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BIO 362
Exam 2 Study Guide
SBU
Spring 2014
Marcu

BIOCHEMISTY 2 Exam 2 Review of Lectures:

Lecture 8: Recording

Primer is a Concept can be DNA OR RNA!!!!

The Enzyme primase is going to synthesize RNA fragments which is 10 -13
nucleotides long these can be used as a primer for DNA Synthesis, Are
primers always 10 15 nucleotides long? NO!!!!! (Primer is a concept, that
tell us where DNA Polymerase is going to recognize, when its
complimentary to the template, it will then extend at the 3end to synthesize
DNA.

Nick Translation: Is a process that can be catalyzed by DNA Polymerase

1, the way it works, when Pol1 synthesizes a piece of DNA, when Pol 1
synthesizes a DNA , when its encounters something in front , it can be a
piece of RNA or a piece of DNA, when it encounters that the 5 to 3
exonuclease kicks in , and it is going to start chewing up what it is in front,
it will open up new template region for the DNA polymerase active sites to

keep putting in new dNTPs(nucleotides). What looks like happened is this

space position is moved from one position to the next.
What is Nick? A nick is paired all the way until it hits the end.OH and
phosphate group not connected.
A gap has a single stranded DNA region that is NOT paired up.
How do you make a phosphodiester bond? You need a nucleoside
triphosphate; the triphosphate will give you the energy you need to make the
bond. YOU NEED THE TRIPHOSPHATE TO MAKE THE
PHOSPHODIESTER BOND!!!! The Enzyme that will fuse the nick together
is called DNA ligase(Which is a different reaction). Introduce DNA
Polymerase 3!!! Important phenonhm in which Cairns took E.Coli Genome,
broke it up gently, spread the genomic DNA onto a piece of membrane,
these cells were grown in Radioactive nucleotides and therefore the genome
was radioactive dNTPs, they put a frame on top of it, they looked under the
microscope and saw a lot of these structures. I and 2 were equal implication

was that DNA Replication starts at a single point and it moves on BOTH
SIDES and the fork progresses until you get two full circles. In order to
synthesize both strands and knowing DNA polymerase must work in a 5
to 3 direction, it implies that one strand must synthesize continuously, and
the other strand must synthesize discontinuously. In order for DNA
Polymerase to work YOU NEED TO PRIME IT. Since one strand is
synthesized discontinuously, it means it needs to be primed frequently.
Okazaki realized when you take Genomic DNA from Bacteria and separate
it out into a gradient, a density gradient a tube with a solution of Sucrose and
load genomic DNA and spin it heavy material at the bottom, they always
found there was these smaller pieces of DNA at the lighter fractions of this

gradient. , this lead to the idea that one of the strand is synthesize in a
discontinuous manner . Enzyme that is responsible for leading and lagging
stand synthesis of GENOMIC DNA in E.coli . DNA polymerase 1 was first
identified because it is the most abundant DNA Polymerase inside the cell.
DNA Polymerase 1 turns out to be extremely slow. If you measure how
many nucleotides it is going to add to a growing template, it turns out it adds
20nt/s. E.Coli Genome is 5 million base pairs, one side you need to
synthesize 2.5 million turns out you need about 2000 minutes if you only
used DNA Polymerase 1. Doubling time of Bacteria is about 15-20 minutes.
Someone identified a Pol 1 mutant, cells were still viable!!!!!, and people
started looking for different polymerases that lead to the identification of
DNA Polymerase 2 and 3. It turns out that Pol 3 is responsible for synthesis
of leading and lagging strand. , This enzyme is much faster and is the main
replicator of the E.Coli Genome; it also has a very high processivity. Pol 1
and Pol 3 have 3to 5 exonuclease activity (proofreading) but Pol 3 does not
have 5 to 3 exonulease activity that Pol 1 has. The 5to 3 exonuclease is
an essential function cells will not be viable if they dont have this. Function
of POL 1 is to removing RNA primers; fill in Gaps, which is important for
DNA repair process, removal of RNA primers, which is an important
process for lagging strand. The function of Pol 3 (HoloEnzyme) in E.Coli is
that it is responsible for both the leading and lagging strand synthesis.
Processivity: The number of nucleotides added to the growing primer strand
per binding event, what this actually means is two different types a
nonprocessive(Distrubutive event) ( this case polymerase would bind to
primer template junction binds one dNTP , adds dNTP forms one
phosphodiester bond and then it leaves and then the cycle goes back again,
that is a completely non-processive process. By Contrast a processive
process, enzyme binds primer-template junction then it keeps adding these
dNTPs to form these long strands a fully processive process will keep going
until it reaches the end. Processivity of Pol 1 is about 10-100 nucleotides, it
means that it will bind at 10 come off and Pol 3 core has an even lower
processivity by itself (1-10 nucleotides). But the Pol 3 Holoenzyme, which
has a few extra subunits, is going to increase processivity to 100,000 ntd per
binding event. This increase in processivity is due to this protein called BSliding clamp. What really enhances the processivity of the POL 3 is this BSliding Clamp.
Question: How does the B-Sliding Clamp improve processivity?
When people analyzed B Sliding Clamp purified it and saw the structure
they found it looked like a ring structure, when you measure distance it is
30Angstrom, which is big enough to accommodate double stranded DNA

helix and even thick enough to accommodate a layer of water molecules,

surrounding the DNA double helix. The idea is that water is lubricating thus
allowing DNA to slide very easily. Structural Biology teaches us how things
work, this leads to the question, how does B-Sliding Clamp assemble onto
the DNA in the first place. B-Clamp is composed of two subunits of the
same protein dimer. Important: In Humans and Eukaryotes, it has a very
similar protein called PCNA, which stands for proliferating cell nuclear
antigen, however, its a trimeric protein, instead of a dimer. Both proteins
need to assemble onto the DNA in the first place.
How the B-Clamp helps polymerase to stay attached to the DNA and
increase processvity. In distributive synthesis, Pol 3 binds to primer-template
junction, extends a few nucleotides and then falls off. But if the B-Clamp is
present, Pol 3 will extend, and even if it falls off, B Clamp will hold on to it ,
and keep it close to the active site, to its primer-template junction and let it
resemble and keep going. The B Clamp will keep Pol 3 long enough that it
will rebind to active site, to increase processivity. You need a second protein
called gamma complex, this is an ATP dependent protein complex, it has
five subunits, it will bind to the B-Clamp, when ATP is around, ATP is
found and causes a conformational change in this complex, and it will
expose the binding site of gamma complex to Beta, when it binds Beta it will
open and put a little crack and allow enough space for the primer-template
junction to enter, when it enters this structure is going to stimulate ATPase
activity of this complex and hydrolyze ATP to ADP , when the gamma
complex has bound ADP it will NO longer have affinity for the B-Clamp.
And the gamma complex comes off and when it comes off, the Beta Clamp
closes itself and therefore got assembled onto the primer-template junction,
once you have that you can attract Pol 3 to bind and extend that region.
DNA Pol 3 HoloEnzyme: When he says HoloEnzyme it has a few more
components, you have two DNA Pol 3 core which can bind Beta clamp, you
have two sets of these things, Gamma complex is rite here and it is linked
together by subunits on Gamma Complex called taue . The Beta Clamp
attached to Pol 3 as so, All players in perspective, put them back onto the
chromosome, What these players are doing at the Replication fork,
What happens is that as Pol 3 moves further up is that Pol 3 will fall off. The
Pol 3 does not have the 5to 3 exonuclease, so Pol 3 can get stuck and then
comes off. B-Clamp stays around after Pol 3 falls off but it turns out that BClamp is important for recruiting Pol 1 and that helps it find RNA primers
that need to be removed. So Pol 3 falls off and it leaves a nick and NOT a
gap!!!!
Summary:

The Chemistry and Catalysis of DNA Synthesis are exactly the same at the
lagging strand and leading strand. However the leading strand is synthesized
continuously, while the lagging strand is synthesized discontinuously. This
is because the movement of the Replication fork is opposite to the lagging
strand synthesis. Therefore lagging strand synthesis must RESTART many
times to fill in the GAPs.
How do you keep distance of two strands the same so it will fit in one
structure, you form some kind of loop, loop will compensate for the distance
changed, thats how the two polymerases will be able to synthesize the
lagging and leading strand.

How are the two template strands separated? In NORMAL

CIRCUMSTANCES it is very difficult to separate them, need to heat it to
95C to melt it out in order to separate them you need this protein called
DnaB, which is a DNA helicase that moves in the 5 to 3 direction!!!!!.
In Eukaryotes, Helicase is on the leading strand (3to 5 direction)
In Prokaryotes, Helicase is on the lagging strand (5to 3direction)
The DnaB, Hydrolyzes ATP, it catalyzes movement along the 5to 3
direction, now you expose this template for the primase to come in, the
primase will lay down short RNA fragments and that will serve as a template
for synthesis, you also have long stretches of single-stranded DNA regions

and that is BAD for the cell, the cell will protect itself by binding singlestranded DNA binding proteins and prevent it from cleaving. END OF
LECTURE 8.
Lecture 9: Special Topics in DNA Replication in Prokaryotes and
Eukaryotes
Learning Objectives:
How the components of the replication machinery (POL 3 core,B Sliding
Clamp, gamma complex,helicase,primase,SSBs) are coordinated during
replication fork progression.
Compare and Contrast the Replication forks in Prokaryotes and Eukaryotes.

How do Pol 3,Pol 1, and Ligase switch? When Pol 3 hits the RNA primer
from previous synthesis and then it falls off, then it will leave behind BetaClamp, that Beta Clamp is recognized by Pol 1, helps recruit Pol 1, however
it doesnt help increase its processivity, helps recruit it there, Pol 1 can now
use its 5to3 exonucleases remove RNA and fill in with new DNA. BUT
WHY WOULD POL 1 NOT keeps going. Pol 1 cannot join strands together;
POL 1 will eventually fall off because it has Low processivity!!!!!!
Pol 3 HoloEnzyme: (Processivity is 100,000 ntd)
Pol 1 Processivity: 10-100 ntds, after 100 ntd Pol 1 will eventually fall off.
So who is fusing two strands together? DNA LIGASE!!!!!!!!!!!!! Dynamics
of Replication fork before diving into Special Topics.
How different components are assembled at the Replication fork, how they
work in a time dependent manner. Lagging Strand (has this big loop) is
primed many times, therefore you have many RNAs primers Arrowhead by
convention 3OH, The beta clamp will be behind Pol 3.
Gamma Complex: Connecting one Pol 3 to the other.
DNA Polymerase 3 is in Prokaryotes therefore DnaB(Helicase will be on the
LAGGING STRAND)!!!!!!!!

