Topic 5 Flashcards
How many times does dna replication happen and when
What does this mean
Happens once and only once during the S phase of the cell cycle
It has to be very coordinated
What pushes the cell cycle forwards (ex. G1 to S)
The cdk and their cyclins
Why is dna replication challenging
There are multiple origins of replication which from replication bubbles
Has to very coordinated because there is potential for the chromosome to break during formation of this bubble
What was the Meselson and stahl experiments
By labeling the dna with isotopes they found that as replication happened, there were bands in the intermediate of N15 and N14
This means that dna was semiconservative
What are the components needed for replications
dNTP (AGC OR T)
primer
What orientation is the newly synthesized strand during replication
5-3
New stuff added to the 3 prime OH end
Is the primer for replication dna or rna
Rna
How does the new dntp get added during replication
The 3’ OH act as a nucleophile attacks the alpha phosphate of the DNTP
this does catalysis of the DNTP
Forms a phosphodiester bond and the nuceleotide form a hydrogen bond with the nucleotide opposite to it.
What are the two important things that have to happen when a nucleotide it being added to a strand
- Recognition of the proper dntp then attaching the with the oh nucleophile
- Has to be able to base pair with the other nucleotide on the other strand
What is the byproduct of replication
The beta and gamma phosphate
What is processivity
The ability of the enzyme to catalyze “consecutive reaction without releasing it substrate”
Should have a very high affinity to its substrate, high processivity
What is an example of a processive enzyme and why
DNA polymerase
Because it catalyzes the synthesis of DNA by using a single active site for any of the 4 DNTP’s
What can dna pol do
Can sterically distinguish between dNTPs and rNTPs (dna vs rna)
Shows kinetic selectivity for adding the correctly base paired dNTP (ex. A TO T and G TO C)
Can start sysnthesis by using either RNA or DNA primer aneealed to the template (ex tac polymerase in PCr adds dna primer)
What is rNTP / NTP
Same thing
Have 2 and 3 oh
What is dNTP
Just 3’ oh and 2’ h
What is a ddNTP and why is it used
Only has 3’ and 2’ H no OH
This terminates replication because it cant make a new phosphodiester bond
Explain the steric constraints of DNA polymerase
The DNA pol forms a pocket that holds the three phosphate of the new incoming nucleotide
On the DNA pol there are discriminator amino acids that recognize the structure of the incoming nucleotide
When recognized, the new nucleotide will line up nicely with the 3’ oh for attacking the alpha phosphate
But if the incoming nucleotide is the ntp (two oh) the discriminator amino acids won’t recognize it and the nucleotide won’t be able to line up for the OH attack.
But hydrogen bonds could still be made
Explain the base pairing constraints of the dna pol
Is the incorrect base is being added, it won’t form the correct hydrogen bonds with the template
Then the 3’ oh cant attack the base and add that nucleotide
No catalysis so no extension of the dna replication
Overall how do dna pols pocket structure help
Helps recognized incorrect sterics (dntp vs rNTP)
Helps recognized incorrect base pairing
What are the three components of the DNA pol structure
The palm
The thumb
The fingers
What is special about the palm of the dna pol
Inside the palm active site, there are irons
These irons let the oh group act as a nucleophile and CATALYZE the reaction of adding dNTPs
also monitor base pairing of the newly added nucleotide
What do the fingers of the dna pol do
Also catalyze the addition of the newly coming DNTP
Enclose the newly added dntp
What does the thumb of DNA pol do
It keeps the correct position of the primer and the active site
Holds the dna pol and the substrate tightly together to allow the reaction to take place
What type of activity does DNA lol have and what does it do
Has exonuclease activity (3-5) to proofread the newly synthesized dna
So if it sees a wrong base it cuts it out from the end
Difference in exo and endonuclease
Exo is when is cuts it out from the ends of the strand
Endo is when it cuts in the middle of the strand
What does DNA helicase do
Unwinds the dna at the replication fork to let the ssDNA act as a template for the primase
What is primase
An rna pol that make the rna primers that anneal to the ssDNA to start replication
Which strand is the leading strand
Lagging
3-5 template
5-3 template
How many RNA primers for leading and lagging strand
Leading is 1
Lagging is many
What removes the RNA primers from the dna after replication compete
What fills the gap
What repair the nicks
RNase H
DNA pol
DNA ligase
In eukaryotes what is special about the dna polymerase
There are dedicated dna polymerases for different things
dna pol epsilon extends the dna leading strand
Pol delta extends the lagging strand
