about ATP.

296
00:25:36,000 --> 00:25:43,000
So this would be a
deoxyribonucleotide triphosphate.

297
00:25:43,000 --> 00:25:51,000
And he was able to make a guess,
because he had to try things until

298
00:25:51,000 --> 00:25:59,000
he found something that would work,
that this was what's used in DNA

299
00:25:59,000 --> 00:26:06,000
synthesis.
So this hydroxyl ultimately attacks

300
00:26:06,000 --> 00:26:13,000
this phosphate here.
And these two other phosphates then

301
00:26:13,000 --> 00:26:21,000
come off as a leaving group.
So if we thought of it as a pea

302
00:26:21,000 --> 00:26:28,000
like this with two more peas here,
these two come off and you get a new

303
00:26:28,000 --> 00:26:36,000
bond formed to the phosphate.
And so what Kornberg then was able

304
00:26:36,000 --> 00:26:45,000
to find by using a DNA template that
had this sort of structure and

305
00:26:45,000 --> 00:26:54,000
[TTATA?] like this,
that he was now able to get an A

306
00:26:54,000 --> 00:27:04,000
added here.  This hydroxyl here
became the new hydroxyl.

307
00:27:04,000 --> 00:27:10,000
And so the direction of synthesis,
this strand is the other way, so the

308
00:27:10,000 --> 00:27:16,000
direction of synthesis of a DNA
polymerase, it's polymerizing in the

309
00:27:16,000 --> 00:27:22,000
5 prime to two 3 prime direction.
This was again an amazing discovery

310
00:27:22,000 --> 00:27:28,000
because it was the first time that
anyone had found an enzyme

311
00:27:28,000 --> 00:27:34,000
that could copy DNA.
Arthur Kornberg got a Nobel Prize

312
00:27:34,000 --> 00:27:38,000
for it.  But at this point actually
genetics came in because there was a

313
00:27:38,000 --> 00:27:43,000
scientist John Cairns who was at
that point down at Cold Spring

314
00:27:43,000 --> 00:27:47,000
Harbor, as I told you the other day.
And John, in spite of the fact that

315
00:27:47,000 --> 00:27:52,000
Arthur had found a DNA polymerase
that had all the properties that you

316
00:27:52,000 --> 00:27:56,000
would expect for copying DNA,
didn't think that was the one that

317
00:27:56,000 --> 00:28:01,000
actually copied the DNA necessary
for cellular replication.

318
00:28:01,000 --> 00:28:05,000
So he reasoned if he was right he'd
be able to find a mutation that

319
00:28:05,000 --> 00:28:09,000
would eliminate the activity of that
enzyme and the cell would still live.

320
00:28:09,000 --> 00:28:13,000
And so they did a screening,
and it was a lot of work, but they

321
00:28:13,000 --> 00:28:18,000
eventually found a mutant of E.
coli that lacked this DNA polymerase

322
00:28:18,000 --> 00:28:22,000
that Arthur Kornberg had discovered.
And the cell was still alive and

323
00:28:22,000 --> 00:28:26,000
was still replicating its DNA.
So it told both John and then

324
00:28:26,000 --> 00:28:31,000
Arthur there must be another
enzyme in the cell.

325
00:28:31,000 --> 00:28:34,000
And so Arthur went back.
And now working in a mutant that

326
00:28:34,000 --> 00:28:37,000
was missing this first polymerase he
discovered he found the one that

327
00:28:37,000 --> 00:28:40,000
really replicates the DNA.
The first one is important,

328
00:28:40,000 --> 00:28:44,000
too.  It's needed for DNA repair.
I'm going to talk to you about that

329
00:28:44,000 --> 00:28:47,000
in next lecture,
but it's not absolutely crucial for

330
00:28:47,000 --> 00:28:50,000
life.  And there's an interplay of
genetics and biochemistry.

331
00:28:50,000 --> 00:28:53,000
And you'll see I'm just sort of
foreshadowing what we're going to

332
00:28:53,000 --> 00:28:57,000
get to when we talk about
the genetics of this.

333
00:28:57,000 --> 00:29:00,000
And I know a couple of you clearly
were frustrated about me showing you

334
00:29:00,000 --> 00:29:03,000
pictures of the people who did this,
but nevertheless since this was such

335
00:29:03,000 --> 00:29:07,000
a historic event a couple of years
ago at Cold Spring Harbor.

