>> Chapter 3 it turns out
is just a juicy material
that I can't help but extend
it by one more lecture
and then next week, we'll be
back to our usual schedule
on each chapter taking
one a week, OK.
So next week will
be on Chapter 4,
the following week
Chapter 5, et cetera.
OK? What I want to talk to you
about today is DNA reactivity
with small molecules,
DNA biotechnology
and then how these all impacts
[inaudible] upon cancer and then
if we have time, we'll be
on Chapter 4, which is RNA.
OK so this topic of
cancer is, you know,
something that actually
[inaudible] I think everyone
in this group, I think
all of us at some point
or another knows someone
who has unfortunately dealt
with this terrible diseases.
It also impacts my students and
impacts students in this room.
Every couple of years of, I'll
have a student who tells me
that they fought off
cancer [inaudible].
So it's one of those
terrible diseases
that strikes all too often.
Anyway, our goal is to
take a 10,000-foot view,
high level view at the biology
of cancer and then zoom out
and start to understand
some of the treatments
that are used to treat cancer.
OK, some office hour
announcements.
All right, I have to make my
office hour a little earlier
because I have to catch a
flight to San Francisco tomorrow
and so my office hour is
going to be at 1:45 to 2:45
and then my Thursday office
hour is going to be canceled,
right and I assume no one
is interested in talking
to me after the midterm.
I know well how that works.
So, OK, so those are
the office hours.
Let's see what else
is happening.
Kritika has office
hour today and Mariam--
I think [inaudible]
office hour on Friday?
[ Inaudible Remark ]
OK, anyway, so that's the plan.
I'd also like Kritika
and Mariam to plan
for one extra office hour
this week before the midterm?
So OK? Good, OK, so they
very graciously agreed.
Thank you guys.
Yeah, so you will get an email
announcing additional office
hours as well, OK?
But I will have an office
hour tomorrow and a little bit
of an extraordinary time
and then after that, next--
the following week, we'll
be back to our usual times.
OK. Announcements.
[inaudible].
OK, midterm 1, that's
coming up in two days
so it's striking really soon.
First, there's going to be
a review session by the TAs
and the review session, like
the midterm is going to cover
through today's lecture.
OK, so everything
that's on today's lecture
or from the very first
lecture is fair game
for the midterm, OK?
And if you want to know what's
going to be on the midterm,
focus on the lectures, OK?
That's what I'll use to drive
the midterm as I write it.
The seating will
be assigned and--
it's already posted, OK, great.
So this has already been
posted to the website.
It's essential that you
sit in your correct seats.
The seating will be checked
and we'll be checking
IDs at the same time.
The way this will work is
halfway through the midterm,
the TAs will make an
announcement to pass your IDs
to either the right or the left
and then they'll walk around
and check the IDs to make sure
that everyone is seated
in the correct seats.
OK, so you also need to
bring a UCI student ID.
Now I realized some of you
don't have your student ID
for some reason, bring a
California driver's license,
a photo ID, we'll
accept that instead, OK?
You don't need any notes,
calculators, electronic devices
and it's really important that
you don't answer your phone
or pick up your notes
or anything
like that during the midterm
for obvious reasons, OK?
Questions about the midterm
or ab out anything else
that you'd like to know about?
You guys are so, so ready
for the-- all right.
Yeah?
[ Inaudible Remark ]
Discussion on Friday.
I think we need to, right?
Because we're going to have--
we already have a new discussion
worksheet that's posted
and we'll get too far behind
if we don't have
discussion on Friday.
[ Inaudible Remark ]
Yeah, I hear you.
OK, so we posted the
discussion worksheet
and it covers the
material that's covered
in today's lecture,
so the answer is yes.
But the worksheet and
the key will be posted.
Why don't we go ahead
and post that today, OK?
The key as well.
Usually we post the key
after the discussion.
This week, we'll
post it before, OK?
Is that all right?
>> Yeah.
>> OK. Other questions?
OK, again, if you want to
know what's on the midterm,
take a look at the
practice midterm
that I've already posted.
Take a look at the problems in
the book, the ones I've assigned
in the book, take a
look at the worksheets,
the discussion worksheets.
When I go to write the
exam, what I do is I sit
down with all that stuff.
And then, I always have some
concept problems as well
and I pull those straight
out of the lecture, OK?
So, the book is chock
full of information, OK?
It's a textbook and it's good
enough for high level audience.
But you can focus down your
studying just by focusing
in on the stuff that's being
covered and that I'm emphasizing
by citing in problems, OK?
So that you don't
necessarily have
to memorize the whole book, OK?
All right, well, I wish
you the best of luck.
It's going to be fun.
You guys did really well in
the past week and I'm going
to be aptly thrilled
to see how [inaudible].
OK. Oh OK, these are the
announcements for this week.
Start reading Chapter 4, work
the odd and asterisk problems.
Again, the midterm
this Thursday.
OK, so I'm done with
the questions
and the announcements, right?
Last chance?
OK. So last time, what I
was showing you is that DNA
in circular plasmids can be
bolded to an astonishing degree.
And I think all of
us were blown away
when we saw those
mighty bases of DNA.
That was plasmid DNA
to which a large number
of other single-stranded
DNAs had been annealed
to give you regions
of double-stranded DNA
that forced the plasmid into
the happy face structure, OK?
Or the little map of
the world structure.
And that's pretty intuitive
force type of stuff.
It's happening routinely in
laboratories around the country.
And it's something that
you could sort of take
for granted at this point.
It's ready-- It's doable.
We also talked about how
you can use plasmid DNA
to program cellular
biosynthesis.
And I told you about the
requirements for plasmid DNA,
that there has to be some
selection marker and there has
to be an origin of replication.
Once you have those
two requirements,
you can encode what kinds
of genes in that plasmid
with all kinds of instructions.
So, the instructions
could include things
like start building this
factory that's going
to produce diesel fuel
or start doing, you know,
this particular function that
causes you to glow green, OK?
So, the ability of program cells
using the plasmids has really
taken off.
And again, this is another
technique that's sort
in the toolkit-- in the toolkit
and it's just commonly accepted
as doable and you can all
simply start applying it
on your proposals.
OK, another thing that
we saw last time is
that DNA polymerase is a
remarkable machine and not
that it cranks out 1000
covalent bonds per minute,
not 1000 per second, OK?
So, it's really chirping along.
That's a very, very fast speed
at a thousand per minute.
And note that it's doing
this with perfect fidelity,
nearly perfect fidelity.
It's one mistake
every 10,000 or so.
And that's truly remarkable.
And what else do I want
to tell you about this?
We talked about the model, the
structural model, et cetera.
Are there any questions
about what you saw last time?
Yeah, over here, Chelsea?
[ Inaudible Remark ]
Yeah, so Chelsea's question is
just the one error every 10,000
or so, does that
include error correction,
and the answer is yes.
So, without error
correction and just rate
of errors will be the prior.
But it's still really
basic, I mean,
and also I should say this
varies quite a bit depending
upon which polymerase
you're talking about.
OK. And reverse transcriptase
is notable
for having a much
higher error rate.
OK, any other questions, anyone?
All right, let's move then.
Last time I end up
by showing you
that you can manipulate the
DNA of organisms as a way
of studying the phenotypes
of organisms.
And on the last slide that we
discussed, I showed you ways
to randomly change DNA,
and I'll talk some more
about that in a moment.
