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LECTURE 7
LECTURE 7
During mitosis, chromatin is ? packed
tightly
when packed tightly, the chromatin ? in length, but ? in width
Decrease
Increases
10nm fiber is the
smallest
30nm fiber is the
largest
DNA is packed into
nucleosomes
what makes up a nucleosome
DNA+histone core
Core DNA is
the DNA wrapped around the histone
Linker DNA is
the DNA between histones
The Histone core is composed of 4 proteins named
H2A
H2B
H3
H4
The four proteins are ? charged
+
There is one other Histone and it is called
H1
What does H1 do?
It binds to the linker DNA
What does it do once bound to the linker DNA
It can help condense the histones
What makes the DNA stick to the Histones
The DNA is - and the Histone core is + charged
First step in forming a histone
H3 and H4 come together to form a tetramer
After the H3-H4 teramer is formed, what happens?
H2A and H2B come together to form a dimer and they join the tetramer
3 things make up a total Histone core
2 H2A H2B dimers
1 H3 H4 tetramer
Complete histone core assembles ONLY when
DNA is present
Two models for nucleosome compaction are
solenoid
zig zag
The solenoid model has its nucleosomesstacked on
the edge
Where is the linker DNA in the solenoid model
burried in the middle of the superhelix, but never passes through the center
The zig zag model has it nucleosomes stacked in
a zig zag array
The linker DNA must ? in a zigzag model
pass straight through the central axis of the fiber
What are the N-Terminal tails crucial for
forming the 30nm fiber
Two ways that the N-terminal tails are modified to regulate Histone function
Acetylation
Methylation
What is the first step in Histone Acetylation
Histone acetyl transferase adds acetyl groups to the N terminal tails
What happens after these tails have been acetylated
Bromodomain proteins associate with the acetylated tails
Histone acetylation means
transcriptionally ACTIVE
What associates with acetylated histone tails?
Bromodomain proteins
What is the first step to Hoistone Methlyation
histone methyl transferase adds methyl groups to the N terminal tails
Once the N terminal tails are methlyated, what happen
Chromodomain proteins recognize the methlyated histones and associate
Histone methlyation means
Transcriptionally REPRESSED
What associates with methlyation?
Chromodomain proteins
After DNA replication, new nucleosome inheritance follows two rules. The rule for the H3 H4 tetramer is
the old H3 H4 tetramer will associate with one of the daughter starnd and the other daughter strand will get a new H3 H4 tetramer
The rule for the H2A H2B dimer
The old H2A H2B dimer is released, so the daughter starnd can get either an old or new H2a H2B dimer
LECTURE 8
LECTURE 8
First two things needed for DNA synthesis
-dNTPs
-Primer template junction
what does the template strand do
provides the ssDNA that will direct the addition of nucleotides
What does the primer do
it is complemntary to the template strand but shorter and it is where you start from
The RNA primer must have ? on its end
3'-OH group
What does this 3'-OH group do
it is extended by the additional of other nucleotides
How is DNA replication powered?
pyrophosphate hydrolysis
The structur and function of the DNA polymerase can be visualized using three strucutres
Palm
Fingers
Thumb
The Palm does 2 things
-Binds metal ions at the catalytic site (where the magic happens)
- Monitors the accuracy of base pairing
The Fingers do three things
- Bind incoming dNTPs
-Then they close and form the bonds once proper dNTPs have been bound
- It bends the template strand to make sure only one dNTP will be added to the next available base
The Thumb does two things
- Maintains the correct position of the Primer:Template junction
- Maintains a strong interaction between the Polymerase and the template strand
What does this strong interaction between the enzyme and substrate do for the RNA polymerase
it allows the polymerase to slide along the template strand and continually bind new dNTPs processivly
What does the proofreading exonuclease do and where does it do it
It removes incorrectly paired bases from the 3' DNA end
Which strand of DNA is replicated?
BOTH
DNA Polymerase functions ONLY in the
5'-3' direction
What does the Primase do
it adds small RNA primers to the template strand
How many primers does the leading strand need?
1
How many primers does the lagging strand need?
many
Okazaki fragments are
the stretches of DNA replicated between RNA primers on the lagging strand
How are RNA primers removed?
By RNAse H
What does RNAse H do
It cleaves the bonds between the ribonucleotides
this makes RNAse a ?
Exonuclease because it can only cleave at the end where there is not bond between DNA and RNA at the primer
What happens after the primer is removed?