336
00:29:07,000 --> 00:29:10,000
This you see the helix model down
there.  There was Jim Watson opening

337
00:29:10,000 --> 00:29:14,000
the symposium.
When I got up to talk I said,

338
00:29:14,000 --> 00:29:17,000
well, I told my students that I'd
let them know what it was like when

339
00:29:17,000 --> 00:29:21,000
I was there, so I took out a camera
and I took a picture of the audience.

340
00:29:21,000 --> 00:29:24,000
And so there are a bunch of Nobel
Laureates and types here who were

341
00:29:24,000 --> 00:29:28,000
sitting there smiling for you guys
in the class.  And there was Arthur

342
00:29:28,000 --> 00:29:32,000
Kornberg giving his talk.
Now, these DNA polymerases are

343
00:29:32,000 --> 00:29:36,000
incredible protein machines.
The crystal structures of DNA

344
00:29:36,000 --> 00:29:40,000
polymerases operating their template
have been solved.

345
00:29:40,000 --> 00:29:44,000
And you can solve,
depending on how many diffractions

346
00:29:44,000 --> 00:29:48,000
you can get, you can get a model
that's more and more detailed.

347
00:29:48,000 --> 00:29:52,000
And there have been very high
resolution models of DNA polymerases.

348
00:29:52,000 --> 00:29:56,000
This blue and white stuff is the
surface of the protein,

349
00:29:56,000 --> 00:30:00,000
and this is sort of a template and
the various parts.

350
00:30:00,000 --> 00:30:04,000
Just to give you an idea here are
these tracings of the shapes of the

351
00:30:04,000 --> 00:30:08,000
electron density.
You can see how the

352
00:30:08,000 --> 00:30:12,000
crystallographers have fit the
nucleotides right in the crystal

353
00:30:12,000 --> 00:30:17,000
into these electron densities.
And here putting it together a bit

354
00:30:17,000 --> 00:30:21,000
in the blue is the secondary
structure of the protein and the

355
00:30:21,000 --> 00:30:25,000
templates and whatnot.
And I don't expect you to see very

356
00:30:25,000 --> 00:30:29,000
much in that, but the point is I
wanted to sort of just set you up to

357
00:30:29,000 --> 00:30:34,000
show you this little movie.
Because DNA polymerases are

358
00:30:34,000 --> 00:30:38,000
incredible machines.
They copy at about a thousand

359
00:30:38,000 --> 00:30:43,000
nucleotides a second and their
accuracy is really amazing.

360
00:30:43,000 --> 00:30:47,000
And I'll tell you all the tricks to
the accuracy in the next lecture,

361
00:30:47,000 --> 00:30:52,000
but I want to show you this little
movie because this is sort of a

362
00:30:52,000 --> 00:30:56,000
simulation of what must happen every
time a nucleotide is added.

363
00:30:56,000 --> 00:31:01,000
Now, we'll see this over and over
again so I'll take it in pieces.

364
00:31:01,000 --> 00:31:04,000
The yellow and the orange are the
secondary structures.

365
00:31:04,000 --> 00:31:08,000
That's an alpha helix.
And certainly one thing you can see

366
00:31:08,000 --> 00:31:12,000
is happening, as we're looking at
this, is the parts of the protein

367
00:31:12,000 --> 00:31:16,000
are moving during this.
So you can see this alpha helix

368
00:31:16,000 --> 00:31:20,000
that's sort of swinging up and
swinging back down.

369
00:31:20,000 --> 00:31:24,000
Now, what's over here is the
template base.

370
00:31:24,000 --> 00:31:28,000
That's the base that correspondents
to the T that I was just

371
00:31:28,000 --> 00:31:31,000
showing you here.
This is the incoming nucleotide.

372
00:31:31,000 --> 00:31:35,000
There is the triphosphate coming
down here.  And,

373
00:31:35,000 --> 00:31:39,000
in fact, you just see those two
phosphates going.

374
00:31:39,000 --> 00:31:43,000
So what's happening here,
this is going to be the end of the

375
00:31:43,000 --> 00:31:46,000
growing chain.
It's going attack right there,

376
00:31:46,000 --> 00:31:50,000
join the phosphate and the
pyrophosphate will leave.

377
00:31:50,000 --> 00:31:54,000
And if you'll take a look,
when you see this movement of this

378
00:31:54,000 --> 00:31:58,000
helix from the beginning state to up
to here, you'll see what happens.

379
00:31:58,000 --> 00:32:02,000
It's squeezing the template base and
the incoming nucleotide together.

380
00:32:02,000 --> 00:32:07,000
What it's really doing is testing
for the correct shape.