Before I do that, I
want to talk about how
to actually program the DNA
to have specific changes.
OK, so, rather than going out
and simply blasting the DNA
and making changes here
and there, which is kind
of an unintellectual
way to do it.
A much more satisfying way would
be to go in and spot this--
you know, spot weld
in specific changes.
You know, make a change here,
make another change here
and have some hypothesis
about those types of changes.
It's a fundamentally different
way of doing genetics than going
for lots and lots of
sort of random changes.
OK, so, to make the
changes specific,
what people do is take
advantage of the fact
that you can cut DNA,
have a little bit
of an overhang shown here in
red and then this overhang,
if it has complementary
Watson-Crick base pairing,
that's As in Ts and Gs in Cs,
with that complementarity,
these two will come
back together
and then you can use another
enzyme called DNA ligase
to finish up the last
covalent bond in here,
where these arrows are.
OK, so simply by bringing
together these two big pieces,
they will find each other,
form the perfect Watson-Crick
base pairing and then reanneal.
Incidentally, a technique
not unlike this was used also
for the-- for a lot of the DNA
biotechnology that [inaudible]
with the folding of DNA.
Remember where I showed for
example patterns of DNA,
not just the smiling faces, but
other ones that's involved a lot
of these sort of techniques
or how you were cutting
and pasting DNA.
OK, the results of
this can be all kinds
of interesting phenotypes,
extra little bugs over here.
There's little wings, sort
of little shriveled
up wings over here.
And what's neat about this is
that you have some hypothesis
going in to the experiment.
You could say I think that this
particular gene is the gene
that causes wings to extend
out, and then you can test
that hypothesis by disrupting
the gene and seeing whether
or not you get these little
short shriveled, you know,
little shrimpy little wings.
And you can use that sort
of knowledge to confirm
and potentially learn even more
about how development
works and other processes.
OK, so let's talk first
about how to make DNA
that has these overhangs.
They're sometimes
called sticky ends.
OK so again, this
is the overhang,
also known as a sticky end.
So to do that, we take advantage
of a special pair of scissors,
which like normal scissors has
a similar sort of symmetry.
This is what it looks
like structurally.
This little hollow region
in here grabs on to the DNA
and like scissors
clamps down around it.
When these cut-- this class
of enzymes are called
restriction enzymes.
When restriction
enzymes cut DNA,
they target palindromic
sequences,
specific palindromic sequences.
Each restriction enzyme
targets typically one
and usually only
one sequence of DNA.
And note that this
is a palindrome.
You all know what
palindromes are, right?
Palindromes are sentences that--
or phrases that are the same,
that have meaning if they're
in both directions, right?
So for example, Napoleon was
said to say when he arrived
on an island of Elba, "Able
was I ere I saw Elba."
OK, so, do that backwards
and you see,
"able was I ere I
saw Elba," right?
Both ways.
OK, so that's a kind of
a famous palindrome, OK?
Madam, I'm Adam, et cetera.
OK, so notice that these
sequences are similarly
palindromic, right?
So this goes CAA
and then over here--
OK, so it goes-- let's see.
So over here, it's-- let's see.
OK, so over here, GAA.
So, this is the palindrome,
TTAA and then over here, TTAA.
OK, so see there's a
palindrome right there,
it's the same going either
backwards or forwards.
So anyway, so these restriction
enzymes have a symmetry
to them just like scissors and
that symmetry in turn dictates
that they're going to be talking
apart palindromic sequences.
When they make the cuts, these
particular cutters are going
to cut this bond over here
and this other bond over here
that are indicated
by the arrows.
And the result will be DNA that
separated and has a sticky end,
two sticky ends, OK, by cutting
apart these two different
phosphodiester backbones--
phosphodiester bonds,
this one and this one.
OK, so here's another
picture of this.
OK, so here's the
enzyme grabbed on.
This is now a side view.
The enzyme is grabbed
on to the DNA.
I've highlighted the
DNA in purple and yellow
to emphasize the two pieces
that are going to come apart.
And here it is, and it's
double-stranded configuration,
and then here it is after
the restriction enzyme,
chops it apart.
And the result now is a
sticky end that will look
for a complementary Watson-Crick
base pairing sticky end.
And when it finds it, it
will then rehybridize,
reform the Watson-Crick base
pair and you can then use this
to glue together
sequences of DNA, OK?
So typically, the way this is
done is you glue that together
and insert it into a plasmid
that includes the
antibiotic resistance marker.
That's the selection marker that
we talked about last Thursday.
And I showed you a large
number of examples of this.
I showed you that
you can use a plasmid
that encodes beta-lactamase,
chloramphenicol
acetyltransferase, et cetera,
et cetera, tetracycline
resistance, et cetera.
OK. So, these sticky ends
turn out to be very useful
for pasting in new DNA.
You could take advantage of
that and do all kinds of things.
You can insert an entirely new
sequences in here that happen
to have the perfect sticky
ends and then they will anneal,
and so now you're
spreading apart the yellow
and purple pieces.
OK, so here's what this
looks like in practice.
Here's your plasmid,
you chop this apart
by restriction enzymes
that gives you a plasmid
that has two sticky ends.
And then chemically
synthesize [inaudible] DNA
or maybe you get the
[inaudible] of DNA by PCRs
or some other technique.
And you set this up so that it
has a nice sticky end hanging
out over it and then you can
reanneal this blue DNA together
at the red plasmid DNA.
And the last step here is
an enzyme called DNA ligase
that makes the last five
prime phosphodiester bond
to make perfectly covalent
close circular plasmid DNA.
And then you send this
back into bacteria.
And there's ways of
pungent holes bacteria
to get this plasmid
DNA to flow in.
And only the bacteria that take
up the plasmid will
be allowed to live.
All the rest are going to
die because you're going
to treat the bacteria with
some of the antibiotic.
And only the ones that have this
plasmid that it goes resistance
from antibiotic are
allowed to survive
at these conditions, make sense?
Yeah.
>> How do you keep
the plasmid DNA
from hybriding [phonetic]
back on to its [inaudible].
>> OK, OK, yes.
So, that's a good question.
It's Anthony, right?
>> Yeah.
>> OK. Anthony's question is
a really good one and it's one
of those things that
drives people nuts, OK?
So the question is what
happens if the DNA--
if the plasmid DNA
reanneals and closes back up?
So, just a little bit technical,
but what we do is we chop off
the three prime hydroxyls--
or sorry, the five
prime hydroxyl over here
on this sticky end, OK,
so there's some five prime
hydroxyl that's hanging off.
Sorry, not hydroxyl,
five prime phosphate
of the five prime hydroxyl,
so we chop that off.
So now, it's just the
hydroxyl with no phosphate.
And that it can't reclose, the
ligase can't operate on it, OK?
So that solves our problem.
The problem is we still end
up with some plasmid
that comes through.
And there's always
some, you know, picking
and sequencing and
stuff like that.
OK, so let's talk about
modifying proteins.
Protein modification is a
tool that's use very routinely
in chemical biology
laboratories.
That's why again, I
encourage you to use
in your proposals very,
very straightforward, OK?
So the way we do this is
we change the DNA sequence
and then use those changes
to the encoding DNA sequence
to resulted changes
to the protein.
And so, one good way of doing
this is something called
QuikChange Mutagenesis.
This is actually
spelled correctly.
It's intensely annoying to me.
A German company came
up with this one.