DNA polymerase binds the free 3'OH end of the DNA and fills the gap
What seals the nick left behind by DNA polymerase?
DNA ligase
How does DNA ligase seal this nick?
By forming phosphodiester bonds between the 3' OH end and 5' P end
What does the unwinding of the DNA?
DNA helicase
Once the Helicase goes through and unwinds the DNA and there is only ssDNA left, what happens
The DNA must remain free of base pairing
How is the DNA reamin free of base pairing?
Single stranded DNA binding proteins bind the bases and dont allow other proteins to bind
These SSBs bind, how
cooperatively - once one binds it helps another and another and another
What helps to releave the tension created by unwinding the DNA?
Topo II bc it cuts both strands
LECTURE 9
LECTURE 9
What is an apoenzyme
and enzyme that require a co -factor, but does not have one
An example of an apoenzyme is
just the DNA polymerase core enzyme
what is a holoenzyme
an apoenzyme with all it's co-factors that
what do these co-factors do?
enhance its productivity
What is an example of a holo enzyme
DNA Polo III
The main goal of DNA Pol I is
to remove the RNA primers
Is DNA Pol I processive?
No
Is DNA Pol III processive?
yes
What helps to increase the processitivity
The DNA sliding Clamp
How does the sliding clamp increase the processtivity
by encircling dsDNA and holding the DNA polymerase onto the DNA primer:Template junction
On the lagging strand, how does the sliding clamp opperate
Once DNA Pol reaches the end of an Okazaki fragment the clamp is released
First step in loading the sliding clamp
The sliding clamp loader starts off closed
Why does it matter that thesliding clamp loader is closed, and what happens
If it is closed it cannot bind the sliding clamp. ATP binds the loader and it opens
what do these co-factors do?
enhance its productivity
What is an example of a holo enzyme
DNA Polo III
The main goal of DNA Pol I is
to remove the RNA primers
Is DNA Pol I processive?
No
Is DNA Pol III processive?
yes
What helps to increase the processitivity
The DNA sliding Clamp
How does the sliding clamp increase the processtivity
by encircling dsDNA and holding the DNA polymerase onto the DNA primer:Template junction
On the lagging strand, how does the sliding clamp opperate
Once DNA Pol reaches the end of an Okazaki fragment the clamp is released
First step in loading the sliding clamp
The sliding clamp loader starts off closed
Why does it matter that thesliding clamp loader is closed, and what happens
If it is closed it cannot bind the sliding clamp. ATP binds the loader and it opens
Once the loader is opened what happens
It binds the sliding clamp
Once the loader has bound the sliding clap what happens
The sliding clamp will open
Pnce the sliding clamp is open what happens
the sliding clamp binds around the DNA q
once the open Sliding clamp has bound the DNA what happens
The clamp loader hydrolyzes ATP and the loader closes, causing the Clamp to close around the DNA as well
LECTURE 10
LECTURE 10
The Polymerase chain reaction is
a powerful way to amplify DNA sequences
First step of the PCR
the template DNA strands must be denatured into ssDNA
Once the ssDNA is made, what happens
Primers are added
why are the Primers added?
PCR cant start without Primers
After the Primers are added, what happens
DNA Pol is added to extend the Primers
This process is then..
repeated over and over
How many times are eukaryotic chromosomes replicated per cell divison
once
Chromosome replication occurs only during the ? Phase
S
Once an origin has been copied, it must..
remain inactive until the cell divides
Eukaryotic DNA replication is initiated by
Pre-Replicative Complex (Pre-RC)
Where is the Pre-RC formed
G1 phase
When is the Pre-RC activated?
S phase
What activates the Pre-RC
CDKs and DDKs
What happens after the Pre-RC is activated by the CDKs and DDKs
DNA helicase begins to unwind the the dsDNA
After the helicase unwinds the dsDNA what happens
a DNA Pol/Primase complex comes in
What does the DNA Pol/Primase complex do
adds a primer and extends and breifly entends it with a short stretch of DNA
Then what happens
The primase exits breifly
Once the Primase exits, what happens
The sliding clamp loader and sliding clamp come in and assemble at the primer template junction and begin synthesizing the lagging and leading strand
Will the DNA Pol/Primase come back?
yes to help synthesize the lagging strand
What are the CDK levels like in G1
Low
If the CDK levels are low in G1, what does this mean
allows Pre-RC to form, but not enough to activate it
How are CDK levels in S phase
high
If the CDK levels are high is S phase, what does this mean
Signals for DNA replication to begin at existing pre-RC complexes
High CDK levels also prevent
the formation of new pre-RCs
CDK remains high until
the cell divides into and enters G1 phase again
When the last RNA primer is removed in the lagging strand, how can DNA Pol fill the gap?