381
00:32:07,000 --> 00:32:11,000
Remember the shape of an A-T base
pair and a G-C base pair is the same.

382
00:32:11,000 --> 00:32:16,000
And if those of you who are
confused about guanine and the

383
00:32:16,000 --> 00:32:21,000
keto-enol thing,
try to draw hydrogen bonds with the

384
00:32:21,000 --> 00:32:25,000
enol form of guanine and see how you
do.  I think you'll begin

385
00:32:25,000 --> 00:32:30,000
to understand a bit.
So at the heart of life is something

386
00:32:30,000 --> 00:32:36,000
that can copy DNA.
And there are these exquisitely

387
00:32:36,000 --> 00:32:42,000
beautiful machines.
The replica machine in E.

388
00:32:42,000 --> 00:32:48,000
coli has 18 proteins and the ones in
our bodies are even more

389
00:32:48,000 --> 00:32:54,000
sophisticated with even more parts.
OK.  But to replicate a DNA

390
00:32:54,000 --> 00:33:00,000
molecule there's another
problem that comes up.

391
00:33:00,000 --> 00:33:18,000
Because DNA polymerases copy --

392
00:33:18,000 --> 00:33:23,000
-- and grow chains in a 5 prime to 3
prime direction.

393
00:33:23,000 --> 00:33:36,000
And they need a 3 prime hydroxy

394
00:33:36,000 --> 00:33:43,000
terminus.  So they won't work if you
just gave it a single strand of DNA.

395
00:33:43,000 --> 00:33:51,000
No DNA polymerase can handle that.
It has to have something like this

396
00:33:51,000 --> 00:34:03,000
where there's a template strand --

397
00:34:03,000 --> 00:34:07,000
-- and there's what's known as the
primer strand.

398
00:34:07,000 --> 00:34:11,000
So there has to be something that
has the 3 prime hydroxyl and there

399
00:34:11,000 --> 00:34:16,000
has to be something that's going to
provide the template that's going to

400
00:34:16,000 --> 00:34:20,000
be copied.  So if we pull strands
apart like this with 5 prime to 3

401
00:34:20,000 --> 00:34:24,000
prime then they'll be 5 prime to 3
prime running in the opposite

402
00:34:24,000 --> 00:34:31,000
direction.
If we have a template like this,

403
00:34:31,000 --> 00:34:39,000
this is OK because the strand here
can be copied 5 prime to 3 prime.

404
00:34:39,000 --> 00:34:48,000
This is the new strand being
synthesized by the DNA polymerase.

405
00:34:48,000 --> 00:34:56,000
But what about the other strand?
The replication fork is moving in

406
00:34:56,000 --> 00:35:03,000
this direction, but if the --
So here is the 3 to 5 prime

407
00:35:03,000 --> 00:35:08,000
direction here.
So if the DNA polymerase is going

408
00:35:08,000 --> 00:35:13,000
to be copying this strand it's going
to be moving backwards to the

409
00:35:13,000 --> 00:35:17,000
direction of the replication fork.
Now, I guess evolution and nature

410
00:35:17,000 --> 00:35:22,000
could have selected for two types of
DNA polymerases,

411
00:35:22,000 --> 00:35:27,000
one that copies 5 prime to 3 prime
and one that copies in the opposite

412
00:35:27,000 --> 00:35:32,000
direction.   But it didn't.
And there are a number of

413
00:35:32,000 --> 00:35:36,000
theoretical reasons that we could
discuss in a more advanced course

414
00:35:36,000 --> 00:35:40,000
perhaps for why that is true.
But, in fact, what it does is it

415
00:35:40,000 --> 00:35:44,000
uses the same polymerase.
So as these things peel apart the

416
00:35:44,000 --> 00:35:48,000
polymerase works in the other
direction, but there's another

417
00:35:48,000 --> 00:35:52,000
problem.  If I just peel it apart
like this there's no 3 prime

418
00:35:52,000 --> 00:35:56,000
hydroxyl.  So it took people quite a
few years to figure out the strategy

419
00:35:56,000 --> 00:36:01,000
that's used in nature.
Nature has a special enzyme that

420
00:36:01,000 --> 00:36:08,000
makes a little piece of RNA.
It's called an RNA primer.  And

421
00:36:08,000 --> 00:36:15,000
what it does is it provides a 3
prime hydroxyl.

422
00:36:15,000 --> 00:36:22,000
And once you have the 3 prime
hydroxyl at the end of the little

423
00:36:22,000 --> 00:36:29,000
RNA chain then the
DNA polymerase --

424
00:36:29,000 --> 00:36:39,000
-- can be made 5 prime to 3 prime.