But, yeah, anyway, so this
QuikChange Mutagenesis works
by having new DNA
sequences shown here in blue
that encode some mutation.
OK, those are the excess.
And then use DNA polymerase to
fill in the rest of the plasmid.
So, that's shown
here in blue, OK?
And the tricky part is you
then treat this resulted
double-stranded DNA with a
special restriction enzyme
that operates only
on methylated DNA.
OK, so DNA that's had methyl
groups transferred to it.
And do you remember earlier
we talked a little bit
about DNA methylation
as a method
for modifying DNA
after synthesis?
I just mentioned it in passing.
But E. coli also do methylation.
And so, this means that this
enzyme will chew apart the green
stranded DNA and also
the yellow stranded DNA
from your original E. coli
and leave intact the blue
in vitro synthesized DNA.
OK, so the yellow and
green is chewed apart
by the special restriction
enzyme
and the blue remains intact.
And then, so what this means
then is when you transfer it
into the cells, a process
called transformation, this--
the cells that take off this
blue DNA preferentially,
because this yellow and
green stuff is chewed apart,
and hopefully you'll get
your mutation preferentially.
And this actually works
at very high efficiency.
You can get I would say 50
to 90 percent efficiency
out of this process
pretty readily.
OK, any questions about
how to make mutations?
OK so, everyone feels
comfortable designing mutation--
mutagenesis [inaudible].
OK, well, this is really
powerful because now you go in
and test specific hypotheses.
For example, in this
protein over here, where red,
this is a heme-cofactor, you
could test the role of residues,
such as histidines
that are interacting
with this heme-cofactor.
So, let's say you wanted to
know what is the imidazole
functionality of this
side chain contributing
to the ability of this protein?
You know, let's just say
that this is some
electron transfer protein
and you have some hypothesis
that an aromatic ring
in the protein is really key
for transferring electrons here
between point A and point
B within the protein.
You can then mutate the protein,
replace the aromatic ring
with just a methyl group, OK?
So you've now taken
out that methyl--
that aromatic ring and you can
then test the variant proteins,
the protein mutants which
we'll call variants,
and test whether electrons
can still move between point A
and point B over here.
That's really powerful.
OK, if it turns out
electron still move,
maybe your hypothesis that the
aromatic ring is critical was
wrong or maybe it turns out
that they can't transfer anymore
and the aromatic ring
is really crucial.
So, this allows you to do
reverse engineering of proteins.
All right, you can take
apart specific pieces
of the complicated
machine called the protein
and then examine what each
one of those little parts does
to contribute to
protein functionality.
OK, any other questions
about DNA biotechnology,
otherwise I'm going to move on.
OK, there's a lot
to talk about here.
This is absolutely
fascinating field.
It's one of those areas
where there's sort
of constant research activity.
There's always new techniques
that are coming along.
And it's also tremendously
fun as well.
I would say this is really one
of the sort of [inaudible] it's
like organic synthesis.
You get to make stuff.
It can be frustrating at times,
but when it works,
it really works well.
OK, I want to switch gears
now and I want to talk to you
about DNA reactivity with
small molecules and we're going
to start with the earliest
type of reactivity of DNA,
which is simply you walking
around in the sun, OK?
So, after this lecture is
over, you start walking back
to your dorm room and
although it's January,
there's still a little
bit of sun out there,
and chances are you'll, you
know, raise your eyes, you know,
to the sky and, you know, be
grateful you're living here
in beautiful California.
During that time, your face,
your skin is being
hit by UV radiation.
And that UV radiation is
causing damage to the DNA
in your skin cells, OK?
And here's what's happening.
If you happen to have
two binding residues,
two Ts that are stacked on top
of each other in a DNA sequence,
these can do a two plus two
photocatalyzed cyclization
to result in a cyclobutane
structure and add
up [inaudible] of your DNA, OK.
The-- Again, this is a
straightforward reaction
and I'll just very briefly
show you the mechanism here.
OK, so it's two [inaudible]
and you're adding light,
which again as usual,
we abbreviate as H nu
and the result here
is a cyclobutane.
OK, straightforward mechanism,
not particularly complicated.
Here's the problem
though, when this happens,
this cyclobutane
distorts the DNA.
DNA no longer has its nice
structure that we're so used to
and this is a problem, OK?
This is something that
your cells have evolved
to deal with, OK?
Because, you know, organisms
on this planet have
always faced UV light, OK?
So even before we were
destroying the ozone hole,
there were still UV light
that was sneaking through.
And so, a series of enzymes have
evolved to fix this problem.
And in goldfish and in
rattlesnakes and other organisms
that spend their
lifetimes out in the sun,
they actually have an
enzyme called photolyase
that reverses the two
plus two photocyclization
that I'm depicting over here.
OK, let me just make this
arrow a little bit better
because it's going to
annoy me [inaudible], OK?
So, in this retro two plus two,
this enzyme harnesses UV light
and then prompts electrons
into the cyclobutane add-up
and breaks it apart, OK?
So the irony here is that it
takes the same energy, UV--
light energy from UV light.
And this time uses that
energy to reverse the reaction
and drive it in the
opposite direction.
Now, to do this, this
enzyme has evolved antennas
to capture that UV light.
The enzyme has two antennas
highlighted here in yellow
and red and I'll show you
chemically the structures
on the next slide.
OK, so one of these antennas
is tuned to absorb the UV light
and pass excited electrons
to an FADH2 cofactor,
which then pumps the
electron into the cyclobutane.
Let me show you what it
looks like over here.
So, here's the antenna
that absorbs the UV
light, it's called MTHF.
Its structure is shown here.
The problem is-- OK, so
this works great, OK?
So the E. coli, the
goldfish, the rattlesnakes,
et cetera, they're fine, OK?
They can spend their
days out in the sun.
Us humans though, we humans
however have not evolved
this enzyme.
We don't have photolyase
in our genome.
We're not capable of
running this reaction, OK?
And so, what we do instead is
we slather on sunscreen, OK?
That's what we've
evolved to do in terms
of we've evolved intelligence,
and intelligence has come
up with ways of dealing
with this.
And notice that the structures
of sunscreen is
para-aminobenzoic acid
or PABA molecule, looks very
similar to a key constituent
of the UV absorbing antenna
used by photolyase, OK?
So again, we don't
have DNA photolyase.
And the sun causes the
DNA damage I showed
on the previous slide.
So, this PABA can-- when it's
on the surface of the skin,
absorb the UV light
and then radiate it
out as simply heat, OK?
So the energy is converted
from being electromagnetic
energy that's going
to excite electrons and
cause the two plus two
photocyclization I showed,
and instead it's going to be--
it's going to be
dissipated as heat instead.
The problem [inaudible]
overtime of course,
PABA has absorbed
enough UV energy
that it's no longer
so effective.
It starts to break down.
And so-- And also
another problem is
that the stuff is soluble
in water and sweat.
And so over time, it
loses its effectiveness.
And so, you kind of have to be
up there continually slathering
yourself with this stuff.
And no one is more
expert in this than myself
because my skin color.
And so, you know, suntan lotion
and I are very good friends.
OK, now let's talk about
other [inaudible] DNA.
Another problem with
the DNA encounter is
that small molecules
can also target DNA.
And one example of this is
a compound called psoralen,
which is found in limes.
And so for example, so it
lines the skin of the limes,
have this compound
psoralen present in them.
And I guess this is most--
best dramatized by a
group of school kids
at a Baltimore day camp who
are making pomander balls.