Primase: Guy that is synthesizing RNA Primer. Primase has weak

interactions with DnaB and this is how primase knows where to lay RNA
primer.
SSBs: Single Stranded Binding proteins, will be coating single stranded
DNA region.
Beta Clamp can also be found at Gamma complex, it is Open waiting to
interact with the primer-template junction.
How does everyone interact with each other? The trombone Model that is
proposed, how all these players are working to synthesize DNA at the
Replication fork.
DnaB in Bacteria will move in a 5 to 3 direction, therefore opening up
single-stranded DNA regions and remember what the fork direction is.
Primase interacts with DnaB and lays down complementary strand of RNA
primers, and when you have primer synthesized this will flip over and will
interact with gamma complex and when it interacts with that complex it will
thread itself through the cavity and put the strand complex into the hole of
the Beta Clamp, once bound ATP Hydrolysis occurs and this interaction
dissociates thus allowing the Beta Clamp to close back up, and when the
Beta Clamp closes it will have high affinity for Pol 3 , this will allow Pol 3
to synthesize at the 3end until it hits 5end of the previous Okazaki
fragment, when it hits the end it will fall off, leaving behind the Beta Clamp
, this is the signal to help Pol 1 to be recruited there, which in turn removes
RNA by Nick Translation with the 5to 3 exonuclease, eliminates RNA
region and synthesizes new strand in the 5to 3 direction.
By Contrast, the leading Strand is continuously synthesized; it doesnt need
to be reprimed multiple times.
If there is a mismatch it will pause, remember flow chart, most of the time
incorrect dNTP will flow back out (1 in 100,000 SN2 reaction slow reaction,
you can still get a mismatch, since polymerase pauses, it will give enough
time for that breathing movement and go the 3to 5 exonuclease. The
exonuclease will chop off the incorrect dNTP. There is proofreading in the
leading strand also, proofreading processes is occurring on Both Strands.
Remember that Pol 3 on the Leading and Lagging Strand are of the same
chemical structure. 14:56
We can learn a lot from Bacteria, Lagging Strand defined by loop structure
In Prokaryotes DnaB is sitting on the lagging strand, moving in the 5to 3
direction, Primase will transiently interact with DnaB structure, thereby
priming synthesis.
In contrast in Eukaryotes, the Helicase is called Mcm2-7 and is sitting on
the leading strand.

In Prokaryotes, the Polymerase on both the leading and lagging strand ARE
THE SAME, they are both Pol 3.
HOWEVER IN EUKARYOTES the leading strand polymerase and lagging
strand polymerase are DIFFERENT.
The Eukaryotic Leading Strand Polymerase is called Pol and the
Lagging strand polymerase is called Pol . The way they identified these
polymerases is they were able to identify mutants, for example a mutant of
Pol was found to have mistakes in the leading strand and when they
mutated Pol they were more mutations in the lagging strand. It turns out
that in Mammalian systems, no polymerase has 5to3 exonuclease. Both
Pol and Pol Have the 3to 5 exonuclease proofreading but doesnt have
the 5to 3 exonuclease.
So how does it remove nicks? It turns out that in Mammalian Systems, the
Okazaki fragments that is synthesize by Pol and when it hits the 5end
RNA region it will plow through generating something that looks like:

When it generates this flap there is an enzyme called FEN1(Flap

Endonuclease) will recognize that flap and cut it thereby removing RNA
primer!!!!! and part of the DNA also!! This is the second major difference
between Prokaryotic and Eukaryotic Replication fork. First major difference
is the position of the Helicase and the second is the mechanism of removing
the RNA Primer.
Functions of Pol 1 (Gap Removal, DNA Repair, enzyme could recognize
mismatch upstream or downstream and put a little nick.
The way Pol falls off has a lot to do with chromatin, DNA is packaged
with Nucleosomes, Pol will stop rite in the middle of the Nucleosome.
Chromatin is professor Luks favorite topic!!!!!!!!!!!

Know the differences in Prokaryotic and Eukaryotic Replication fork, for

example DnaB is called Mcm2-7 in eukaryotes, you want to know that
there are two different polymerases at Replication fork in Eukarytoes and a
third polymerase which is associated with primase. Primer is not just
RNA!!! There is a third Polymerase that is associated with primase!!!
In Eukaryotes, primase will start first and the Pol a will add a tiny bit of
DNA. KNOW FEN1(Flap Endonuclease) will recognize that flap and cut it
thereby removing RNA primer!!!!! And part of the DNA also!!
MAKE SURE YOU KNOW THE POSITION OF THE HELICASE IN
PROKARYTOES AND EUKARYOTES,
The Clamp Loader in Eukaryotes is RFC(Replication factor C).
In Eukaryotes, single stranded binding proteins is called RPA(Replication
protein A)
The B-Sliding Clamp in Eukaryotes is called PCNA (Proliferating Cell
Nuclear Antigen).
Learning Objectives for Special Topics
Explain how Eukaryotic Origin of Replication are regulated to fire once and
only once!!!!
Explain how Cyclin dependent Kinases (Cdks) active the pre-replicative
complex
Describe the end replication problem
Explain what the functions of Telomeres
Explain how the lengths of telomeres are regulated.
How fast is DNA Replication? We know that the E.Coli genome is 5 million
base pairs, (REMEMBER THERE ARE TWO REPLICATION FORKS)!!!!
5 million /1000bp/s=5000 and because replication fork will go in both
directions 2500 seconds is about 40 minutes, InVivo its a little faster,E Coli
can replicate its genome in about 20 minutes You can have a plane moving
along the DNA with that kind of speed.

How long does it take to replicate the Human Genome? Which are 3 Billion
base pairs, Speed of Polymerase Reaction: 1000 nt/sec. If you have one
origin of Replication going in both directions 3000,000,000
/2/1000=1.5,000,000 seconds/60 seconds which gives you 25,000 min,417
hours = 17 days. 3 days after In Vitro fertilization you have 8 cells (The
math doesnt add up!!!! You need to have more origins of replication,
Turns out that in Human Cells, there are 10,0000 to 100,000 origins of
replication. All origins in Eukaryotes are 30 kB apart and are coordinated to
fire during S Phase (Stage where DNA Replication occurs). After it fire
once, the DNA Sequence that specifies the origin of replication, it will no
longer fire and will keep off DNA sequence until you form complete
genome. Another way of looking at it, So you have 5 different origins of
Replication , Origins 3 and 5 fire first, making new DNA , forks moving in
both directions , Later on Origin 2 fires and Origin 1 fires, Origin 4 never
fire but the fork actually passed through Origin 4 and also needs to make
sure it no longer fires!!!!! So there is a lot of regulation!!! How does it know
that once it fires once it wont fire again. Origin 4 never fired, but Origin 1
fired late, Origin 2 and 4 are passively replicated. So most likely you have
Back Up origins of Replication!!!!!
From Slides: No Origin can initiate after it has been replicated,
therefore, it must be inactivated until the next round of Cell Division,
Not all potential origins need to activate to complete replication.

How do you know that there is an Origin of Replication? Sequences,

Origins of Replication have specific DNA Sequences; can use

Bioinformatics to look for sequences that are similar to each other, these
Origins of Replication are marked by specific proteins!!!!!
KNOW CELL CYLCE G1,S,G2,M

Superimposing Cell Cycle Stages to the process of DNA Replication

Sequences in Origin of Replication are recognized by a lot of proteins inside
the cell, one protein complex is called ORC!!!!! Origin Recognition
Complex recognizes the DNA Sequence of the origin and is loaded first.
These things are loaded during G1 phase, once ORC is loaded this will
recruit other proteins including Cdc 6 and Cdt 1 these are helicase loading
proteins. Once ORC is loaded this is going to recruit other proteins Cdc 6
and Cdt 1(THESE ARE NOT HELICASE, but they ARE PROTEINS that
help load Helicase. Mcm2-7 mammalian version of DnaB that is a ring like
structure, How do you load Ring-like structures onto DNA strands, When
Cdc6 and Cdt 1 are loaded they can recruit MCM 2-7 to LOAD onto double
stranded DNA. So the helicase is loaded onto the Double stranded DNA
Will it split two Strands apart? NOOO not engaged in proper way. The point
of theses proteins on the Origin of Replication pre-RC (Nothing is going to
happen, the loading step you load the pre-Replicative Complex at the
Origin of Replication to PREPARE THE CELL for DNA Replication. At G1
phase the cells are actually preparing a lot, to prepare the cells for DNA
Synthesis.

What happens next in transition from G1 to S-phase there are a group of

proteins called S-phase Cylin dependent Kinases: Cdk and Ddk these
kinases will add phosphate groups to a lot of target proteins, will add to
Cdc6 and Cdt1 and Mcm2-7, when Cdc 6 and Cdt 1 are phosphorylated
they dissociate from the Pre-Rc Complex. Cdc 6(with phosphate) will be
degraded because of the phosphorylation mark, the Mcm2-7
phosphorylation will cause a conformational change, so Mcm2-7 will now
encircle single strand (Mcm 2-7 will now encircle LEADING STRAND).
This activity is determined from activity of these Kinases called
Cdks!!!!!!!!!!!!!
At G1, Cdk kinase activity is LOW!!! Therefore Cdc6 and Cdt 1 are
unphosphorylated and Mcm 2-7 is unphoshorylated. This allows proteins to
assemble onto Origin of Replication complex, and when you transient to Sphase, all the Kinases turn on, when it turns on it phosphorylates Helicase,
therefore engaging it into the active conformation, and now can assemble the
replication fork allowing Pol and Pol to engage with DNA by allowing
synthesis to occur. When you have HIGH KINASE ACTIVITY you
INHIBIT formation of Pre-RC and activate Pre-Rc to allow it to fire, this
ensures that Replication fork fires once and only once!!!!!! Pre-Rc
assembles at an origin only in G1!!!!! And activates after entering S-Phase.