425
00:36:39,000 --> 00:36:47,000
So as you peel open the replication
fork then little pieces of RNA are

426
00:36:47,000 --> 00:36:55,000
used to make a new strand of DNA and
it goes this way.

427
00:36:55,000 --> 00:37:03,000
Now that obviously doesn't give you
a new intact DNA strand.

428
00:37:03,000 --> 00:37:08,000
And part of the clue to this working
out what was going on at DNA

429
00:37:08,000 --> 00:37:13,000
replication was the recognition that
newly synthesized DNA was made as

430
00:37:13,000 --> 00:37:18,000
little pieces.
And then later it got joined into

431
00:37:18,000 --> 00:37:23,000
longer pieces.
And the person who discovered this

432
00:37:23,000 --> 00:37:28,000
was Okazaki.  So these fragments of
DNA that are synthesized initially

433
00:37:28,000 --> 00:37:34,000
are called Okazaki fragments,
after the person who discovered this.

434
00:37:34,000 --> 00:37:38,000
It was rather puzzling because when
you tried to look at the synthesis

435
00:37:38,000 --> 00:37:42,000
of DNA you're looking at a long
molecule, and you found some of the

436
00:37:42,000 --> 00:37:47,000
newly synthesized material was in
short pieces.  And as you watched

437
00:37:47,000 --> 00:37:51,000
over time it got longer.
So the cell, I think you can sort

438
00:37:51,000 --> 00:37:55,000
of see from first principles what
has to happen here then.

439
00:37:55,000 --> 00:37:06,000
That in order to come and make --

440
00:37:06,000 --> 00:37:10,000
This strand is pretty easy to do,
but what the cells have to do now is

441
00:37:10,000 --> 00:37:14,000
they've got these little
RNA primers.

442
00:37:14,000 --> 00:38:25,000
And then they remove the RNA by an

443
00:38:25,000 --> 00:38:32,000
enzyme that's capable of degrading
the DNA or clipping it

444
00:38:32,000 --> 00:38:38,000
at the junction.
And that then leaves the cell in

445
00:38:38,000 --> 00:38:43,000
this sort of situation where there
are little tiny gaps in between

446
00:38:43,000 --> 00:38:48,000
these pieces of DNA.
But at the end of each one of these

447
00:38:48,000 --> 00:38:53,000
is a 3 prime hydroxyl.
So another polymerase or one or

448
00:38:53,000 --> 00:38:58,000
another polymerase in the cell can
fill those little pieces

449
00:38:58,000 --> 00:39:04,000
of DNA out.
And then there's one little nick

450
00:39:04,000 --> 00:39:10,000
that needs to be sealed.
And so what you finally end up with

451
00:39:10,000 --> 00:39:16,000
is a 3 prime hydroxyl here,
a 5 prime phosphate that's at the

452
00:39:16,000 --> 00:39:22,000
other end, and then these are joined
together.  This is one nucleotide

453
00:39:22,000 --> 00:39:28,000
here and the other here.
These are then joined together to

454
00:39:28,000 --> 00:39:34,000
give the ordinary phosphodiester
bond that links --

455
00:39:34,000 --> 00:39:41,000
-- the two nucleotides together like

456
00:39:41,000 --> 00:39:46,000
that.  And the enzyme that does that
is an enzyme called DNA ligase.

457
00:39:46,000 --> 00:39:51,000
You can almost think about it as
DNA Scotch tape that will take a

458
00:39:51,000 --> 00:39:56,000
little nick in DNA,
if we've got a phosphate and

459
00:39:56,000 --> 00:40:01,000
hydroxyl, and it will
join them together.

460
00:40:01,000 --> 00:40:06,000
So this process of replication,
which can go at about a thousand

461
00:40:06,000 --> 00:40:12,000
nucleotides a second with this
amazing degree of accuracy,

462
00:40:12,000 --> 00:40:17,000
uses two different DNA polymerases,
both of which biochemically can only

463
00:40:17,000 --> 00:40:23,000
go in one direction.
But you can see they have to be

464
00:40:23,000 --> 00:40:28,000
somehow oriented so that one of them
is able to move in this direction

465
00:40:28,000 --> 00:40:34,000
and the other one is able to move in
that direction.

466
00:40:34,000 --> 00:40:38,000
The key part in this sort of the
course is to try and understand this

467
00:40:38,000 --> 00:40:42,000
5 to 3 prime and to get this basic
idea that nature had to do something.