OK, and I bet no one in
this audience knows what a--
does anyone know
what a pomander ball?
Anyone? OK, well, I'm not too
surprised, I didn't know either.
Pomander balls are these limes
that have cloths stuck in them
and they're often
wrapped with ribbon.
So, this is an example, OK?
So, it's a lime and then some
little kid has stuck a bunch
of cloths on to the outside
and you're probably wondering
why would anyone want
to do this?
OK, so evidently,
people then throw those
in with their underwear
in their underwear drawer
and it makes everything
smell kind of nice, OK?
Now, that's the idea at least.
So anyway, a bunch of kids in
Baltimore were at a day camp
and they were making these
pomander balls using limes.
And so they're rubbing this
lime juice and lime oils
on their skin and what ended up
happening was their skin broke
out in all kinds of
lesions just sort of erupted
in these terrible red
lesions as the psoralens
in the lime reacted
with your DNA.
I'll show you pictures
in a moment, OK.
And let's take a
quick close look
at the chemistry
that's happening here.
So, what's happening is-- oh,
actually this is predictable.
Does anyone want to--
has a guess as to how this
compound interacts with DNA?
James?
[ Inaudible Remark ]
Intercalator, yes.
Pi stacking, exactly.
So, this is one of these flat
aromatic compounds that's going
to slip into the pi
stack of the DNA, right.
OK, so now imagine this,
you're rubbing your skin
with this stuff and now
it's slipping into your DNA,
that's kind of scary, isn't it?
OK. And here's what it
looks like structurally.
So, here's the psoralen.
There it is in the pi stack
and it has [inaudible] perfectly
positioned to react with DNA
and form again those
two plus two
after photocatalyze two plus
two cyclization reactions form
these cyclobutanes.
And again, the problem here is
that the DNA is now
cross-linked.
It's distorted.
It can no longer be
used for replication.
It could inappropriately
cause transcription, right?
Because now, you have this
hydrophobic mass in the middle.
So when a transcription
buffer comes along
and starts scanning
the sequence of DNA,
it gets all confused
and that's bad news.
It can start inappropriately
turning on genes
that shouldn't be turned on, OK.
And what happens is you get
these terrible red rashes.
OK, so I have some graphic
pictures in the next few slides.
And if you're kind
of a softy like me,
turn your head away, OK.
But if on the other hand you're
planning to go to, I don't know,
you know, obstetrics or
something like that or,
you know, you really
like gory stuff,
the next few slides
are for you, OK?
I can't resist, all right?
It's part of-- the fun part
of learning all these stuff.
OK, so here's what
it looks like.
What it looks like is you
get these sort of red rashes
that look like that and
maybe this isn't so dramatic.
But what ends up
happening is on the skin,
you basically have
lesions, wherever it is
that that lime stuff, you know,
comes in contact with your skin.
OK, so this happens a
lot with farmers who are
out picking limes or celery
or other fruits or vegetables
that has psoralens
in them, right,
'cause that's the
part that's exposed.
It also happens to
bartenders, right,
who are mixing margaritas
in the sun.
But it also happens to
college students, right.
So, if you happen to
be doing belly shots
and here's the little-- the
juice of the lime that's,
you know, kind of
like, you know,
running across this
person's abdomen.
You end up with these
bizarre streaks.
Basically everywhere the
psoralen manage to get to
and that can be a lot of places.
So, for this reason,
college students are
at particular risk for this.
So, that's one of the
several PSA announcements
that I'm going to have today.
In fact, this whole lecture
should be subtitled how
to protect your DNA, OK.
So, number one no belly
shots out in the sun.
Do it indoors.
Let's talk about the processes
that are inactive
when DNA is damaged.
OK, so in order to divide, cells
go through a complicated cycle
and it's a little bit like the
military in that all cells have
to either advance up the ranks
or they're called
from the population.
They must either advance or die.
And so, cells have a cell cycle.
The timing of the cell cycle
depends on the cell type.
Some cells that you know are
dividing much more rapidly
than other cells.
The cell cycle is
depicted briefly here.
And again, I'm giving you
kind of a 10,000-foot view.
The book provides more detail
and then other classes
provide even more detail.
OK, so at some point in the
cell cycle, the cells have to--
in order to be able to
divide, the cells go
into a synthesis phase when
DNA is being replicated.
And during this process, there
are a number of checkpoints
and other sort of sensors
that check to see whether
or not the cell is ready to
advance to the next stage.
In this case, the G2
stage of the cell cycle
and these checkpoints
are highlighted
by these red arrows over here.
OK, so many anticancer
drugs target this S phase
of the cell cycle.
For example, you can
imagine something
like psoralen would
massively disrupt this S phase
of cell cycle, the
DNA is cross-linked.
The two strands cannot
be separated,
which is requirement
for DNA replication.
So, let's talk about how
many of these compounds work.
I've shown you the two plus
two photocatalyzed cyclization.
A more common mode for reaction
with DNA involves using DNA
simply as a big nucleophile.
It turns out that the DNA
bases are hugely nucleophilic.
OK, the backbone, negatively
charged, not so nucleophilic.
But the bases themselves,
lots and lots
of opportunities act
as a nucleophile.
The reason for this is that
there's lots of lone pairs
that are orthogonal to these
aromatic rings that are sticking
out from the aromatic rings,
like this one over here
on this adenine function--
adenine base.
OK, so where this arrow is
pointing, there is a lone pair
that is sticking straight
out away from the ring.
And that lone pair is not
participating in aromaticity.
It is totally ready
and available to react
with any electrophiles that
it happens to encounter.
So, for this reason, many,
many electrophiles are
fantastic carcinogens.
OK, if we look at
carcinogens as a class,
they are largely electrophiles,
some other classes as well.
But in general, you want
to avoid electrophiles
if you can, OK.
And I'm going to show
you some examples.
Here's an example that I
showed in an earlier slide.
OK, so, in an earlier slide,
this was from Thursday's
lecture.
When I showed you that you
could treat fruit flies
with random mutagens and
that would result in--
I forgot what it was.
I think it was like extra eyes
that were growing
out of the fruit fly.
The way that that reaction
worked is here is the
nucleophile of DNA
attacking the sigma star bond
between this methyl group
and this good leaving
group, a sulfonate, OK.
And so, what happens now is this
DNA has been modified, right?
You now have a new
methyl group over here.
And the problem with
that is now instead
of having a lone pair there
that transcription
factor can recognize,
now you have a hydrophobic
functionality
that might be going
out to recruit transcription
factors improperly.
And so, it might
start turning on genes
that otherwise might
not get turned on, OK.
Yeah. Here's some examples.
So, here's again the structure
of DNA that's the B-form of DNA.
This is the major group.
This is the minor group.
The arrows are pointing
to the most nucleophilic
of the lone pairs.
And all of the most nucleophilic
lone pairs are lone pairs
that are on aromatic rings
but are not participating
in aromaticity, OK.
Notice that both of these rings
have six pi electrons, two,
four, six, two, four,
and then six,
and this lone pair over here.
This lone pair, the one that
has an arrow pointing to it
and this other one with
that arrow pointing
to it don't participate
in aromaticity.
OK, they're kind of spectators
in the whole aromaticity
business.
And because they're
not participating,
they're really available
to act as nucleophiles.
They're super nucleophiles.
The problem of course is that
they also have a role to play
in terms of encoding sequences
and that role can be messed
up when they are modified.