What is dna polymerase switching
Once a polymerase has done its job, the next one comes in to replace it
What does dna lol aplha (primase) get replaced by in dna pol switching
Why
In the leading strand, it gets replaced by pol epsilon
In lagging, replaced by pol delta
Because the dna pol aplha had low processivity conpared to epsilon and delta
What is the role of the sliding clamp protein
How does this help
Keeps the polymerase close to the dna template
This increases the processivity of the dna pol
What is the origin of replication
Sites on the dna that unwind to intimate synthesis
What is the replicator
The dna sequence that directs the initiation of DNA replication
It serves as binding sites for the initiator proteins
Has an AT rich sequence which can denature and unwind easily
What are initiator proteins
A bunch of sequence specific proteins that come and bind to the dna to start replication
What is the replicon
The new strand of the dna that has been replicated from a origin of replication
What is the ORC
The origin replication complex
Made of 6 different proteins that recognize the elements of the replicator
These proteins then recruit other replication proteins (like helicase) to the replicator
They mark the site of replication to occur
What elements on the replicator are recognized by the ORC
The B1 and A elements
What does the B2 element of the replicator do
Recruits proteins for dna unwinding (like helicase)
What needs to happen for the ORC to interact with the DNA strand
Atp hydrolysis
Does ORC binding always mean the strand will separate
No
How many replicator sites are there on the dna
What does this mean
Multiple
Many replications of the dna can occur at the same time
Explain how replicator inactivate and activate during dna replication
Ex. Replicators 3 and 5 activate and replication starts
Then replicators 2 and 4 are passively activated by the replication from 3 and 5
3 and 5 inactivate
Replicator 1 gets activated independently (by itself)
What happens to each replicator after the dna has been replicated at that site
They become inactive to prevent multiple replications
What is the role of G1 and S in the initiation of replication
In G1 there is replicator selection where the cell needs to identify the sequences that direct the initiation of replication
The ORC has to come and select the sequences that start replication BUT replication doesn’t start yet
In S, the origin activation happens where replication actually occurs
What type of selection is it in the G1 and S to make specific things happens as different times
A temporal selection
What is helicase loading and when does it happen
The helicase gets added by the orc
Occurs in g1
What are the steps in helicase loading during late g1
The replicator is recognized by the ORC
Using atp, ORC recruited the helicase loading proteins Cdc6 and Cdt1
Cdt1 recurits the Mcm2-7 helicase
Hydrolysis of atp let the cdc 6 and cdt1 leave the replicator
Then the helicase wraps around both of the dna strands
On what way does the Mcm2-7 get added to the replicator
In a head to head fashion (two subunits)
What actually triggers the unwind g of the dna after helicase is recurred
The hydrolysis of atp
So this hydrolysis doesn’t happen until s phase
Once the Mcm2-7 helicase is loaded, what happens
This is all happening in s phase now
There are two kinases
DDK phosphorylates the helicase
CDK phosphorylates the sld2 and 3 proteins
Once they get phosphorylated , these proteins make the CMG complex
What is the CMG complex
After the kinases CDK and DDK phosphorylate
Two proteins get recruited to the Mcm2-7 helicase
CDC 45 and GINS which are helicase activating proteins
All together cdc45/Mcm2-7/GINS makes the CMG complex
After the CMG complex is formed what happens
First the dna pol epsilon is recruited (leading stand)
The helicase is activated
Then unwinding takes place and the helicase now sits on a single stranded dna (used to be on dsDNA)
DNA pol alpha (primase) and delta are recruited after unwinding
After helicase is unwinding dna what happens
The dna pol aplha (primase) and dna pol delta (lagging) are recruited
Before helicase activate (in g1) what did the helicase look like
What about after (s phase)
Low cdk activity , so the helicase is on dsDNA as a head to head double hexamer (two subunits)
The cdk activity increase, the head to head separate, each subunit is on one ssDNA
Since replication can happen only once during each cell cycle , what has to happen to the replicator site
The replicator has to be inactivated at the end of the replication
This is important to maintain the genomic stability
What is required for loading of the helicase
What about to initiate dna replication
Low CDK activity, which is in g1 NO ACTIVATION of helicase
High activity in S phase, this helps activation of the helicase
Ultimately, what controls the activation of the helicase and this activation of replication