In Summary,Cdks have two roles during S-phase:

1. Cdks activate the Pre-RCs that are already formed
2. Cdks inhibit the assembly of any new Pre-RC
3. Cdks are not active in G1

When a replication fork passes through a Pre-RC that has not yet been
fired, it disassembles the Pre-RC(Origins 2 and 4) . End of Lecture 9
47:03
Lecture 10: Special Topics in DNA replication and DNA Topology!!!!
Learning Objectives:
Describe the End Replication problem
Explain what the Biological Functions of Telomeres are
Describe the components of Telomerase
Describe the enzymatic activities of Telomerase
Explain the Molecular Mechnishm of telomerase
Explain how the length of telomeres is regulated.
Telomere sequence and length in Eukaryotes
A telomere is a region of Repetitive DNA Seqeunces at each end of a
chromosome. It functions to protect the chromosome end from
deterioration and from fusion with neighboring chromosomes. Telomeres
are composed of hundreds of repeats of a TG-rich 6bp sequence
(5TTAGGG 3 IN HUMANS)
Lecture 10 Recording Notes:
How do special cells in Eukaryotes replicate the end of a chromosome, a
lot of bacteria have a circular chromosome, so they dont really have an
end, but in Eukaryotes they have a linear chromosome, this becomes a
problem when you have to replicate the end of a chromosome. The
Enzyme that has evolved to fix that problem, Telomere region of DNA at
the ends of linear chromosomes, consists of a lot of repetitive DNA
Sequences in different organism these repeat are uniquely different. In
humans we have the repetitive sequence 5TTAGGG 3 this unit can be
repeated 800 times to 2,500 times. Telomere has two major functions
this special sequence at the end helps prevent detoriation because of
DNA Replication, number 2 is that end normally will be recognized as a
break and that break will initiate a chain of events that will tell the cell to
fix double stranded break. However cell will distinguish real
chromosome end and accidental break cause by DNA damaging agent.
This TELOMERE will prevent the chromosome from undergoing fusion
that is triggered by DNA Repair Machinery!!!!!
END REPLICATION PROBLEM: Why do you need Telomeres? Why
would end of linear chromosome deteriorate as time goes on?
In Eukaryotes, you have multiple origins of Replication. Understand
why the leading strand and lagging strand are placed where they are.
Why on the other side of the Replication fork the leading and lagging
strand flip. Make sure you know which arm is leading strand and which

arm is lagging strand, you need to know where deterioration is coming

from. Leading Strand is synthesized completely all the way till the end!!
Eukaryotic machinery does not have Pol 1, it has Pol which is
responsible for lagging strand. Leading strand is synthesized completely
till the end. Pol will plow through the Okazaki fragment and make a
flap, Okazaki fragment will flip out, when you have flap an enzyme
called FEN1 is going to cut and remove it, eventually RNA primer will
be targeted by an Rnase and you will end up with a single-strand Gap
(that is sticking out and you wont have anything to prime the synthesis.
Now daughter cells will go through cell cycle and will replicate again.
Top Strand contains all Genetic information, but the bottom strand when
used as a template; you can see that it is lacking some genomic
information, so you are losing some genomic information. Every time
you duplicate the genome, you will lose tiny bits at the end and
eventually you would chew into genes, if you do that you will lose
function of the gene. That is the END Replication
problem!!!!!Appreciate the End Replication problem. Lagging strand
Synthesis is unable to copy the extreme end of linear chromosomes. The
genetic information in the chromosomal termini will be lost in the next
round of replication and repeated rounds of replication will cause the
ends to get progressively shorter. This is called the End Replication
problem.

Enzyme called Telomerase, which will extend this 3end, it looks like it
doesnt have a template when it extends it, and it has a template that is
incorporated into the enzyme to do this. Once you extended the 3end to

make it longer, now you put in primers and DNA Polymerase can come
in and extend DNA Sequences, therefore protecting it from being
degraded like artificially provided an end to prevent deteriation of itself.
MAKE SURE YOU KNOW who is synthesizing which strand, little
green strand is the RNA primase puts in primer puts in primer template
junction that will be recognize by DNA Polymerase

How telomerase enzyme works at molecular level, remember end of

telomere consist of these 6-mer repeats, 6 nucleotides of repeating sequence
in this case it has TTGGGG and it repeats. Telomerase has three
components!!!!!! One is a DNA polymerase (5to 3) similar to pol 1
pol3active site, also has Telomerase RNA which is used as template for
synthesis it is (150-1300nt long), only a small region is used as a template. It
has RNA: DNA Helicase activity, once it synthesizes new strand it will
separate it, then enzyme can move on to the next position. Telomerase RNA
(variable in different organism) contains a 9-mer(9 nucleotide) sequence that
is complementary to the 6-mer repeats, the template is 9 , but what it is
putting on the repeat is 6 Nucleotides. It will use its first 3 Ribonucleotides,
CAA will pair up with the end(3 nucleotides at the end of the telomere) and
exposed single stranded RNA template(which will be recognized by DNA
polymerase domain of telomerase to synthesize extended strand . Anneal
first exposing six nucleotides and that will add nucleotides and you have
now extended telomeres 6 nucleotides long. Next it basically needs to repeat
this process needs to translocate, these are all Hydrogen bonded, Watson and
Crick base pairs , double helical structure which is stable. In order to
separate that you need Helicase activity will allow, Telomerase enzyme to

move and expose single-stranded DNA region to synthesize next 6

nucleotides. You can actually mutate and change sequence of telomere.
20:59

Other sequences of telomerase are not used as template; Telomerase forms a

loop that can be recognized by a protein component, RNA Polymerase will
transcribe Telomerase RNA. Once it is transcribed it will interact with
other protein component. How does the cell know not to export Telomerase
RNA to cytosol to make protein, you have this unique structure that is going
to be bound by telomerase protein, once it is bound it will fold into
Telomerase enzyme.
IMPORTANT: When you mutate Telomerase RNA template, you see the
telomere sequence can change thats how they proved telomerase RNA is
used as a template for synthesis. You can put mutations in the
Telomerase RNA, this will change the sequence of the Repeat!!!!!!23:14
Once telomere end is extended to certain length, it is going be recognize by
some proteins, these proteins will bind and prevent it from being recognized
by double stranded breaks that will inhibit recognition by DNA Repair
machinery.
When will you see double stranded breaks? Ionizing Radiation will break
covalent bond in phosphodiester bond, when two strands are broken, the
strands will fly off, once it flys off this is a problem.
When will you get double stranded break in Replication fork during
Recombination you want to make breaks intent ially and once you make that
break you want to recombine with Homologous situation of programmed
breaks.

When will you have higher frequency of making double-stranded breaks?

when you have paused polymerases when you have mistakes, for example
cigarette smoke, blocking process of replication fork. PCNA(Sliding
Clamp), MCM2-7(Eukaryotic Helicase), Helicase will keep going and
leave a region of single stranded DNA, when you have DNA damage you
have increased chance of double stranded break. When you have these kinds
of breaks you will immediately recruit repair proteins. When protein
recognizes repeating sequences, it will coat whole Telomere with a special
protein that will prevent telomere from being recognized by the Repair
Machinery and inhibit DNA repair process. Last thing you want to do is
fuse ends of two chromosomes together.!!!! and you see that a lot in
cancer cells. Another model that showed how chromosomes ends is different
from DNA breaks, is that these repetitive sequences can invade themselves
and forming a t-loop, people have said that this inhibit DNA Repair
Machinery. Telomerase activity is pretty low in Somatic Cells(NonSex
CELLS),
Why dont they need Telomerase activity? Somatic Cells dont divide
quickly. When you dont have Telomerase activity it means that Telomere is
going to shorten.(Plays an important role in aging) Germ cells, Stem Cells,
Cancer cells, need to divide quickly when they have too. 90% of Cancer
cells have Telomerase activity that is because Telomerase will help
overcome END Replication problem!!!!!! Do Somatic Cells have
Telomeres? Yes but they dont have telomerase activity.

Learning Objectives:
Understand how DNA Supercoiling affects replication and transcription.

Know how to use linking number (Lk) to describe DNA topology and
calculate Lk of circular DNA and linear DNA with constrained ends.
Know how the two components of Lk: Twist(Tw) and
Writhe/Supercoiling(Wr) are related to each other and know how to
determine the Tw and Wr values of DNA.
Describe how Type 1 and Type 2 Topoisomerases change the linking
number of DNA.
DNA Topology: DNA Supercoiling how it affects DNA Replication.
What is DNA SuperCoiling? Coiling of a Coil, DNA double Helix is a coil.
Relaxed DNA (10.5bp/turn) is NOT a supercoil!!!! Supercoil is a coil of a
coil.
Why is DNA Supercoiling important to the understanding of nuclear
processes? As you are separating the two strands and fixing constrained end
is that the number of turns is going to change but is going to compressed to
be contained in a shorter distance, the number of bp/trn is getting smaller
this is not stable, DNA is not happy it will relax the cell by making these
supercoils, it is going to change the number of base pairs per turn BACK
10.5, DNA will try relax itself by making supercoils.
In Transcription, in front of RNA Polymerases you will see over wounding
of DNA, BUT in case of Transcription you dont have free DNAs that will
get relaxed, you end up with Underwound DNA in the back of RNA
Polymerase
In Both Replication and Transcription you end up with Overwound DNA in
the front!!!!!! DNA Polymerase and RNA Polymerase cannot progress
without dealing with this issue. Enzyme Topoisomerases is responsible for
taking care of this situation. E.Coli has a circular plasmid (chromosome) it
doesnt have an end that can freely rotate. In case of chromosome, he said it
has an end in theory that end can rotate but since its so big the mass will
prevent it from turning. Chromosomes are organized in Scaffold forms,
Chromosomal DNA attached to some kind of protein, which is going to
create loops; point is you have constrained ends that will keep it from freely
rotating. DNA Replication ABSOULTLY dependent on these enzymes
called Topoisomerase, which are responsible for releasing Torsional Stress.
There are drugs that will inhibit Topoisomerases you will block DNA
replication, these enzymes prefentially target cancer cells over regular cells.
Linking Number (Lk) is a topological property that describes the number
of times one circle wraps around the other. DNA is a double helix and wraps
around many times. In order to demonstrate just imagine DNA is just two
parallel strands. Two Unlinked circles give a Lk=0. By Convention, one
right-handed helical turn/twist in the closed dsDNA circle gives a linking

number of +1. Closed Circles with Three Helical Turns will cross each other
3 times and give a Lk= +3.

Gives you two hundred Helical turns and an Lk=200!!!!

Lecture 11: DNA Topology
Learning Objectives
Understand how DNA Supercoiling affects replication and transcription
Know how to use Linking Number (Lk) to describe DNA topology and
calculate Lk of circular DNA and linear DNA with constrained ends
Know
how
the
two
components
of
Lk:Twists(Tw)
and
Writhe/Supercoiling(Wr) are related to each other and know how to
determine the Tw and Wr values of DNA.
Lecture 11 Recording:
Introduce two more parameters to understand DNA Topology!!! The
enzyme that is responsible for relaxing DNA change Topology to enhance
activity for Replication and Transcription!!!! When you have one righthanded helical turn, by convention you have a linking number of 1 and you
can appreciate its one helical turn, by imagining that you straighten out, the
red strand, then you flip the red circle over, and you also straighten out the
black strand. You can see that one circle is making a full circle around the
other. One helical turn you have a linking number of plus 1!!!!!!

Need to know where does a Helical turn start!!! Three helical turns gives a
linking number of +3!!!!!

Linking number must be an integer!!!! DOES NOT HAVE DECIMAL

PLACES, You either pass through the circle or NOT!!!!!

Dont have half a linking number!!!! Can think of the Linking number as the
number of times the black lines passes t0hrough the soap film!!!! Linking
number of a closed circular DNA is the number of times one strand passes
through the imaginary surface (soap film) formed by the other strand.

You can diffuse sharp turn into small turns that distribute equally throughout
the entire molecule this is called a supercoil but they are arranged in slightly
different ways, there is an intertwining and solenoid supercoil. Distinguish
between a positive and negative supercoil!!!!!