OK, so it turns out--
and I don't want to
panic you too much.
It turns out actually even
if you encounter a really
large amount of electrophiles,
you're not automatically
going to get cancer, OK.
And there's actually an
absolutely fascinating case
study on this.
In this case-- let's see.
So, in the case that
if we're going to--
that we're going to see, you
know, there was an ex-boyfriend
of this woman here who
wanted to poison his family
by giving them all cancer.
And he unfortunately had
access to chemical mutagens.
He worked in a laboratory.
I believe in Kansas.
And he had an access to
this compound over here.
And so, he started
or actually-- sorry.
It's not this compound.
It's another simple
electrophile.
And what he did was
actually add large quantities
of an electrophile to
milk and to lemonade
that he found in
the refrigerator.
OK, he stops by the house,
he looks for an opportunity,
goes to the refrigerator and
he dumps in large quantities
of these chemical
mutagens, shakes the stuff up
and then disappears, OK.
And then, later when other
people come to visit,
she serves them this
cold lemonade
that has the mutagen, OK.
Here it is.
Here's a picture of him.
This is Sherrie's
former boyfriend,
here is Sherrie again.
What he served or what he--
oh here's a picture of his,
you know, disgusting
little chamber of horrors.
But what he did was he actually
gave them these N-nitrosamines,
which are coded potent
DNA alkylating agents.
And why don't we take
a look very briefly
at the mechanism of this.
OK. So this is the
nitrosamine and in the liver,
it goes through an
interesting activation step,
where eventually it gives you
this diazo compound shown here,
OK.
Notice that this has nitrogen
as a leaving group, OK?
So earlier I showed you
sulfonate as a leaving group.
You can't get any
better than nitrogen
as a leaving group, right.
This is N2, bubbles off as gas,
fantastically stable
leaving group.
It does not get any
better than this.
Notice too that the electrons
get to bounce their way
over to a positively charged
nitrogen, they love doing that.
OK that's, you know--
that's electron paradise.
And so this makes just a very,
very potent DNA alkylating
agent.
OK, so it's obviously something
that you don't want to adjust.
All right.
Let's get back to our story.
What happened to the
family over here?
It turns out that
they got-- they died.
Actually, Duane Johnson and his
infant nephew began bleeding
inside and just basically
died in tremendous agony.
However, they didn't
get cancer, OK.
So they died.
It was horrible.
But they did not get cancer, OK.
So none of the victims
got cancer,
not all the victims died.
There's this fascinating
book called "Toxic Love"
that could tell you
more about this.
Here's what we learned
from this episode.
OK. So, first, DNA is
a terrific nucleophile.
On the other hand,
it's very hard
to convince the cells
to induce cell death.
And it's really hard
to cause cancer,
even when you have
enormous quantities
of these noncarcinogenic
materials.
OK, so there's enough
sort of error correction
in your DNA machinery and
your cellular machinery
to fix basic changes to the DNA,
to fix modifications to the DNA.
I'll show you that on the
next couple of slides.
But this is great news, OK.
This means that you can
all safely walk back
to your dorm rooms, knowing that
your DNA is getting cross-linked
by the two plus two
photocatalyze cyclization
that I'm showing over
here and also knowing
that you're not going
to die of that, OK?
So your cells are very
good at fixing these types
of [inaudible] the DNA, OK.
And in fact, actually this
stuff is totally ubiquitous
in our daily lives.
How many people had
bacon for breakfast?
All right.
Anyone else, sausages?
Anyone had sausage breakfast?
What do you guys
eat for breakfast?
OK. Well, at least one
person had some bacon.
I'm a bacon fan myself.
Here's what happens.
The problem is that
sodium nitrite is used
as a preservative for
bacon, for bologna,
for sausages, et cetera.
And the problem is
that by heating
and also other acid-catalyzed
reactions which can happen
in the stomach, this sodium
nitrite can get rearrange
to eventually form a
terrific electrophile, OK.
So as the primary
preservative of packaged meats,
you get this fantastic
electrophile.
The good news is that this
is probably not something
that we have to spend a lot
of time worrying about because
at the same time, vitamin
C, which is also ubiquitous
in our foods, can
actually reduce this.
It's an antioxidant,
meaning it reduces things.
It can reduce this electrophile
giving us a much more harmless
molecule, nitric oxide, OK.
So here's ascorbic
acid, that's vitamin C
and it can react very quickly
with this reactive nitrite
and prevent the formation
of nitrosamines, OK.
Here is the nitrosamine over
here and for the reasons
that are shown here, this
is why you do not want
to have nitrosamines round.
Again, this is a nitrosamine.
It eventually will rearrange
to give you a great
DNA alkylating agent.
But vitamin C reacts with the
nitrite preventing the formation
of those nitrosamines.
OK so, the thing that
I'm trying to tell you is
that DNA will do a lot
of reactions with a lot
of molecules, but there's
no reason to panic.
OK, because the chemistry
on this stuff is
remarkably complex.
There's a lot of other
molecules around.
And furthermore, the cells have
all really effective mechanisms
for repairing damage to the DNA.
And we'll take a look at
that on a future slide.
Before we do, let's take
a look at some carcinogens
that aren't found
in our daily lives.
OK, at least I certainly
hope not.
Many of these are used to treat
cancer, various kinds of cancer,
not so commonly now because
this serves our front lines
of cancer-- any cancer
treatments some time ago.
So, these classes of
compounds are known
as DNA alkylating agents.
For the reason that these
are electrophiles that react
with DNA and leave the
nucleophilic DNA alkylated.
So they're going to come
along and modify the DNA.
Some of these are ones that
are kind of familiar to us.
OK. So these nitrosoureas over
here had a similar reactivity
to the nitrosamines that I
showed a couple of slides ago.
Others like these nitrogen
mustards we'll talk some more
about, OK, or the
cyclophosphamide.
OK. So let's start at the top.
Mesylates.
Mesylates, great
leaving groups, OK.
So this compound busulfan
can alkylate the DNA.
Notice that it has not one but
two mesylates, that's a problem
because that means that it
can react with both strands
of the DNA and that's
always bad news, right.
Because now, the DNA
is cross-linked, right.
It has the two strands
that otherwise should
be held together
by hydrogen bonding are now
covalently welded together
permanently or semi-permanently.
Similarly, this bis-chloromethyl
ether,
also a potential
cross-linking agent, right.
Two leaving groups--
Two chlorides which can
act as leaving groups.
MOM chloride that has
one leaving group, OK.
Bad news, alkylates DNA, gets
into cells very effectively.
These are starting to
look like compounds
that you've encountered
in the laboratory, right.
We routinely use for example
chloroform, methylene chloride.
We use lots of alkalotic
halides for example, OK.
These are things that you
encounter in insecticides.
Let's see.
Methylbromide for example is
routinely used to treat grapes
and to use for tenting house.
You know, when you see
a tent over a house
for termite control,
that's actually--
it's working using a DNA
alkylating agent that's sprayed
into the tent.
OK. These guys over here,
the nitrogen mustards,
we'll talk more about
in a future slide.
This one again is just like
the nitrosamines that I showed
on the previous slide which is
to say this eventually
rearranges
to give you a diazo
leaving group.
OK. Now, here's the thing.