468
00:40:42,000 --> 00:40:47,000
It was fairly easy to copy one
strand because that was sort of the

469
00:40:47,000 --> 00:40:51,000
direction of the polymerase movement
was the same as the replication for

470
00:40:51,000 --> 00:40:55,000
it movement, but the other strand
had to have been much

471
00:40:55,000 --> 00:40:59,000
more a problem.
And so when you get down to a

472
00:40:59,000 --> 00:41:03,000
biochemical level,
though, it's very conceptually easy

473
00:41:03,000 --> 00:41:07,000
to say, oh, you've got complimentary
strands so we just take it apart,

474
00:41:07,000 --> 00:41:11,000
we take the photograph and the
negative and we make the opposite

475
00:41:11,000 --> 00:41:14,000
one and now we've got two copies.
When you get down to the

476
00:41:14,000 --> 00:41:18,000
biochemical details there is this
major biochemical issue of whether

477
00:41:18,000 --> 00:41:22,000
the polymerase can go in the 3 prime
or the 5 prime direction.

478
00:41:22,000 --> 00:41:26,000
And nature has chosen to do it all
or has been selected to do it all

479
00:41:26,000 --> 00:41:30,000
somehow with a polymerase
going in one direction.

480
00:41:30,000 --> 00:41:35,000
There are many other aspects to DNA
replication.  And one of the tricks

481
00:41:35,000 --> 00:41:40,000
that I find most fascinating is that
these polymerases,

482
00:41:40,000 --> 00:41:45,000
once they get on DNA they stay on.
And that's part of the secret

483
00:41:45,000 --> 00:41:50,000
because it takes about a millisecond
to add a nucleotide,

484
00:41:50,000 --> 00:41:55,000
but if it comes off the DNA it has
to get back on.  Then it

485
00:41:55,000 --> 00:42:00,000
takes about a minute.
So the whole trick to being a very,

486
00:42:00,000 --> 00:42:04,000
very fast DNA polymerase is to
somehow hang onto the DNA.

487
00:42:04,000 --> 00:42:08,000
So what biochemists did was they
purified the actual enzymatic

488
00:42:08,000 --> 00:42:12,000
activity that could carry out this
process, and then they started to

489
00:42:12,000 --> 00:42:16,000
look for other protein factors that
would help the process to work

490
00:42:16,000 --> 00:42:20,000
better.  And they discovered
something called a processivity

491
00:42:20,000 --> 00:42:24,000
factor which made the polymerase
stay on the DNA.

492
00:42:24,000 --> 00:42:28,000
And people wondered for a lot of
years how that worked and why did

493
00:42:28,000 --> 00:42:33,000
this system work so well.
And finally the crystal structure of

494
00:42:33,000 --> 00:42:38,000
the processivity factor was
discovered.  And if I go back to

495
00:42:38,000 --> 00:42:43,000
this sort of diagram where this is
the piece of DNA that's copied,

496
00:42:43,000 --> 00:42:48,000
what it turned out was that the
processivity factor is basically a

497
00:42:48,000 --> 00:42:53,000
doughnut that kind of gets clamped
around the DNA like that.

498
00:42:53,000 --> 00:42:58,000
So it's sort of like taking a
washer with a place where you can

499
00:42:58,000 --> 00:43:03,000
pry it apart opening it up,
putting it around the DNA like this.

500
00:43:03,000 --> 00:43:07,000
And then the polymerase,
more or less since this is

501
00:43:07,000 --> 00:43:11,000
topologically linked to the DNA,
is like a washer sliding on a wire.

502
00:43:11,000 --> 00:43:16,000
This DNA polymerase hangs onto that
and it doesn't come off.

503
00:43:16,000 --> 00:43:20,000
And I think there's a little
picture of it.

504
00:43:20,000 --> 00:43:25,000
Here's a little movie.
There's the DNA going through and

505
00:43:25,000 --> 00:43:29,000
this is one of these clamps.
It's virtually the same structure

506
00:43:29,000 --> 00:43:34,000
in a bacterium and inside of us.
But, in fact, the amino acids are

507
00:43:34,000 --> 00:43:38,000
almost all different.
But the underlying structure of the

508
00:43:38,000 --> 00:43:43,000
protein is almost identical.
And there are special machines

509
00:43:43,000 --> 00:43:48,000
called clamp loaders that pry open
this clamps, clamp them around DNA,

510
00:43:48,000 --> 00:43:52,000
and that's part of the secret to how
these polymerases are able to

511
00:43:52,000 --> 00:43:57,000
polymerize DNA so fast.
There are a lot of other pieces of

512
00:43:57,000 --> 00:44:02,000
this machinery.
If you go on you'll hear more about

513
00:44:02,000 --> 00:44:06,000
them.  I just want to give you one
of the most recent insights.