You can transform a Supercoil to one helical turn (Twist) if you separate the
two strands apart, you can see the red strand and black strand are
topologically linked together.
Linking number can be expressed in different ways it can look like a
supercoil or it can be a Helical Turn. Lk does Not change when the circles
are Bent or deformed, will only change if you cut the two strands.
For Example if you start with a molecule with a Lk=+1, Now if you cut it
the two molecules are no longer linked together. Whenever you have
something that is cut, the Lk is undefined!!!! At this point you can take one
end and twist it, in this example you introduce two more twists (three helical
turns for that molecule and still the Linking number is undefined because the
two molecules arent linking together, but once you reseal (re-ligate) that
molecule the Linking number becomes defined again, and now because you
have three helical turns the Linking number is now three!!!!!

You can also do that opposite by cutting at molecule with Lk=+3 then
untwisting to decrease Linking number!!!! The ability to cut the DNA and to
add or remove helical turns/supercoils in DNA is biologically important.

Tw(Twists): The number of Helical turns

Wr(Writhe): Number of Supercoils
Linking Number is the Sum of twist and Writhe!!!!! You can see how it
moves from one form to the other!!! Once you cut something the Linking
Number is undefined. In a closed DNA Circle or linear DNA with
constrained ends, Tw and Wr are interconverble.
When you a circle that is laying flat on a surface, basically there is NO
supercoil at all and immediately tell that the molecule has a Writhe of Zero,
Basically the linking number is equal to the twist!!!!! Molecule can be
overwound or under wound and still the writhe is zero!!

By Convention, Lko refers to the Linking Number in Relaxed B-Form DNA

and NO Supercoil!!!!
As you are moving forward in the Replication fork the number of Base pairs
per turn is getting shorter Int: 10.5 bp/turn and then After: 8 base pairs/turn,
you have fewer base pairs per turn(this overwound) This will turn into
Supercoil!!!! When it turns into a SuperCoil it will be less twisted, when you
increase the Writhe the twist is going to decrease!!!! So initially you have an
overwound end but once you form supercoil, you relax it. The Supercoil will
eventually block replication machinery and you will want to remove
supercoil for the Replication fork to move freely.

So we have TopoIsomerase to take care of that. So at this point Linking

Number does not change. But once Topoisomerase comes in the Linking
number will change. If no supercoil, writhe is zero!!!!!!!!

If you introduce a Tw the molecule does not have 10.5 bp/turn, so it wants to
relax itself and in order to do that it forms a supercoil!!!! Once it forms a
supercoil the molecule is no longer twisted. The twist is now turned into a
supercoil!!!!! And the twist becomes more of its Natural state. Remember
natural tendency of the molecule is to go back to 10.5 bp/turn. DNA
molecule does not like sharp turn like that, the twist can cause strain, and
there is an equilibrium that turns twist into supercoil. Twist and Writhe
doesnt have to be an integer, you can have half a twist!!! How molecules Lk
number is changed to three and three to one, now we are applying the
concept to a real DNA molecule!!!!
Ok so you put a nick in the DNA strand the Linking number is undefined,
take the end and go clockwise and remove two helical turns and then reseal
thus changing the linking number remove two helical turns thus the linking
number becomes 198!! Didnt change supercoil yet.

These two molecules above are topologically equivalent because they have
equal linking number!!!
Two Negative Supercoils (Writhe is -2) In order to have Negative Writhe;
you need to compensate with twist. You need to increase Twist to get
constant Linking Number!!! When you remove one supercoil the twist has to
decrease, because you increased 1 writhe.
So there are two ways of making supercoil, positive and negative supercoils.

Theses

two cases

above you stressing DNA Molecule and the molecule doesnt like to be
stressed, Wants to relieve Torsional Stress thus forming Supercoils. The
base pairs per turn goes back to 10.5bp/turn, Sharp turns also place stressed
on the DNA also.
How can you tell the difference between negative and positive supercoils?
Need to tell if you are looking at interwound or solenoid version of
SuperCoil, when it is interwound, you see a right-handed supercoil, you are
looking at a negative supercoil, when you look at solenoid and it is left
handed it is a negative supercoil.
LUKS RULE:
Thumb is pointing to yourself, you see this a right handed, which is a
positive supercoil!!!!!!

Left-handed is NEGATIVE supercoil!!

So first determine if its interwound or solenoid supercoil, once you

determine if its interwound find the end of the molecule and do the left hand
and right hand rule.
IF ITS LEFT HANDED YOU ARE MOVEING UP AND THAT
NEGATIVE!! When you look at a solenoid or torodial left handed is always
negative supercoil!!!!
When its right-handed its Positive!!!!!!!!
Come Back to the cell, as DnaB is moving forward, over winding DNA,
when you do this too much you are going to accumulate SuperCoiling in
front of the polymerase and eventually block movement of the Replication
fork, cell needs a way to relieve Torsional Stress to allow Replication fork to
move forward. So they are two types of TopoIsomerase in the cells that can

relieve Torsional Stress and do so in two very different ways. Focus on

molecular and Biochemical Mechanism on how these two enzymes work!!!
Topoisomerases: Either Catalyzes the Unwinding and Over winding of
DNA thereby changing the Linking Number because they cut the DNA and
twist it and religate it. These Enzymes are targets for a lot of Cancer drugs
because cancer cells need to replicate quickly and Topoisomerase activity is
essential for these Cells. A lot of Drugs target these molecules.
Type 1 Topoisomerase: Breaks one of the two DNA strands and once its
cut it brings one strand through the other, thereby removing a helical turn.
Type 2 Topoisomerase: Breaks BOTH DNA strands and once it breaks 2
strands it passes strand of DNA over.
Type 1 topoisomerase will change Lk=+1 ONE AT A TIME
Type 2 topoisomerase will change Lk=+2 in increments of two!!!!!!!!

Focus on Type 1 topoisomerase, Molecular Mechanism first, first step

Topoisomerase first binds double stranded DNA, pay attention to the lines, it
will then cut one strand, after it cuts it will hold on to the two ends and let
the other strand pass through, the enzyme will then undergo a
conformational change and the other strand goes through, when the other
strand goes through, it will seal broken strand and doing so it will change the
linking number by 1. This enzyme doesnt require ATP Hydrolysis, make
sure to indicate whether its single stranded or double stranded.
Type 1A attaches to the 5end, Type 1b to the other end.(DIFFERENT
BRIEFLY MENTION
LUK EMPHASIZES: When it breaks the strand, one end forms a covalent
bond with the enzyme (transiently), the other end is held on by NON-

COVALENT INTERACTIONS!!!!!!!!!!!Final outcome is will hold two

strands and pass the other strand across.
In step 1 there was a Tyrosine Residue on the enzyme that is going to attack
phosphodiester bond, when it does that it cleaves a phosphorus oxygen bond
and leaves a 3Hydroxyl Group. Tyrosine will hold on to the strand, this
creates an opening that allows other strand to go through this bond is called
a 5phosphotyrosyl linkage.

Once you passed other strand through, you need to reform this bond, the
3OH group will attack phosphate group and reform the phosphodiester
bond!!!!(Reforms Tyrosine) These molecules are stabilizing by Argine
Residues or Metal Ions. YOU NEED TO KNOW THAT THERE IS A
TYROSINE THAT WILL FORM A TRANSIENT LINKAGE, that will first
cut DNA and then religate it. There is a conformational change after loop
passes through.

Lecture 12: DNA TOPOLOGY (CONTNIUED) DNA Lesions, mutations,

and repair!!!!!
Learning Objectives of Part 1
Describe how Type 2 Topoisomerases changes the Linking number of
DNA!!
Draw the molecular and Biochemical Mechanisms for Type 2
Topoisomerases.
Explain the Electrophoresis gel assay for DNA topoisomers and interpret the
experimental results!!!
Understand how gyrase generates-ve supercoil in DNA plasmids
Describe the structure of a NUCLEOSOME AND CHROMATIN
Explain how nucleosome assembly and disassembly influences DNA
Topology, transcription and DNA Replication.
Lecture 12 Recording Notes:
Assay you would use to measure DNA Topology.
What is an ASSAY? A test that measures certain properties of a molecule
for example, an assay that measures how many supercoils are in a circle.
Bacteria, there is an enzyme called Gyrase!!!! Which is a Type 2
Topoisomerase, which will introduce negative supercoils inside the cell.
Why do you want to generate negative supercoils inside the cell in Bacteria?
In Eukaryotes you dont have an enzyme like Gyrase , the enzyme is Type 2
Topoisomerase which wouldnt actively put negative supercoils in DNA.
Talk about Chromatin, how Chromatin will change Topology of DNA,
might play a role in dealing with Torsional stress you introduce during DNA
Replication and Transcription. Type 2 topoisomerase difference between
Type 1 and Type 2 TopoIsomerase is the MAJOR DIFFERNCE TYPE 1
ONLY CUTS ONE OF TWO STRAND, when it cuts one strand it lets the
other strand pass through, therefore can remove a helical turn by doing that
you change the linking number by an increment of 1. By Contrast Type 2
Topoisomerase will cut two strands each time, when it does that it breaks the
entire double strand of DNA and passes another double strand of DNA
through by doing that it changes the linking number by 2. This Enzyme ATP
is required for the process by contrast Type 1 you dont need ATP
Hydrolysis. Make sure you know the difference between Type 1 and Type 2
Topoisomerase. Molecular Mechanism first!!!!! So a Type 2 Topoisomerase
would hold on to 1 double stranded DNA make a cut in the middle and
passes another dsDNA through it. So the supercoil in the picture below is an
interwound supercoil.
The molecule below first has a -2 writhe!!!!

Look at molecule below, pull strand apart and you see a circle now!!!!

How the linking number changes after it goes through Topoisomerases 2,

think of cutting Rubberband and passing it through the back, We changed
the linking number from +2 to 0 or from 200 to 198.
Reaction catalyzed by type 2 topoisomerase, What else is going on the type
2 topoisomerase has different domains, it has a domain called N-Gate and a
domain called C-gate, serves as a gate which will allow one molecule of the
dsDNA to enter. When a dsDNA binds the N-gate closes, you can no longer
allow a second strand to enter!!!This is important because you only want
strand to pass through that break!!! That gate prevents multiple strands from
coming in. Once N-gate encloses strand(pink strand) , the type 2
topoisomerase will undergo a conformational change that will allow a
Tyrosine Residues to attack phospodiestrer bond on the blue strand, when
you do that it will break one strand of the double , and a second tyrosine will
do the same thing , you have two tyrosine residues attacking the two strands
on the blue double stranded DNA.Seperated Strands with Tyrosines holding
on to the Two Ends of the two terminai of the blue double stranded DNA,
now you can pass other dsDNA through the C-gate pink DNA is by c-gate
region, at this point blue strand will get resealed. Tyrosine will get kicked
out and dsDNA will reform at this point the pink DNA is released!!!!