Many of the compounds I showed
on the previous slide are
also used as anti-cancer drugs
and the idea there is that
you're going to give these
to patients and hope
that the cancer cells,
which are dividing very,
very rapidly in the patient,
more actively and more
avid to take up the drugs
than other normal cells
in the patient, OK.
And for the most
part, this works, OK.
I mean, it's true that these
things are also incredibly toxic
and they'll-- you know, they'll
kill other rapidly dividing
cells in the poor patient,
but at the same time,
they are preferentially
loaded into cancer cells, OK.
So the compounds I showed on
the previous slide also appear
in many anti-cancer
compounds, OK.
So this is the dimesylate
compound.
This is a combination
of a nitrogen mustard
and also a nitrosourea.
This is a nitrogen mustard.
And these all have
little niche markets.
For example, this nitrogen
mustard, which is used
to treat a rare form of cancer,
is found on the skin,
specifically hands.
And it's a tiny little market.
It might be 50 million
dollars a year,
but it's used as an ointment.
And so patients who have
this will get this ointment,
rub it on their hands
specifically in the area
and use this to kill any rapidly
dividing cells that happened
to be in that area, OK.
And that's actually kind of nice
because that it's
specifically targeted just
to that area as an ointment.
Paracelsus, who is an
absolutely fascinating character
in the history of science,
had this great quote
that goes something like-- and
I won't read it to you in German
because my German
is not up to his.
It basically says, you
know, show me any compound
and I'll show you
two sides to it.
One side, it's a toxin.
The other side, it can
be a treatment and cure.
All this depends on the dose.
OK. So at high doses, this
can be a toxin, at low doses,
these things could be
used as treatments.
OK, that sort of the
paradox of these things.
So the goal of chemotherapy is
to induce cell program
suicide called apoptosis
in rapidly dividing cells faster
than you induce new cancer,
new carcinogenicity
in normal cells.
OK, that's the big challenge
for aging cancer treatments,
at least up until the last I'd
say 15 to 20 years ago, OK.
In the last 15 or 20 years,
chemists have started coming
up with compounds that
are much more specific
at targeting what makes
cancer cells cancer cells.
The compounds I'm talking
to you about today are ones
that simply literally any
DNA that they have to find
and the future really is
targeting the specific
attributes of cancer that
makes that cell a cancer.
OK. So let's talk a little bit
about how your cells repair
themselves after an affront.
You know, you're walking
home and you decide to chat
with your friend out in
the sun and so you end
up with some cross-linking
bindings.
OK. This is the photocatalyzed
two plus two
that I'm showing over here.
OK, so the way this works is
that your cells have
a constellation,
a fleet of DNA repair proteins
that are constantly
circulated, OK.
The analogy would be tow
trucks that are driving
around on the 405 looking
for broken down cars
that are disrupting traffic and
then as soon as they find one,
they just pull it off the road,
rather than disrupting traffic.
OK. So you have these
DNA repair enzymes
that are constantly scanning
your DNA looking for things
like cyclobutane
atoms and looking
for this sort of affront.
When they find this,
they don't simply snip
out the mistake over here.
Instead, what they do is they
snip out all the big segment
of DNA, big chunk, 10
base pairs on this side,
10 base pairs on that side.
And this part is excised
using a restriction enzyme
and DNA polymerase
comes along and builds
in the correct sequence of DNA.
So it's an excision
of their mechanism.
OK, that you chop
out a big chunk
and then DNA polymerase
moves in.
The reason you can't simply
remove just those couple
of phases is that DNA polymerase
likes to get a right start, OK.
It doesn't work so well
just on couple of bases.
It really needs 20 or
something before it can start
cranking along.
It just doesn't-- It's
not-- It's a professional.
It's doesn't like
to do small jobs.
Any questions on
what we're saying?
All right.
OK. So monofunctional
alkylating agents,
things like this MOM chloride
compound that I showed
on a previous slide are
relatively harmless.
They're not super lethal because
they can be so readily fixed.
This is not to say
that you should go out
and start inhaling
MOM chloride, OK.
You want to stay as far away
from that stuff as possible.
Here's the mechanism.
In this MOM chloride,
what's happening is you're
actually forming an oxonium ion
intermediate and this oxonium
ion intermediate is a fabulous
electrophile, right?
Notice that the oxygen
bears a positive charge
and we know oxygen by
virtue of its position
on the periodic table does not
like having positive charge.
It's electronegative.
And so this makes this an
exceptional DNA alkylating agent
and here it is on
attacking or being attacked
by the nucleophilic DNA.
And again, this could be readily
fixed using excision repair.
And the problem is if you
have too many changes,
you can actually end up
overwhelming the repair system.
So if there are too many
places that are alkylated,
then it's just hard for they--
on DNA repair machinery
to keep up.
It's just too much to handle.
The other problem is that some
DNA alkylating agents had not
one but two strands of DNA.
And this excision
repair mechanism assumes
that you'd have a second strand
of DNA that's available to act
as the perfect copy and
to provide a template
to fix the broken strand.
What happens if both the strands
have been damage, say by x-rays
or something like that?
You're in trouble.
That's a real problem, OK.
So, why functional cross-linking
agents are insanely toxic?
Busulfane is a lot more
lethal and damaging to DNA
than two equivalents
of methylmesylate,
the monofunctional
DNA alkylating agent
that we've been seeing today.
OK, so cross-linking
DNA far more damaging
than monofunctional adducts.
And again that's because
the cross-linking,
they hold together the two
strands of DNA and prevents you
from having the template
strand to act
as a copy during DNA repair.
OK. So here's I think a
couple of examples of this
and this is-- in this
example, this is a nitrosourea.
What's important about this
is it gives us again the diazo
leaving groups, that's
the nitrogen
as a leaving group,
this diazo compound.
Analogous to what we saw
with the other nitrosoureas,
but then this other chloride can
hang along to form this adduct
of DNA over here and the second
strand of DNA can attack this.
And this gives you now
two strands of DNA bridged
by an ethyl functionality.
OK. So now, they're
covalently held together.
And again, this is bad news.
OK. Excision to repair
works pretty well except
when it encounters these
double-stranded breaks.
These sort of double
strand problem, you know,
where you get cross-linked,
your double strand breaks.
For that matter, excision repair
can also be lethal if it's not--
if there's a genetic abnormality
that encodes a protein
that's no longer functional.
OK. And there's actually
a fantastic movie
about this called
The Others by--
and includes a great
performance by Nicole Kidman.
Has anyone seen this?
OK. Spoiler alert.
The kids have this disease
in which they have
abnormal DNA repair enzymes.
OK. If you haven't seen this,
you should still see it.
I haven't totally
ruined the movie for you,
[inaudible] but not total.
OK. So missing or deficient
DNA repair enzymes cause
severe disease.
OK. We again got kind of
graphic image on the next slide.
So, the disease xeroderma
pigmentosum
for example is caused
by an incorrectly
coded DNA repair enzyme
and this poor girl has this
and the problem is again you're
constantly being affronted
with damage to your DNA even
when you're outside in the sun,
UV light causes the
DNA to be cross-linked
by this photocatalyzed two
plus two photocyclization,
you absolutely require
functional excision
repair enzymes.
Without those, you
cannot live on our planet.
It's just too much
damage going on.
And otherwise, you end up
with this [inaudible] disease.
OK. So let's get back to the
story with the crazy guy,
the psychopath who tried
to murder the family
by giving them all cancer and
none of them died of cancer.
It turns out that when
we look at cancer cells,
we find hundreds
if not thousands
of mutations to the DNA.
