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BIO 362
Exam 2 Study Guide
SBU
Spring 2014
Marcu
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
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.
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)!!!!!!!!
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:
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.
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
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
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
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.
Need to know where does a Helical turn start!!! Three helical turns gives a
linking number of +3!!!!!
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.
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!!!!!!
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.
Look at molecule below, pull strand apart and you see a circle now!!!!
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.
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
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.
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.
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.
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.
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.
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.
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.
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
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
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.