Enzyme will ensure that only one double stranded will enter the Gate, and
one strand is released, and the conformational change requires ATP
Hydrolysis. ATP Hydrolysis is absolutely required for Type 2
Topoisomerase. Now zoom in how reaction occurs. Tyrosine in
TopoIsomerase 2 acts as a Nucleophile, looks very much like exonuclease
mechanism, in exonuclease the Nucleophile is water, this case the its the

Hydroxyl Group of the Tyrosine, You need Metal ions, because 3Hydroxyl
Group is a terrible leaving group. TopoIsomerase 2 also has Mg as cofactors.
Important DNA Polymerase, Exonuclease and TopoisomeraseS have
this 2-metal ion mechanism. One of the metal stabilizes 3Hydroxyl Group
which is the leaving group here, distributing the negative charge, making the
3OH a better leaving group, also helps deprotonate the Tyrosine, so it is Owhich is a better Nucleophile. Mg also helps distribute charge on oxygen
attached to the phosphorus making the reaction better. The Mg will not just
be floating around, what is the best residue to coordinate Magnesium?
Aspartate!!!! Aspartate is D and Glutamate is E (one extra carbon) all these
coordinate Magnesium putting it in the rite orientation, to allow reaction to
occur efficiently. Another example of Two Metal Mechanism!!!
Once you have the 5phosphoTyrosol linkage (transient) form and the strand
passes through what will happen is the hydroxyl group will attack phosphate
again and then kicking tyrosine off as a leaving group. Talked about Type 1
and Type 2 Topoisomerase!!!!

Introduce two different Methods, too study and measure activity of this
enzyme as well as measuring DNA Topology of Circular (plasmid DNA).
First Method is Electron Microscopy, seeing it under the Electron
Microscope, you can see a DNA Plasmid, which basically has No supercoil
(Circular DNA), or a molecule with 4 super coils (More Supercoil or less
supercoil just by looking at it. Much easier method to quantify determines
how much supercoil is present in DNA. This would be using Agarose Gel
Electrophoresis assay; small pieces of DNA run faster and long pieces of
DNA run slower!!! C will run faster in the Gel because it is more compact,

when you have a big circle it really slows it down. IF YOU HAVE MORE
SUPERCOILS IT WILL RUN FASTER, COMPLETELY RELAXED
CIRCULAR DNA will be VERY SLOW!!!! Linear DNA of the same length
will be in the middle.

Summary: Most of the Enzymes will relax supercoils, majority of

Topoisomerases relax DNA supercoils, either negative or positive
supercoils. In Bacteria you have a very special Type 2 TopoIsomerase (DNA
Gyrase), which is found in Bacteria that will actively put in negative
supercoils in DNA.

If you transform a circular piece of DNA onto E.Coli , how pneumonia will
take up DNA from smooth strain and pick it up and utilize it, same
procedure where you take Bacteria , mix with a piece of circular DNA which
will allow the cells to pick it up and put it inside their cytoplasm, and what
will happen then is that Gyrase will act on this circular DNA and put a lot of
negative supercoils into that circle and when you do that and run it a gel
what you see in slide is what you will see!!!!!!You see a band at the bottom
of the gel of the highly negatively supercoiled DNA.
Why do you still see Relaxed DNA? Nicked circles (when you open up the
cells, you used, once you break bound you are going to nick it, and the other
strand because of the stress is going to relax, when it relaxes it becomes a
circle again therefore when you purified plasmid DNA. Plasmids are
artificial circular DNA, you used to put in Bacteria, a lot of time carries drug
resistance genes to help purify proteins. So a lot of times you will see a nick
circle.
Now take Plasmids from E.Coli and then you mix it with Type 1
TopoIsomerase, and after five minutes you precipitate DNA and run DNA in
a gel or let it go longer 60 minutes and run it in the gel. Anything in between
are TopoIsomers that have been cleaved Unwound.Reseleaded, you have a
distribution of molecules. The type 1 topoisomerase can cut one strand,
relax,reseal,bind again, cut and reseal and end up with molecules with
different linking numbers.
Which molecule has the highest linking number? Nicked circle has
undefined linking number. If the circle were sealed then would have
highest linking number from example. Bacteria are going to put in huge
amounts of negative supercoils.
Inside bacteria, DNA is supercoiled, now moving onto Eukaryotes,
Eukaryotes dont have DNA Gyrase!!!, it has type 2 topoIsomerase which
will relax or + supercoils. BUT IT DOES NOT HAVE ENZMYE THAT
WILL ACTIVELY PUT IN SUPERCOILS IN THE CELL!!!!!!!! 29:35
However Chromosomes in Eukaryotes is packaged into chromosomes and
the structure is highly organized, organized in these condensed chromosome
structures. Chromatin associated with some kind of scaffold to make these
kind of loop-like structures, basically many DNA circles, if you zoom in you
will see DNA is wrapping around a core of Histone proteins into these so
call Nucleosomes structures!!!!!! These Nucleosomes are packaged on top
of each other to form this 30nm FIBER. This compacts the DNA even
further, this allows 1meter of DNA to be compacted into a nucleus of
micrometer size. Very highly compact DNA at the same time extremely

dynamic ,because in order to access underlying DNA sequences , you really

need to make sure Histones can come in and out to allow access.!!!!

Zoom into the structure of the Nucleosome you will find 146 bp DNA that is
wrapping around a core of Histones with 8 proteins, has 2 dimers of (H2AH2B) Two separate proteins that form a dimer, one dimer on each side of the
molecule, one in front and the other on the backside. And the center is a
tetramer by (H3-H4)2 .
Nucleosome structures are organized in a Repetitive manner. Luks lab
studies how these Nucleosomes are organized in the Cell, turns out they are
organized in a very specific manner, very specific position inside the cell
and that position changes when you want to turn on or off a gene. Take
molecule apart to see what it looks like.

Yellow and Red is the H2A-H2B dimer, remove a dimer in front and the
back. Now remove (H3-H4) tetramer, now you can see a 146bp DNA is
wrapping around core. Solenoid form (Left handed coil) Negative supercoil
in a solenoid, left handed turn is a negative supercoil.

Why do Bacteria like to put Negative Supercoils into DNA,

remember what happens in DNA Replication and when have RNA
polymerase (transcription), what happen in front of the fork and what
happens in front of RNA polymerase? Are you OVERWINDING OR
UNDERWINDING? In front you are overwinding DNA and in the back you
are Underwinding.
Knowing that the bacterial Genome is negative supercoiled is this
Helping Transcription?
During Transcription, when you move forward or DNA replication when
you move forward, you are overwinding, if DNA is already somewhat
Underwound, isnt that you are preparing the Genome for Transcription and
DNA replication, therefore Gyrase is important for Both process. In
Eukaryotes you dont have Gyrase!! DNA is packaged into Nucleosomes,
when you kick off a Nucleosome (you basically have a negative supercoil)
and the negative supercoil is in Equilibrium between Under wound DNA
with negative supercoils. Once you remove Histones it no longer stabilizes
Solenoid Structure, now free to go between supercoil state or Underwound
State, makes underwound state to promote Transcription. Movement of
Transcription machinery will generate Positive Supercoil that will actually
kick out Histones!!!!

Lecture 13: DNA Alterations and Repair!!!!!

Learning Objectives of Part 1
Distinguish between Transition and Trans version Mutations
Draw the Adenine Tautomer and show how it could pair with cytosine to
cause a mismatch.
Explain the two-step process that converts mismatch into a mutation
Describe step-by step how the Mismatch Repair Pathway removes the
incorrect nucleotide!!!!
Explain how MMR knows which of the two nucleotides in a mismatch is
the incorrect nucleotide!!!!!

Lecture 13 Recording Notes:

DNA Replication how enzyme has proofreading mechanism and the error
rate to be low. Proofreading is not enough (1 out 10million) is not enough
for replication of our Genome. Talk about the Mismatch Repair System that

ensures that duplication of Genome has high enough accuracy. Distinguish

Mutations versus DNA Lesions!!!! One of the Mechanisms that is
responsible for fixing mismatches. Polymerase Insertion Fidelity has one
mistake in 100,000 nucleotides and the accuracy is improved when the
enzyme has the proofreading mechanism. , 3 to 5 exonuclease, which will
stall polymerase, and this, increases the accuracy to 1 mismatch in 10
million nt). In vivo accuracy is 1 mistake in 10 billion nt, this is really
dependent on DNA Repair Mechanism and this is called Mismatch Repair
System!!!!!!
So lets say there is a Mismatch, then in the process of generating a mutation,
a lot of times is a two step process, it involves incorporation of a mismatch
has not been repaired after second round of replication, THEN IT WILL
TURN INTO A MUTATION, and that will be read by the Transcription
Machinery to make a protein which has a mutation in it. So if you have this
Repair System then you want to fix this problem, at which step is repair
Mechanism fixing this problem. YOU WANT TO FIX IT AT THE B STEP,
WHEN MISMATCH OCCURS.
Think about an enzyme that is trying to correct this mistake, it only
recognizes the mismatch, it doesnt know what happened before and it
doesnt know what happened after. If you have a repair system, you need to
know number 1 there is a mismatch! If you think about the structure of
DNA, its always 20A in diameter but if you have a mismatch, then you
probably have a little bulge, also major and minor groove are going to
change also, with the Mismatch that pattern is going to change, that system
needs to be able to recognize that. At the Mismatch, its best position to
recognize that error because its structurally different!!!!! Remember the
protein doesnt have eyes to see what is going on, what the enzyme sees is
the structure, how the structure differs from the Normal Sequence. So
Number 1 it is Recognition of the Mismatch, what is the other challenge of
this system, what does it need to know, when it needs to correct this error.
WHICH IS THE ORIGINAL STRAND.

How does the Mismatch Repair System know which was original correct
strand? TWO VERY IMPORTANT CRITERIA that Mismatch repair
system functions at!!!!!!
Introduce Nomenclature, Two Types of point mutations, Transition and
Trans version.
In Transition Mutation if you have Pyrimidine on one strand and changed
to other Pyrimidine such as changing from C to T, a small ring changed to
another small ring, but a different ring then its a transition. When its a
trans version, changing from a purine to a pyrimidine, Thats a Tran
version mutation or pyrimidine to purine.

To have these kinds of Mutations it is usually a two-Step process, first,

either has a Mismatch or damaged base and in Second Round of Replication,
it incorporates the wrong base pair. DNA Alterations, lesions, you can
damage base pair in such a way that is no loner pairs with correct base pairs.

You can have big adducts that attach to it, then you would block replication
fork progression, Tran lesion polymerase will come in, you would most
likely incorporate error, you will probably have lots of mismatches. When
you block you put in Tran lesion Polymerases and you incorporate error that
way also. Once in a while DNA Replication Machinery , will incorporate
wrong nucleotide into strand. Bases have a very low probablity to
interconvert between Tautomer forms. In Adenine, nitrogen can get
deprotonated and fire electrons go down to form a double bond and other
nitrogen pairs up with Hydrogen. Now the Adenine Tautomer kind of looks
like a guanine now pairs up with Cytosine, which leads to a Transition
Mutation.
When Adenine Tautomer goes back to the original form Adenine , this will
cause a mismatch and the structure is going to change and cause it to be
recognizes by the Mismatch Repair System.