OK, so when we start
sequencing tumors,
we find a tremendously
heterogenous mixture
of mutations, where each
tumor is slightly different.
And furthermore when we take
biopsies in different spots
of the same tumor, we also find
tremendous differences amongst
the cells which otherwise
superficially look identical.
OK. So to form a cancer requires
hundreds if not thousands
of mutations to the DNA.
It takes an enormous change
to the DNA to do that.
Furthermore, those
mutations have
to target three different
control aspects of the cell
and I like to think of
these as an accelerator,
a clutch and the brakes.
OK, I'm a car guy, so for me,
this analogy works really well.
If you don't drive stick shift,
you might not know that back
in the day when all cars were
manual, they had a clutch.
The clutch was this extra
pedal that you would push
down to switch gears, OK.
And earlier I showed you
that cells have to progress
through a cell cycle and
that there were checkpoints
through that cell cycle.
So I like to think of those
checkpoints as shifting gears.
OK. So that the cell cannot
shift between different phase
of the cell cycle,
it cannot proceed.
However, if you have
mutations to those checkpoints
to the proteins that are
controlling the test for whether
or not the cells should
be allowed to progress,
you could end up with
mutations that allow the cell
to inappropriately progress
in the cell cycle allowing
runaway cell division.
OK. So those are mutations,
the clutch which are
the checkpoint proteins,
often kinases.
In addition, all cells have on
the cell surface growth factors
and growth factor receptors,
which are responsive
to the external environment.
Mutations to these
growth factor pathways are
absolutely required.
That's like a mutation
to the accelerator.
These growth factors are
telling the cell start dividing,
start producing this, start
doing this, get going on this.
So that's the mutation
to the accelerator.
And finally, all cells
have tumor suppressor genes
and we'll look in a
moment at one called p53,
which I like to think
of as the brakes.
This is-- These are the-- This
is the machinery that shuts
down the cell when it starts
running out of control.
And when this happens,
these tumor suppressors
can trigger apoptosis,
cell suicide that
prevents cancer.
OK. So, these hundreds to
thousands of mutations have
to affect all three
of these pathways.
If just one is affected,
then there's a good chance
that the cell will
developinto apoptosis and arrest
and prevent the cancer.
If on the other hand you
have mutations to all three,
then pathways can start
running out of control.
OK. So, let's see.
If we take a very
brief look at p53,
which is the tumor
suppressor protein.
Here's a cell, here's
the self-destruct button
and there are a whole series
of different questions the cell
is constantly asking before it
could go into cell division.
And at any given time the answer
to any of these questions,
is it big enough, is
there enough room,
are there two copies of DNA,
is the DNA probably lined up.
If the answer is no, the cell
will be immediately sent it
to apoptosis, where it
basically blebs apart.
Bleb is a fancy word
for explode.
OK. It starts bleeding out
these blobs off the surface,
bleb and blob are
two great words.
OK. They sound like each other
because that's really
what's going on here.
So p53 is a hair trigger sensor
that can turn on apoptosis
and mutations to the p53 gene
allow DNA damage to accumulate.
So mutations to p53 are
bound in like 60 percent
of colon cancers for example.
This is a very, very
common set of mutations
that are behind a large number
of different cancer types.
And in fact, it's almost
mandatory for some cancers
to get going because the
functional p53 will shut
down most of their
runaway cell division
that would otherwise
cause cancer.
OK. So for this reason, you
know, things that you do
that affects the p53 gene
are really, really serious
and for example, we-- scientists
have been able to link mutations
to p53 directly from smoking.
OK. So if you're out
smoking, there's a good chance
that you're starting to
cause mutations to p53.
This is literally
the smoking gun.
OK. This is what causes the
link between cancer and smoking,
which until then, until
this was done in the--
linked in the mid-'90s or
so-- cancer companies, no.
Cigarette companies were able
to promote their cigarettes
and say there's no
definitive evidence
that cancer causes smoking.
Here is the definitive evidence.
What happens is you end up
with mutations to the residues
that are highlighted in yellow.
So these are residues that are--
have a positive charge
and interact
with double-stranded DNA.
Notice that p53 does a
lot of things to the cell.
One thing it does quite
importantly is act
as a transcription factor
to trigger apoptosis.
And if these positively
charge residues are mutated,
the p53 can no longer
bind to the DNA
and it could no longer
trigger apoptosis.
So smoking affects p53 and it
produces mutations specifically
at the molecular level to
these residues here and that
in turn results in cancer.
OK. If you're smoking
now, you should stop.
OK. If you know people
who are smoking,
you should persuade
them to stop.
I cannot think of
anything worst.
My father died of lung cancer.
I am passionate about this.
Stop smoking now.
You might as well just
put your mouth on to
like the exhaust pipe
on a bus or something.
Right. It's as crazy as that.
All right.
Now, what is it about
smoking that causes cancer?
We actually know quite
a bit about why it is
that unburned things
cause cancer
and it goes back a long ways
along the history of biology.
And I guess we have to go back
to the father of epidemiology,
the great Sir Percival Pott.
Percival Pott shown here
was a physician in London
and he noticed that his chimney
sweeps had a ridiculously high
level of testicular cancer.
OK, so chimney sweeps
are these guys
who in 18th century London would
actually climb into chimneys.
OK. So this isn't Santa Claus.
This is actually, you know,
real humans that are climbing
into the chimneys as a
way of cleaning them out
and they would carry
brushes that look like this
and they would just be covered
in soot, OK, because they're
in the chimney, where
there's all these sort
of unburned sooty stuff.
In the unburned sooty stuff,
what we find are chemicals
that cause testicular cancer
and other types of cancer.
And I have a picture
of testicular cancer
on the next slide.
Sorry, I couldn't resist.
If you have a diversion to this
sort of thing, avert your eyes.
I'm convinced though by
showing you these images
that at least one of you--
one of my students someday is
going to benefit from this.
I'm really hoping
that I'll prevent
at least one person from--
or maybe I have helped one
person detect cancer very early.
OK. So here's a picture
of a cross-section
through a testicle.
Check out the size over
here, this is in centimeters.
So this whole region in here
is I guess one giant cancer
that has grown.
So you end with this, you
know, just tremendous growth
of cancer in testicles.
Now, here's the good news.
Good news is if it's
caught early,
this is totally treatable.
OK. This is the sort of thing
that if you catch it early,
it could be stopped by both
chemotherapy and also surgery.
OK. And so men in the audience,
you should all be thinking
about monthly self-check exams
and get more information here.
And again, this is another
one of my PSA announcements.
I really hope someday I'll have
prevented someone from dying,
that would be the coolest thing.
OK. So testicular
cancer-- oh, oh.
I have another picture.
OK. This one is a
little bit gorier.
These are actually-- These
are the cancers highlighted
over here, a little
hard to see in there.
But again, this is
totally treatable.
OK. So Sir Percival Pott
is noticing that in London,
a very high percentage of
his chimney sweeps are coming
down with cancer,
testicular cancer
and he's wondering what
is it about the soot.
He hypothesizes that the soot is
what's causing this testicular
cancer in chimney sweeps.
We now know that in unburned
carbon or unburned stuff,
there are a lot of carcinogens.
And I want to show you
that on the next slide.
OK. So this benzopyrene is found
in high concentrations
in unburned carbon.
OK. So-- question over here.
[ Inaudible Remark ]
Let's see.