IT CANT
this means that since it cant fill the gap at the end of every single round of replication
linear chromosomes gets shorter and shorter
Some cells have ? that are used to ?
Proteins that are used as primer to synthesize the last okazaki fragment
Another way to get around this problem is by utilizing
Telomeres and Telomerases
At the very end the ? end extends farther then the ? end
3' end extends farther than the 5' end
What extends the 3'
Telomerases
Telomerase is actually called a
riverse transcriptase
It is called reverse transcriptase because
it uses an RNA strand to synthesis a DNA strand
Once the Telomerase synthesizes the DNA from the RNA
it moves 6 nucleotides forward and does the same thing
Now normal replication can take place by
Primase
DNA Pol
Primer degradation
LECTURE 11
LECTURE 11
What is a Transitions mutation
is the change of a pyrimidine-pyrimidine, or purine-purine
What is a Transversion mutation
pyrimidine-purine , or purine-pyrimidine
The mismatch repair system does what
Increase the accuracy of DNA synthesis
Whats the first step in this process
MutS (scans) dimer scans the DNA of kinks produce by mismatch bases
What does MutS do once it finds a kink
It binds and distorts it even further
Once this kink is more distorted, what happens
MutL (localizes) localizes to the site
Once MutL gets to the site, what does it do
it recuirts MutH
What does MutH do once it gets to the kink
It nicks near the mismatch
What happens once MutH nicks near the mismatch
UvrD is a helicase and it unwinds the DNA from the nick to the mismatch
What is the final step
DNA Pol III and ligase close the gap
With the help of ? , MutH will know which strand is the newly synthesized one
Dam Methlyase
What does Dam Methlyase do
it adds methyl groups to A with the 5'-GATC-3' sequence of the parent strand
how does this relate
When DNA is newly synthesized Dam methylase has not methylated it yet
So that means MutH binds
to the non methlyated strand of the Hemimethylated DNA (Hemi = MutH)
LECTURE 12
LECTURE 12
Three types of CSSR Recombination
Insertion
Deletion
Inversion
During insertion, what happens
DNA is inserted into specific sites
During deletion, what happens
Deletion of a DNA segment
The arrows are point in what direction for deleltion to occur
Same way
What happens in inversion
The DNA segment is inverted, or flipped
For inversion to occur the arrows have to be facing
towards each other
What do Transposons do
They move certain genetic elements from one DNA site to another
Transposons are present in the genome of
All organisms
They do/do not require duplication?
Do not require duplication
Why are Transposons the most common source of new mutations in organisms?
They show very little sequence selectivity
Three classes of Transposons
DNA Transposons
Viral-like retrotransposons/retroviruses
Poly-A retro transposons
If the Transposon encodes for Transposase, what happens
It is autonomous
What does autonomous means
on its own
If DNA Transposons encode for ? and ? they will be autonomous
Transposase
Inverted repeats (Arrows facing the opposite way)
If a DNA Transposon does not have Transposase ...
It will not be non-autonomous (stuck)
Viral-like Retrotransposons/Retroviruses have ? repeats and encode for ?
Inverted repeats (like DNA Transposons)
Reverse Transcriptase
Poly-A Retrotransposons have ? repeats and encode for
DO NOT have inverted repeats (theirs face the same way)
Encode for reverse transcriptiase and endonuclease activity
Poly-A Retrotransposons have a distinct ? and they are know as ? and ?
Termini
5'UTR & 3'UTR
LECTURE 13
LECTURE 13
The most simple type of DNA Transposition is
Cut and Paste transposition
First step of Cut and Paste
Transposase binds the inverted repeats
After Transposase bind the inverted repeats..
It brings the ends together
bring the end together forms the
synaptic complex
Once the synaptic complex is formed the transposons are
cut out
When the transposons are cut out it forms
2 free 3'OH groups
what do these freed 3'-OH groups do
attach the phosphodiester bonds in the middle of the new insertion
this new insertion is called the
target DNA
Once this target DNA is loaded onto the old DNA the 3' ends serve as
primers
the remaining nicks are
ligated