How it recognizes that unique structure and how does it distinguish the
newly synthesized strand, which has the error, and parental strand that
shouldnt have the error. Mismatch repair can also correct a small loop,
which is sticking out. Mismatch could be either in leading and lagging
strand. Now say you have this that adenine Tautomer, you have this
mismatch and is recognized by something and that something is a protein
complex called Mut S MutL this will recognize changes in diameter in
dsDNA and also bases. Mut S forming a dimer almost looks like it is
surveying by sliding through the hole. Mut S: Sensor Mutl: linker. This

thing is binding and recognizing that Mismatch, satisfying first the criteria of
this system, which is recognition!!!!! , In order to work what is the second
thing MMR Mismatch repair needs to know?, which strand is parental stand
,which strand should be template , which strand is newly synthesized ,
which should contain the error, How it does this depends on E.Coli Genome
, which in bacteria the DNA is methylated . And is methylated by DAM
Methylase this will find GATC sequence and put methyl marks on Both
sides on the A. Is the Methylation mark going to change Watson and Crick
base pairing with T? No it will not affect Watson and Crick base pairing.
However, there is an extra chemical group facing the Major groove DNA. If
you have both sides, two methyl groups rite next to each other in major
groove then you know this is the DNA before synthesis, Hemi-Methylated
site: DAM Methylase hasnt had the chance to put methyl group on the other
A yet, therefore you will form this Hemi-Methylated Site. Then there is a
protein that binds to this Site called Mut H!!!, it will recognize it, and this
Mut H is actually an Endonuclease, it cuts in the middle of the strand,
cleave phosphodiester bond, MUT H it binds to Hemimethylated site , it
would just bind to it and not do anything until it interacts with MutL-MutS
Complex when they interact it will stimulate endonuclease activity of MUT
H.
Which strands will Mut H CUT? It will cut the daughter strand. Mut H binds
first but is not doing anything then stimulate activate endonuclease activity
and then will cut, once it is cut then enzyme UvrD which is a Helicase is
going to separate two strands toward the direction of the Mismatch. An
exonuclease will also be recruited, as its doing that it will chop up, so you
generate a long region of single stranded DNA, usually around 1000 bps on
average why is it so long, isnt it better to cut it closer, the reason why this
cut region is so high, is because the probalitiy of getting this GATC
Sequence is not high. YOU NEED TO HAVE GATC Sequence to get HemiMethylated site. Somewhere in the genome you will find GATC site, but the
question is how far is it. You can have a GATC side on the right side and a
GATC side on the left side. It will find the closest Hemi-methlyated GATC
site and cut that site once its cut , it will denature strand using Helicase
activity (UvrD) and exonuclease will come and chop it Up. So you have this
long region of single stranded DNA so single stranded binding protein is
going to bind to it and from our understanding Pol 3 will come in and
synthesize that strand and replace it with the correct sequence. IN THE
MISMATCH REPAIR SYSTEM POL 3 IS THE ONE THAT IS COMING
IN!!!!! When SSBS bind , you somehow need to know there is single
stranded DNA and that there is no base pairing , sugar phosphate backbone

is going to fold a really sharp angle to single strand DNA. Needs to bind to
an angle to SSBS.Double stranded DNA cant bind SSBS because it is too
much of an angle for DSDNA.
On average base pairs cleaved here are greater than 1000 bp. The distance
depends on where you find GATC Site. If Mut H cannot recognize site, it
will need to go to the next one.
Key: Is you need Mut S-MutL to bind and recognize that mismatch and
same time have MutH binding to it and this first complex activates the
second to make the cut.
DAM Methylase: Methylates A when you see this specific GATC
Sequence, only methylates at this particular sequence. Exonuclease is going
to pass this mismatch chew away from Mut H, will pass Mismatch and
opens template strand for Pol 3 synthesis. 32:00
Lets say there is a mismatch, lets say you have two hemimethylated GATC
Sites, what will happen it will bind the closest one. KNOW MOLECULAR
DETAILS FOR MISMATCH REPAIR PATHWAY!!!!

\There are other types of DNA Alterations, you can have mismatch, you can
have changes in the Bases such as Deamination and depurination which is
caused by water. Water is going to change chemical property of the base, or
changing whole nucleotide by cleaving the base. And talk about BULKLY
ADDUCTS/Thymine dimers that are formed when you have carcinogens or
other mutagens that is changing the structure of DNA. Need to know how
these DNA alterations are corrected inside the cell. Using different kinds of
pathways for deamination and depurination(Use will use Base Excision
Repair System) and for Bulky Adducts one way to correct is by Nucleotide
Excision Repair.
Nucleotide Excision Repair: Cuts one base leaving the Sugar-phosphate
Backbone behind.
Cutting Nucleotides one at a time to expose correct template.

First type of damage is deamination. Amine group can be replace with

oxygen if water attacks carbon and forms Uracil.
Why evolution has selected Thymine, but not Uracil as DNA nucleobase? If
Cytosine can easily turn into Uracil, then once it changes no way to know if
original sequence is C or U. There is an enzyme that will recognize Uracil
Base and cut it out.

Depurination, which is the removal of the purine, water, attacks the C1

carbon and causes the base to leave, leaving a hydroxyl group. How could
this affect insertion fidelity, you no longer have other base to pair up in the
active site you can accommodate all four different bases, you dont have
base pairing to align the alpha phosphate for Nucleophile attack, but it will
still happen, although a lot less frequently, if you have a basic site you have
chances of incorporating all 4 nucleotides there.

The third type of DNA alteration is Bulky Adducts, Benzo pyrene molecule
in coal/Tar, when enter Liver you have an enzyme call P450, it is going to

put oxygens and it allows amine group to attack carbon forming these
covalent adducts this can block progress of Replication fork and force Trans
lesion Synthesis to occur.
Nitrogen Mustard is going to link two bases together Uv will do something
similar by linking two bases together. Downstream of Insertion strand on
template strand, there seems like there is a big kink or angle on the structure
of polymerase there is a big link or angle on the structure of polymerase
there is a very important twist on the template strand, preventing base from
pairing up with incorrect nucleotides, These Thymine dimers will block
progress of Replication fork!!!!
Base Excision Repair:
In Bacteria, what will happen is there enzyme Glycosylase that will sense
small changes in the structure in the Bases, for example if you have a Uracil,
you have a lacking of a Methyl Group, and you sense that and it will cut
Uracil Out. Once you cut that Base you will have a hole! AP endonuclease,
a purine endonuclease or pyrimidine endonuclease and will find that site and
make a cut on the sugar phosphate backbone and will allow Pol 1 and Ligase
to work. Uracil DNA Glycosylase will find Uracil Base domain that almost
looks like a thumb that will push Uracil out into the active site to get cut.
Glycolyse will cut out base, and then AP endonuclease will cut in sugar
phosphate backbone, then Pol 1 will come in to fill the GAP!!!!
Nucleotide Excision Repair: Is for Repairing Bulky damaged Bases, first
will get recognized by Uvr A protein they will bind, then UvrC which is an
Endonucleases will cut it in two sites. UvrD will unwind, expose,template
Pol 1 comes in again and fixes it.

Lecture 14:BioMedical/Research Applications

Learning Objectives
Define Genome, transcriptome,proteome,protooncogens, and tumor
suppressor genes
Explain how regulation of gene expression ultimately defines Cell Fate!!!
Explain how to use Northern Blot Analysis to measure mRNA level on a
gene by gene basic
Explain how to use Microarray analysis to measure gene expression in a
genome wide scale.
Lecture 14 Recording:
Two different Methods to measure Gene Expression Levels, one is a geneby-gene method and the other is a genome wide scaled measurement which
involves Micro Arrays.
What is a Genome? Is REALLY the information, complete Genetic
information that is required to specify that organism. In Eukaryotic Cells, a
genome is the entire complement of genetic information of the
Unpaired, Haploid chromosome set. That is the complete DATA SET.
Therefore since genome is Not really DNA, DNA is the material THAT
CHEMICAL THAT ENCODES GENETIC INFORMATION!!!! IN
EUKARYOTES AND PROKARYOTES Genomic information is encoded
by Double Stranded DNA. In viruses, genome can be encoded by
ssdna,dsDna,ssRNA,or dsRNA.
In Eukaryotes in addition to the Nuclear Genome you also have organelles
such as mitochondria, chloroplast, that also have their own genome.
DNA is the material that stores information in parallel if you think about 3D
printing, the material that makes up the HARDDRIVE, the coding system
A,T(U),G ,C in Biological systems and that parallel in 3D printing is 0,1.
In terms of Biological Systems, Genome is entire complement of
information in parallel 3D printing is the information of the entire hard
drive. The physical Hardrive is more related to the Nucleus. Genomic
DNA in humans is about 3.2 Billion base pairs contain approximately
25,000 genes, A lot of information that is important to define organism is
not only in the DNA that encodes the Genes. But also in the Regulatory
elements that regulate expression of these genes. If you actually look at the
Genome look at sequences you will find that only 1.5% of these sequences
are actually defining protein-encoding genes!!!!! The rest of the sequences
are either introns, non-coding RNA, regulatory DNA,unknown
noncoding DNA.
Why do you need all these DNAs that are not used for coding proteins? In
the olden days people thought it was junk DNA, but it turns out that these

DNA play an important role in regulatory expression of these protein-coding

genes. The 1.5% of protein coding genes play very important roles, for
example Cytoskeleton formation, immune proteins, Nucleic acid binding
proteins, and some of those are transcription factors!!!!! all this information
are present in an organism, they are turned on , and turned off at the rite
place, in order to determine Cell Fate of CELLS. These Non-Coding
sequences play an important role in regulating expression of these cells.
Totipotent cells: Are cells that have potential become all different Germ
Layers.
Puripotent Stem Cell: Can differentiate into other stem cell types and
ultimately those cells types lead to formation of other cell types in the body.
A totipotent Stem Cell has the same number of genes as that of a
Neuron. DNA Sequences are exactly the same, there is nothing changed in
the Genome, the Totipotent Stem cell genome and neuron genome is exactly
the same!!!!!!THE ONLY DIFFERENCE IS THEY ARE EXPRESSING
GENES AT DIFFERENT LEVELS.
So lets say a Totipotent stem cell and Neuron they both have 3.2 Billion
Base pairs, 25,000 protein-coding genes. The genes responsible for Cell
proliferation are turned on the Totipotent Stem Cell, because Stem CELLS
NEED to be able to self regenerate, Telomerase would be turn on in a
Totipotent stem cell. By Contrast Neuron doesnt want to proliferate at this
point, it has fully differentiated and dont want to proliferate and the cell
proliferation genes should be turned off. The genes are still there, they are
just NOT TURNED ON!!!!
In Totipotent Stem Cell, Stem cell specific genes should be turned on and
off in Neuron.Neuro-Specific Genes should be off in Totipotent Stem Cell.
In totipotent Stem Cell you want to make sure that Neural Specific and
Muscle Specific gens are off and progressing through differentiation of the
cells here. THEY NEED TO KNOW WHEN TO TURN ON AND WHEN
TO TURN OFF.
Within same organism different cell types express and repress different sets
of genes at different times and levels to produce specific functional
OUTPUTS!!!