That was a picture of an
operation to remove cancer.
[ Inaudible Remark ]
Let's not [inaudible]
too closely.
We'll talk more about it
later [inaudible], OK.
All right.
So these compounds over here
are definitively cause cancer.
OK. So for example if you take
benzopyrene and you rub it
on the backsides of these rats,
they come down with these
horrible cancerous lesions,
those bumps over here and
these work by mechanisms
that you can predict, right.
These are flat aromatic
compounds, how do they work?
Flat aromatic-- yes.
On three, let's do it together.
One, two, three.
>> Intercalators.
>> Intercalators, yes.
So these flat aromatic
compounds fitted to the DNA,
they slide straight
into the pi stack.
But in addition, they
also can alkylate the DNA.
And this is less
obvious and I want
to show you this
on the next slide.
OK. So when you smoke
cigarettes or for that matter
when you smoke alternative
things, there are unburned bits
in there and those unburned
bits are benzopyrene.
OK. And the benzopyrene
that gets
into your liver is epoxidized.
OK. So your liver will
try to process this stuff.
OK. So you can imagine, this
stuff is not soluble in water
and it's really bad news
to have insoluble fragments
of stuff floating
around your bloodstream.
So the liver does its very
best to deal with this
and the liver strategy for
solubilizing insoluble matter is
to oxidize it and introduce
hydrophilic functionality
that would make it
soluble in water.
OK. And so here is
a-- here is an enzyme
in your liver oxidizing
the benzopyrene.
Here's a successful
oxidation to make a diol.
Here's a second oxidation
that creates instead
of a diol, an epoxide.
Epoxide has a strained
three-membered ring.
This strained three-membered
ring is a
fantastic electrophile.
OK. So now this is
really bad news.
You now have this
intercalator that's sliding
into your pi stack that has
the perfect alkylating agent,
electrophile delivered right up
against your nucleophilic DNA
and that's really the problem.
OK. This is an enormous problem.
OK. What ends up happening is
the DNA gets modified covalently
by this benzopyrene.
OK. So for this reason, you
know, countries that eat a lot
of barbecued food, that have
a lot of sort of burned stuff
in their diets tend to
come down with high levels
of stomach cancers, OK,
likely because they're counting
benzopyrenes in their diet.
Your goal should be to try
to eliminate benzopyrenes
as much as you possibly can.
There's lots of stuff out
there that people are paranoid
about that they think
is bad for them.
Here's one that we
genuinely know is bad for you
and here is something
that you can do
to help yourself live a lot
longer simply by avoiding it.
All right, here's another
one, this is aflatoxin.
Aflatoxin is produced
by molds that grow
on grains like peanuts, OK?
And in the United States, all
the peanut butter is tested
for the presence of this mold,
this Aspergillus flavus mold.
But in other countries where
the public health systems aren't
in the sort of food safety
mechanisms aren't as vigorous,
this is more of a problem.
But the problem is that
the mold looks like this
and it gives off this aflatoxin,
which again is modified
by cytochrome P450.
I should have pulled it.
This is cytochrome
P450 in the liver.
And that introduces an epoxide.
This epoxide is a
fantastic electrophile
for the nucleophilic
attack of the DNA.
And once the DNA is modified,
this eventually leads
down to DNA strand cleavage.
OK so, modified DNA
eventually leads
to DNA that's been
chopped apart.
That DNA that's been chopped
apart is no longer available
and if you get hundreds
of these mutations,
eventually, you get cancer, OK?
Bad news. All right, let's talk
about the nitrogen mustards.
OK so earlier, I showed you
compounds that had a nitrogen,
ethyl group and a chlorine.
Here's a variant where
it has a sulfur instead
of a nitrogen, OK?
And both of these
compounds, they both go
through a common mechanism,
which is the heteroatom
in the center of the compound
can act as a nucleophile
to form a terrific electrophile.
And in this case,
the nucleophile attacks
and is alkylated.
If this nucleophile is DNA,
then the DNA can be cross-linked
giving you two strands
that are covalently
welded together.
OK, so earlier I showed you
for example I believe
it was busulfan,
the compound I told you about
that you rub on your skin
for this very rare
case type of cancer,
that's used as a
chemotherapeutic.
So these were also used as
war gases in World War I,
which is completely insane,
OK, that you would actually,
you know, do this to
anybody on the planet.
But in any case, these
compounds cause cancer
by forming fantastic
electrophiles, which then react
with the nucleophiles
bound in the DNA.
OK, so again, here is the
nitrogen mustard equivalent
and these are compounds that we
saw earlier in today's lecture.
And all of them go through
a common intermediate,
this aziridinium ion over here.
And notice what a great
electrophile this is, right?
The nitrogen has
positive charge on it
and nitrogen hates
having positive charge.
So when the nucleophilic DNA
attacks here, the electrons tend
to bounce their way
to the positively charged
nitrogen setting up sort
of the perfect electrophile
for modifying DNA.
OK, any questions about
the nitrogen mustards
or sulfur mustards,
DNA alkylating agents?
OK. I want to switch
gears now and I'm going
to take the last five minutes
to start talking a little bit
about RNA just to kind
of wet your appetite
as we talk about RNA.
There are no other
questions about DNA, right?
Questions about cancer,
anything like that?
So RNA, we've-- chemical
biologists have come
around to recognize RNA's
tremendous importance
in cell biology.
As an example of this, this is
actually a structure of a DNA
that was posited by Phoebus
Levene, who I'm going
to show on the next slide.
And, you know, this is
around like 1920 or so.
And structure of DNA and RNA was
not very well understood, OK.
So he posited that this DNA
structure was the structure
of DNA.
Of course, this is
totally wrong, right?
We know what the correct
structure of DNA is.
But what the Levene did right is
he also assigned pentose ribose
sugars in nucleic acids.
And he specifically showed
there is deoxyribose in the DNA
and also figured out
that DNA is a nucleoside
to denote the glycosidic bond.
OK so, it's possible that
science can be totally wrong,
yet get some details correct.
OK so, this structure to us
looks totally nuts, right?
It's only, you know, four bases
of DNA arranged in a circle.
But on the other hand,
when you look more closely
at the details here, you
start to find all kinds
of interesting features,
like for example
that the bases are held
on by glycosidic bonds,
that this has a phosphodiester
backbone,
that the connectivity
here is five prime
to three prime, et cetera, OK?
So in a similar way, we kind
of have sort of a reevaluation
of the importance of RNA.
When it was originally
discovered, it was thought
to be merely a transfer,
sort of a go-between,
between DNA and proteins.
And in last 20 years, our
appreciation of RNA's key role
in the cell has expanded
enormously.
OK. So we now know that
RNA can act as a soldier,
a sailor, and tinker and spy.
And first as a soldier,
RNA is actually a very
effective catalyst
for cleaving its own sequences.
It could actually go out
and cleave sequences
of other RNA strands.
As a sailor, transfer
RNA delivers amino acids
to the ribosome, here's transfer
RNA delivering aminoacyl amino
acids-- aminoacyl tRNAs
to the ribosomes during
protein synthesis.
As a tinker, the ribosomal RNA
can act as a catalyst machine
to synthesize proteins.
And finally as a spy,
messenger RNA encodes proteins.
So RNA is capable of all
kinds of things and this means
that when we come
back on Tuesday,
we're going to have a
lot to talk about it
and I look forward to that then.
Thanks.
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