The CDK activity
After s phase what happens to the complex on the replicator
The used helicase is disassembled after DNA replication
The CDK activity stays high in S and G2 and M to prevent the loading of a new helicase that hasn’t been used
This restricts it to one replication per cell cycle
Where is high and low cdk activity
In g1 low for loading
S g2 M high
What is the replication of the circular DNA in prokaryotes
The circular DNA can be completely replicated
but it links with the parent strand (two Ds circles)
So the two daughter DNA molecules get separated by Topo II
What is the end replication problem of a linear chromosome
At the end of the lagging strand there are primers
these primers are removed by the polymerase but the sequins is still short since the primer is now gone
As generations continue to replicate , the stand continues to get shorter and shorter due to this primer
Slowly this will disrupt the passing down of genetic material from generation to generation
What is the first possible solution to the end replication problem
Instead of using an rna primer, use a protien primer at the end of the lagging strand
Explain how you can use a protien primer to fix the end replication problem
And what organism can use it
The oh of the protein amino acid (COOH)
Can be used as a nucleophile to form a phosphodiester bond with the incoming nucleotide
Basically acts as the 3’ oh of the primer to get the sequence started
No sequence is lost since the protien primes the last nucleotide
Used by linear bacterial chromosomes and viruses
What is the second solution to the end replication problem
What can make it happen
You can use the repeating TTAGGG telomeres sequence as an origin of replication for the 3’ end of each chromosome
The telomerase would be the DNA polymerase for the lagging strand
What is telomerase
A ribonucleoprotein complex
In it there is a protein and rna component
An rna subunit : RNA serves as a template to replicate the end of the telomeric sequence on the chromosme (3’ end)
There’s no real exogenous dna template needed
A reverse transcriptase subunit: a dna polymerase that turns the rna sequence onto the dna sequence
What are the similarities in telomerase and dna polymerase
Needs a template to direct the nucleotide addition
Extends at the 3’ OH end
Uses the same nucleotide precursors
Act in a processive manner
What are the differences in telomerase and dna polymerase
Telomerase has an rna component
The telomerase does not need a exogenous template because the rna itself is the template
Telomerase Can use a ssRNA template to make ssDNA (can reverse transcribe from rna to dna)
Telomerase has RNA:DNA helicase activity to displace its RNA template from the DNA to have repeated rounds of synthesis
How much of the telomeric sequence does the rna subunit of telomerase encode
What does this mean
1.5 x copies of the telomeric sequence
Half of the telomerase RNA sequence is able to complement with the 3’ end of the DNA (make new terlomeric dna at the 3’ end)
What is the process of telomerase fixing the end replication problem
What usually uses this solution
Telomerase is on the single strand 3’ telomeric end of the dna
Through reverse transcription it fills in rest of the sequence and makes a telomeric dna sequence at the 3’ end
It then trans locates to the last four bp it just made and does reverse transcriptase activity again to make more telomeric sequence
Eukaryotes
How does the extension of the telomere sequence help the end relocation problem
Since the lagging 3’ to 5’ strand usually loses dna
The extra telomeres sequence in the 5’-3’ end gives extra space for the primase to lay the primer in the laying strand
Both strands are being extended in the end
The 5-3 strand still has a 3’ overhang which can protect the telomeres
What is good about the repeating telomere extension
The telomeric dna is non protein coding
Meaning the extra telomere sequence doesn’t interfere with cellular functions
Since we don’t want the telomeres to replicate and extend infinitely how do we stop it
In s cerevisiae
In s cerevisiae
The cdc 13 recruited the telomerase
When there is an excessive amount of telomeres sequence, telomere binding proteins get recruited to inhibit the telomerase
Rif1/2 and rap1 are the telomeres binding proteins that inhibit the telomerase
Since we don’t want the telomeres to replicate and extend infinitely how do we stop it
In humans
The POT Telomere binding proteins inhibit the telomerase activity
And recruits shelterin which protects the end of the telomere from dna repairing enzymes
Needs to protect it because we don’t want the telomeres to extend and get degraded or recombine
What type of formation does the end of the telomeric sequence take and why and in what organisms
In humans They form t-loop structures which is when the telomeric strand invades into the double helix
This folding protects the end of the chromosome