Bring us back to Central Dogma of molecular biology DNA to

mRNA(Transcription) ,mRNA to protein(translation) and the protein itself
can be modified in a way that can also regulate its function(PostTranslational Modifications) that will ultimately give specific functional
outputs at the molecular level.
In Reality, if you want to think in the context of the genome, you have sets
of genes 25,000 that each are transcribing at different levels this gives rise to
very specific levels of mRNA,sets of mRNA that are all regulated by
transcription, as well as the rate of degradation, these mRNAs are then fed
into the translation system and produce protein and the translation system is
also regulated, so you have different levels of proteins also. These proteins
and mRNAs are ultimately defining functional output, which can cause
cell proliferation or having these stem cell specific genes, and allow cells to
assume stem cell state and suppress and regenerate differentiation signals.
The whole full set of mRNA that it transcribed is the Transcriptome !! And
the entire complement of proteins being expressed is the Proteome!!!
This complete set of mRNA and proteins ultimately determined the function
of the cell!!!!!!!
In Neurons, some of these Genes are down-regulated they are low, because
transcription even translation is down regulated the expression of these
mRNA to make these protein levels even lower and ultimately produces
these functional outputs, here in Neuron cells are not proliferating. Stem Cell
Specific Genes are off, and the Neural Specific Genes are ON. However
when things go wrong, such as in Cancer Cells, Genes that are supposed to
be OFF are turned ON. And Genes that are supposed to ON are turned OFF.
For example this gene is normally low in Neuron Cells but now is misregulated and turns on and genes that are overexpressed and cause cancer are
called (Proto-Oncogenes) which normally want to suppress to a low level,
and cancer cells are Unregulated. What could those genes be, what are those

genes normal function for proto-oncogenes they can be growth factors, you
dont want these to replicate themselves, if you upregulate this the cells that
are supposed to be, NOT DIFFERENTIAED, NOT DIVIDED,WILL
START DIVIDING.
Is telomerase a proto-oncogene or a Tumor Suppressor Gene? Telomerase us
a proto-oncogene!!!!!, normally you want to suppress its expression levels,
you only want to turn telomerase on when you want cells to duplicate itself.
So telomerase is a proto-oncogene. Other proto-oncogenes like
transcription factor MYC that will turn on a lot of Cell Cycle Genes, WNT
and RAS, signaling molecule that will also stimulate cells to diffentiatte.
Tumor Suppressor genes are cells that suppress cell proliferation, famous
ones are p53 and pRB.

In Research, it is very important to identify these genes, and to see genes

that are NOT supposed to turn on are TURNED ON!!!!
How you measure gene expression levels of Genes?
One Method is Northern Blot Analysis , in order to do that you would purify
messenger RNA from normal tissue and Tumor Tissues, the Question is
which gene is Unregulated in the TUMOR!!!! In order to do this you need to
know what to look for. Lets say you think that MYC Gene is
UPREGULATED and that it the Hypothesis, you see tumor is growing
Rapidly and suspect MYC gene is Unregulated, so you do this experiment
by extracting mRNA, separating all RNAs on the Gel, why do you need
denaturing gel, since RNA has a lot of secondary structure, therefore you
want to unfold it, so that it will separate on Gel and separate by Linear
Length of RNA. What is the Chemical you would use to denature RNA?

You wouldnt use alkaline because it will cleave RNA, THE CHEMICAL
YOU WOULD USE TO DENATURE RNA IS FORMALDHYDE!!!! Why
would this denature RNA? Formaldehyde is a really good Hydrogen Bond
Acceptor, will bind to all bases, and prevent Hydrogen Bond Formations. So
you would use Formaldehyde as the denaturing gel!!!!!! Formaldehyde
would denature RNA so these pieces will be resolved in the gel. Lets say the
red messenger RNA represents the MYC gene, you want to know if the
MYC gene is Unregulated . You separate it on the denaturing gel in which it
is still invisible to your eye, you cant see it, molecules arent labeled, you
dont see them. After you separate the Gel, you put a piece of membrane; the
membrane is very sticky to Nucleic acids. What you do next is you put a pile
of paper towels above it, and the paper towel will soak liquid up and create
little current that goes from bottom to top. RNA will transfer from the Gel to
the Membrane, when it hits the membrane, it gets stuck, membrane is very
sticky. RNA is now on the membrane, and you take this membrane and
incubate it with a DNA probe, that has complementary radioactive DNA
sequence as the MYC mRNA. This complementary sequence as you remove
the Formaldhyde and let is bind slowly what will happen is that this probe
will bind to this particular position where mRNA is located. This is still
invisible, but the way we make the probe is we either fluorescent molecule
or could put in radioactive dNTPs!!!! To make this DNA probe. When you
take this and hybridized it to the membrane, the radioactivity will
concentrate at a certain position in the gel. IF YOU HAVE LOW LEVELS
OF MYC mRNA here, you will hybridized LESS to the DNA probe, If you
have more you will hybridize MORE to the DNA Probe! If MYC is
overexpress in the Tumor, what you would see is after Hydrization, is that
you would have more Radioactivity in the position you expect to see MYC
mRNA in the tumor Sample!!!!
Important: The Limitation to Northern Blot is you need to know what gene
you are looking for. You need to be able to guess correctly that Myc is
Unregulated. But what if you dont know what gene is the tumor!!! WAY
TO DETECT is to take Radioactive membrane and expose it to film and the
film will be irradiated and change color and observe increase in
Radioactivity Signal in Tumor Cells!!!!. In order to let two strands
Hybridize, you really need Annealing procedure, DNA and RNA can also
Hybridized. DNA and RNA interactions are even stronger then DNA-DNA
interactions. DNA-RNA Hybrid same principal applies to the annealing of
DNA and RNA.

Limitation of Northern Blot is you need to know which gene is Unregulated,

which is NOT! You have to guess for example Myc gene is Unregulated. If
you dont know which gene is Unregulated, you need to use an unbiased
approach, therefore you would use MICROARRAY ANALYSIS. How to
use Microarray genome wide approach to measure, which genes are on and
which genes are off. To do that you ALSO PURIFY mRNA from CELL
TYPES. Lets say on one hand you have Normal Tissues and the other hand
you have tumor tissues and extract mRNA. Lets say you have a mRNA that
is expressed at low levels in Normal Cells and lets say this gene is
Unregulated in Tumors (but you dont know which one it is yet) and you
want to find out which one is Unregulated, so you take a set of mRNAs and
label one set of mRNAs with a green dye and the other set of mRNAs with a
Red dye, then you mix these two sets of mRNAs and hybridize to a glass
slide on which each dots represent a complementary DNA sequence of a
gene, for example one dot has a complementary DNA sequence of Gene 1
and Gene 2 and so on and so forth. THE IDEA IS THAT IF A GENE IS
BEING EXPRESSED EQUALLY IN BOTH THEN GREEN AND RED
DYES WILL HYBRIDIZE EQUALLY AT THAT SPOT ON THE
MICROARRAY SLIDE.

How do you label these mRNAs? How do you convert mRNA to something
that is fluorescently labeled? To do that you use an enzyme called Reverse
Transcriptase, which is actually a DNA polymerase but uses RNA as a
template, it will convert mRNA sequences into DNA, if you put in dNTPS
which are labeled with dye which is green these fluorescently labeled
Nucleotides will get incorporated into cDNA sequence and you can use
another color of dye say Cy5 red. Now you have two groups of cDNA that
represent collection of mRNA of two cell populations.
Reverse Transcriptase is a DNA polymerase how do you prime DNA
synthesis on these mRNA, what is the primer you would use to do that. The
mRNAs are all single stranded and you dont have primer, mRNA in
Eukaryotes when they finish transcribing at the very end they add these poly
A tail, this is very important inside the cells, very important inside the cells
to stabilize mRNA inside the cell. We take advantage of this is we use that
as way to prime mRNA for synthesis of cDNA. What sequences would you
use to prime the poly A tail, you would use a stretch of Ts that is going to
Hybridized to provide 5end for synthesis of complementary DNA strands,
therefore you have these cDNAs from Tumors and cDNA from Normal
Tissue. When you zoom in Microarray on each of the spots, is a piece of
DNA that is covalently linked to the glass slide, that sequence corresponds
to gene 1, when you incubate cDNAs to these glass slides, it will find correct
target and hybridized to these probes like this.

If you have equal number of molecules of tumor cells and Normal Cells, is
you will have equal numbers of the red and Green molecules, hybrized to
that certain glass spot. By CONTRAST, if you have something that is Up
regulated in certain types of cells then a certain color will be MORE ON
ONE SIDE, you would have for example more red color or green color!!!
Depending on the experiment. Then lets say you take a MicroARRAY slide
and use a laser scanner to excite a green dye and red dye and allow to give
off a red dye and green dye. There are a lot of yellow spots (Both Green and
red being excited) yellow spots both genes are being expressed, relatively
equal. Once in while you would see a Red spot and some green spots which

are differently expressed genes. And to quantify the data, the machine will
give you a quantification of the signal. Suppose Gene 1 has a very strong
Red Signal and very yellow signal. The machine will give you a number;
Gene 2 is green because red signal is relatively low, Gene 3 that has a strong
Red signal. You also have a situation where you have Dark Spots (they are
expressing neither the normal tissue of tumor tissue gene. But you see you
have very low amounts of signal, when you have a signal that not
necessarily mean the gene is being expressed. A lot of times you see
NOISE!! How can you tell something is noise and the other isnt noise?
Gene 2 has fourfold decrease in tumor and gene 5 has four-fold decrease in
Tumor also. How do you differentiate real signals from weak signals, you
need to look at significance of weak signals!!!
Terrible p-value gives you low significance, in order to tell if a ratio is
significant or not you need statistical determinations.
One way to represent Microarray data is to use X-Y plot.
Tumor Suppressor Gene is normally suppressing cell proliferation; they
should be high in Normal cells and down regulated in Tumors. Gene 2 is a
tumor suppressor gene. Gene 5 is not statically significant, signal becomes
spread out because it is basically noise on the microarray (fluations so it is
really spread out) Another way to look at the DATA is by looking at the
Ratio, lots of times we take log Ratios. When you see something that is
positive you immediately see it is a tumor suppressor and anything that is
negative will be a potential proto-oncogene. Another advantage of Using a
log scale is that you exaggerate small numbers. Big Numbers arent
expressed as much in log scale. You basically have a better summary when
you take log scale.
It is sometimes difficult to compare X-Y plots, you want to see which genes
are overexpress or under express in these samples, it is difficult to identify
which genes are common between different tumors. Another way to look at
this data is to look at Heat Maps. What the Heat Map is doing is that instead
of plotting ratios as numbers it plots colors. When you look at heat map, you
need to pay attention to gradient scale (What does each color mean)
In example yellow value means a 2 value (Normal expression is higher then
tumor) when it is Blue it means the opposite. Advantage of using Heat Map
is that you can collapse the Data in one single line. You can literally read
one line and see which genes are upregulated and which ones are down
regulated. You can then look at other tumor and see which genes are
expressed at similar levels in different tumors. Advantage of Heat Maps is
that it collapse entire data set into a single line

Practical Example: IN NIH there are about 60 different Human cancer cells
lines that we mixed mRNA from and compare individually to normal cells.
For example in Leukemia cells a lot of genes have similar overexpression
patterns, which is not found in other cell types and you tell that maybe these
genes are responsible for Leukemia progression.

