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Cmmb 411 > Topic 5 > Flashcards

Topic 5 Flashcards

1
Q

How many times does dna replication happen and when

What does this mean

A

Happens once and only once during the S phase of the cell cycle

It has to be very coordinated

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2
Q

What pushes the cell cycle forwards (ex. G1 to S)

A

The cdk and their cyclins

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3
Q

Why is dna replication challenging

A

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

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4
Q

What was the Meselson and stahl experiments

A

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

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5
Q

What are the components needed for replications

A

dNTP (AGC OR T)

primer

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6
Q

What orientation is the newly synthesized strand during replication

A

5-3

New stuff added to the 3 prime OH end

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7
Q

Is the primer for replication dna or rna

A

Rna

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8
Q

How does the new dntp get added during replication

A

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.

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9
Q

What are the two important things that have to happen when a nucleotide it being added to a strand

A
  1. Recognition of the proper dntp then attaching the with the oh nucleophile
  2. Has to be able to base pair with the other nucleotide on the other strand
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10
Q

What is the byproduct of replication

A

The beta and gamma phosphate

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11
Q

What is processivity

A

The ability of the enzyme to catalyze “consecutive reaction without releasing it substrate”

Should have a very high affinity to its substrate, high processivity

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12
Q

What is an example of a processive enzyme and why

A

DNA polymerase

Because it catalyzes the synthesis of DNA by using a single active site for any of the 4 DNTP’s

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13
Q

What can dna pol do

A

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)

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14
Q

What is rNTP / NTP

A

Same thing

Have 2 and 3 oh

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15
Q

What is dNTP

A

Just 3’ oh and 2’ h

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16
Q

What is a ddNTP and why is it used

A

Only has 3’ and 2’ H no OH

This terminates replication because it cant make a new phosphodiester bond

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17
Q

Explain the steric constraints of DNA polymerase

A

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

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18
Q

Explain the base pairing constraints of the dna pol

A

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

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19
Q

Overall how do dna pols pocket structure help

A

Helps recognized incorrect sterics (dntp vs rNTP)

Helps recognized incorrect base pairing

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20
Q

What are the three components of the DNA pol structure

A

The palm

The thumb

The fingers

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21
Q

What is special about the palm of the dna pol

A

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

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22
Q

What do the fingers of the dna pol do

A

Also catalyze the addition of the newly coming DNTP

Enclose the newly added dntp

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23
Q

What does the thumb of DNA pol do

A

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

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24
Q

What type of activity does DNA lol have and what does it do

A

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

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25
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
26
What does DNA helicase do
Unwinds the dna at the replication fork to let the ssDNA act as a template for the primase
27
What is primase
An rna pol that make the rna primers that anneal to the ssDNA to start replication
28
Which strand is the leading strand Lagging
3-5 template 5-3 template
29
How many RNA primers for leading and lagging strand
Leading is 1 Lagging is many
30
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
31
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
32
What is dna polymerase switching
Once a polymerase has done its job, the next one comes in to replace it
33
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
34
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
35
What is the origin of replication
Sites on the dna that unwind to intimate synthesis
36
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
37
What are initiator proteins
A bunch of sequence specific proteins that come and bind to the dna to start replication
38
What is the replicon
The new strand of the dna that has been replicated from a origin of replication
39
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
40
What elements on the replicator are recognized by the ORC
The B1 and A elements
41
What does the B2 element of the replicator do
Recruits proteins for dna unwinding (like helicase)
42
What needs to happen for the ORC to interact with the DNA strand
Atp hydrolysis
43
Does ORC binding always mean the strand will separate
No
44
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
45
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)
46
What happens to each replicator after the dna has been replicated at that site
They become inactive to prevent multiple replications
47
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
48
What type of selection is it in the G1 and S to make specific things happens as different times
A temporal selection
49
What is helicase loading and when does it happen
The helicase gets added by the orc Occurs in g1
50
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
51
On what way does the Mcm2-7 get added to the replicator
In a head to head fashion (two subunits)
52
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
53
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
54
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
55
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
56
After helicase is unwinding dna what happens
The dna pol aplha (primase) and dna pol delta (lagging) are recruited
57
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
58
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
59
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
60
Ultimately, what controls the activation of the helicase and this activation of replication
The CDK activity
61
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
62
Where is high and low cdk activity
In g1 low for loading S g2 M high
63
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
64
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
65
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
66
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
67
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
68
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
69
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
70
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
71
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)
72
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
73
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
74
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
75
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
76
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
77
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

Cmmb 411 (12 decks)

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