514
00:44:06,000 --> 00:44:10,000
I mean this, as you might guess,
since DNA replication is at the

515
00:44:10,000 --> 00:44:14,000
heart of life it's been studied very,
very hard, ever since the discovery

516
00:44:14,000 --> 00:44:19,000
of DNA helix.  My colleague,
Alan Grossman, made quite a

517
00:44:19,000 --> 00:44:23,000
discovery just probably three or
four years ago.

518
00:44:23,000 --> 00:44:27,000
He took that green fluorescent
protein that we've seen a few times,

519
00:44:27,000 --> 00:44:31,000
and he actually joined the gene
encoding green fluorescent protein

520
00:44:31,000 --> 00:44:36,000
to the backend of a piece
of the DNA polymerase.

521
00:44:36,000 --> 00:44:39,000
So wherever the DNA polymerase went
now there was a little fluorescent

522
00:44:39,000 --> 00:44:43,000
molecule.  And he looked to see
where it was in the cell.

523
00:44:43,000 --> 00:44:47,000
And I, like many other people,
had for years taught, and this is

524
00:44:47,000 --> 00:44:50,000
why, you know,
I have respect for the fact that I'm

525
00:44:50,000 --> 00:44:54,000
just teaching you the current model.
For much of my career I taught, so

526
00:44:54,000 --> 00:44:58,000
DNA polymerase is sort of like a
train going down the tracks a

527
00:44:58,000 --> 00:45:03,000
thousand molecules per second.
And we're doing all this stuff with

528
00:45:03,000 --> 00:45:09,000
the leading and with the two strands.
So let me just put those words up

529
00:45:09,000 --> 00:45:15,000
while I'm up there.
This one is called,

530
00:45:15,000 --> 00:45:22,000
this strand that's easy to replicate
is called the leading strand.

531
00:45:22,000 --> 00:45:28,000
And this one where you have to do
the primer and whatnot is called the

532
00:45:28,000 --> 00:45:33,000
lagging strand.
In any case, what I had taught was

533
00:45:33,000 --> 00:45:37,000
that polymerase was like a train
running on tracks.

534
00:45:37,000 --> 00:45:41,000
You could calculate how fast it
would move.  What Alan,

535
00:45:41,000 --> 00:45:45,000
to his amazing I imagine,
found was when he looked to see

536
00:45:45,000 --> 00:45:48,000
where the DNA polymerase was,
it wasn't spread out all over the

537
00:45:48,000 --> 00:45:52,000
cell as if you thought it was a
thing running on tracks.

538
00:45:52,000 --> 00:45:56,000
In fact, it was in the center of
the cell.  And then late in cell

539
00:45:56,000 --> 00:46:00,000
division it split into two spots
that went to the midpoints of what

540
00:46:00,000 --> 00:46:04,000
would be the daughter cells.
And so what he ended up realizing

541
00:46:04,000 --> 00:46:09,000
from that was that instead it was
more as if the polymerase was a

542
00:46:09,000 --> 00:46:14,000
factory and it pulled the DNA
through it rather than it traveling

543
00:46:14,000 --> 00:46:19,000
down the tracks of the DNA.
And that was a very surprising

544
00:46:19,000 --> 00:46:24,000
discovery that went against all the
dogma and all the pictures in the

545
00:46:24,000 --> 00:46:30,000
textbook. And it was
a discovery at MIT.

546
00:46:30,000 --> 00:46:34,000
That was published in,
I think it was 2001, something like

547
00:46:34,000 --> 00:46:39,000
that, a very recent discovery.
Things keep changing.  That's again

548
00:46:39,000 --> 00:46:43,000
why I keep emphasizing I cannot
teach you a fact in biology.

549
00:46:43,000 --> 00:46:48,000
I can teach you the best
understanding we have that explains

550
00:46:48,000 --> 00:46:52,000
the experiments to date.
But somebody may make a discovery

551
00:46:52,000 --> 00:46:57,000
tomorrow.  That means we'll have to
change our understanding.

552
00:46:57,000 --> 00:47:00,000
OK?  So good luck on the exam.
I'll see you on Monday, OK?