Lecture 15: Molecular Cloning, Restriction Endonuclease and PCR

Describe the key feature of an artificial plasmid
Describe the experimental steps involved in cloning a gene(Insulin) into a
plasmid DNA vector.
Explain the mechanism of restriction endonucleases and how these enzymes
can create sticky ends for ligation.
Explain the concept of Polymerase chain reaction (PCR)
Explain how protein expression vectors regulate protein expression.
Lecture 15 Recording:
Make bacteria make Insulin gene for you. What is a plasmid?

Polylinker sites artificial sequences people put in the plasmid. Advantage of

these polylinker sites is that they are basically a whole bunch of restriction
endonuclease sites, can use commonly used enzymes to cut specific site
here, the advantage of having Polylinker site is that you can insert your
favorite gene such as the Human Insulin gene, if you also have
complementary site there. Reason you need selectable marker is that you
want to trick bacteria to keep that plasmid, otherwise why would the bacteria
want to keep the Human Insulin gene. You want bacteria to express that
protein for you, you want to put in something the bacteria wants and to do
that you put in these Selectable marker genes!!!!!! Such as Ampicillin
resistance, kanamycin resistance these Antibiotic resistance genes in the
plasmid. For some bacteria you also want to use Protein-expression vectors,
your Human Insulin gene must somehow be controlled and should be read
by the bacteria transcription machinery and in order to do that you need a
promoter that is recognizable by bacterial transcription machinery, you want
something you could turn on or off controlled otherwise bacteria keep
expressing these proteins and sometimes these proteins can be toxic to cells.
You dont want that to happen so want promoter you can turn on or off on
command. These are properties of artificial plasmids.

So you got the Plasmid, what do you do next? Remember here the goal is to
trick bacteria to make more of this plasmid, In theory, you really only need
one molecule of this circular plasmid, if you can get bacteria to pick up this
single molecule and if you grow it in flask and get billions of cells, every
single cell in theory should carry this plasmid because plasmid has origin of
replication, replication machinery will see that origin and replicate
everytime when it duplicates itself by doing that you are tricking the bacteria
to make a lot of your DNA meaning you are making a lot of your favorite
gene. This is one way of using plasmids is by making a lot of it. This process

that introduces this single molecule of Plasmid into Bacteria is called

Transformation. The experiment that touches upon this process was the
Avery and Griffith experiment. Avery and Griffth took rough bacteria mix it
with DNA from smooth bacteria ,telling on how Rough Bacteria picked up
DNA and this process is Transformation!!!!! Bacteria can pick up small
piece of DNA and bacteria can pick up this DNA very efficiently.
To transform a cell is to introduce (stably) a foreign DNA inside the cell.
What transformation means, Typically transform bacteria, two methods we
can use , that is chemical transformation , electroporation, poking holes in
bacterial membrane by using chemicals like Lituim or Electroporation
where you shoot strong current through the cell and poke holes through
the cell and force them to pick up DNA. You can transform cells with this
kind of plasmids. In humans cells you can also have transformation, on one
hand by making these plasmids you can make more of your favorite DNA
but you can also use this approach to make your favorite protein. To do that
you also introduce that plasmid by transformation into bacteria, use special
strain, so it will recognize promoter and then turn it on in bacteria when you
need to. Once you have a big flask of cells and they all have your plasmid of
DNA then you can add a drug to it and turn on Transcription of that gene so
that it will translate into proteins. You can use this approach to make a lot of
DNA and can also use this approach to make a lot of protein.
EMPHASIZED these genes are initially off, because a lot of times these
proteins can be toxic to bacteria, you dont want that to happen until you
have a big flask of growing cells then you turn on gene and then you get a
lot of protein, without worrying about killing the cells. To important
applications of that process. How you insert your favorite gene (Human
Insulin Gene into these plasmids? Molecular Cloning techniques!!!
Conceptual steps you need to go through in order to clone gene into plasmid,
so you start with plasmid vector, which has origin of Replication, which has
a drug selectable marker and perhaps poly-linker site that you can insert
favorite gene into, and other hand you have this DNA you want to insert, a
lot of times the DNA is coming from genomic DNA from humans or cDNA
from humans you only want to put small fragments of that DNA into the
plasmid vector to do that you use these enzymes called Restriction
Endonucleases, which will recognize specific DNA sequences in the DNA
and cut it and will generate specific DNA site that will allow you to ligate
them together thats the idea basically cut and paste to generate this product.

What are Restriction Endonucleases? Enzymes that will recognize sort of 6

mer sites(A lot of them recognize 6 mer sites, many of them are palindromic
sequences . For example this enzyme called EcoR1 will recognize site (often
recognize Major groove of DNA. Find structural motif and put one cut on
the sugar phosphate backbone and put another cut other side of sugar
phosphate backbone, there cleaving the double stranded DNA. Now if you
cut your favorite piece of DNA, with same Restriction Enzyme then you will
create so called Sticky End, which is this short complementary single
stranded region, which can then Anneal to each other,although not very
tightly., but once you have small sticky end, this will stick to each other,
then you introduce ligase which will seal it and regenerate plasmid. Some
Restriction EndoNucleases, do Not create sticky ends, they generate Blunt
end enzymes, they can still be used by DNA ligase, but chances of finding
pairing and allowing ligation is significantly less efficient. But still doable as
long as you have enough DNA molecules to force reaction to occur. This
how you would basically cut a piece of DNA with Restriction Endonuclease
and ligate it with ligase.

If you zoom into the sugar-phosphate backbone, this is what you would see,
you know enzymes are going to cut DNA, what is the nucleophile

responsible for cleaving sugar-phosphate backbone water (OH-) is attacking

5G phosphorus atom, so why isnt your DNA just spontaneously breaking
apart? Kinetically it takes hundreds of Thousands of year to break down
these bonds. Like Exonuclease, you also have Mg stabilizing the attacking
nucleophile OH group, this ensures you are not just using water molecules,
but you are using a Hydroxyl Group. Mg is also going to stabilize transition
(distribute charge on the phosphate group,also additional side chains like
Argine and Lysine (which are both positively charged also distribute
charge.

How Eco RI works as well as other enzyme? Now you know you can cut on
the sugar phosphate backbone. So you have DNA you want to insert into
plasmid, so you can use Restriction Endonuclease to cut lets say both sides
of double stranded DNA you have sticky ends on both sides of your double
stranded DNA and you have sticky ends on the end of your plasmids, you
can now ligate that into plasmid circle.

LUK TOLD US that we can use the Restriction EndoNucleases to cut those
Genomic DNA into pieces and you select the one that contains your favorite
gene and insert it there. The problem here is that the genome is very
complex most likely you get thousands of Resticiton endonuclease sites.
How do you select your favorite piece of DNA to put in your vector? It is
difficult to select a piece of DNA but amplifying it would be the answer.
Amplify your favorite gene using PCR.

PCR would start from Genomic DNA and use primers, so you would
amplify that region, you can use small amount genomic DNA and use
primers so you would amplify that region, and will be able to give you large
amounts of the same thing, will significantly enrich your what you want to
insert here and the probability of getting your piece into the vector is much
higher. Design your primers, and know what is in the reaction and you just
dont mix everything. UNDERSTAND ALL COMPONENTS OF PCR
REACTON. First step in this process is you start with a template, you design
your own primers with two specific sequences, First you will heat up to 95C
so double stranded DNA will get denatured into single strands, these base
pairing will get exposed now when you cool it because you added 2000 x
excess of primer, the probability of primers that they will find and reanneal
to these sites would be higher then reannealing with original strand. Once
you have primers annealed a certain position and the other primer annealed
to other position then you can use DNA polymerase to synthesize a second
strand!!! Why do we used this special polymerase called Taq Polymerase!!!,
polymerase purified from Thermus aquatus so it doesnt get denatured at
95C, olden days people used DNA polymerase 1 without exonuclease for the
PCR process , every time they went through the process they added new
enzyme, allows us to do PCR without using new enzyme. Products of first

round and then second round you heat it again (denature strands) and then
primers will anneal again and synthesize strands like so.

Components of a Typical PCR Reaction:

You need buffer to maintain pH, you dont want pH way to high or way to
low,so you dont want to denature DNA or you even can denature protein.
You want to maintain your reaction conditions; therefore you want some
kind of buffer. Why do you want MgCl2? Want it is a cofactor for the
polymerase in theory enzyme should have metal, why do you need extra
metals? The metal is loose in the enzyme and doesnt bind very tightly.
Every binding has a dissociation constant Kd of the metal for the enzyme
(the number is very high which means that it binds very loosely, you need a
high concentration for the metal to bind. In order to drive metals for the
enzyme you want to put a lot of MgCl2. Also buffer might have EDTA these
are molecules that will bind to metals and sequester the metal and make it in
available. If you have enough EDTA in the buffer, you will sequester your
Mg and make reaction not work.
Great Exam Question: Why would PCR not work?
In the cell there is an enzyme that cleave pyrophosphate to monophosphate
to drive reaction to the right, since you dont have that enzyme there
wouldnt it be less efficient, but you provide dNTPs at high enough
concentration you are already driving the reaction to the right. Increase
concentration of dNTPs. You need dNTPs substrate for DNA Replication.
Forward primer reverse primer and your template .Taq Polymerase very
efficient. You DONT WANT EXONUCLEASE IN THE TAQ
POLYMERASE because you will have a lot of chewed up product, but now
there will be more errors so Taq polymerase is not a very accurate
enzyme.!!!!!General PCR Program: Use heat at 95C to denature, then 55

(general temperature to anneal., in general you want to use temperature 5C

below the melting temperature of the strands ,5C below melting temperature
of your primer. You designed as your annealing temperature, 72C is the
optimal temperature Taq polymerase will extend that strand, the time you
need for extending the strand depends on how long that piece of DNA is,
you know how to calculate how much time you need by knowing what is the
extension rate. Extension rate of Taq Polymerae is 1000 nucleotides per
minute therefore you would need to 2 minutes for 2K genes!!!!