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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
Q

Difference in exo and endonuclease

A

Exo is when is cuts it out from the ends of the strand

Endo is when it cuts in the middle of the strand

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26
Q

What does DNA helicase do

A

Unwinds the dna at the replication fork to let the ssDNA act as a template for the primase

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27
Q

What is primase

A

An rna pol that make the rna primers that anneal to the ssDNA to start replication

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28
Q

Which strand is the leading strand

Lagging

A

3-5 template

5-3 template

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29
Q

How many RNA primers for leading and lagging strand

A

Leading is 1

Lagging is many

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30
Q

What removes the RNA primers from the dna after replication compete

What fills the gap

What repair the nicks

A

RNase H

DNA pol

DNA ligase

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31
Q

In eukaryotes what is special about the dna polymerase

A

There are dedicated dna polymerases for different things

dna pol epsilon extends the dna leading strand

Pol delta extends the lagging strand

32
Q

What is dna polymerase switching

A

Once a polymerase has done its job, the next one comes in to replace it

33
Q

What does dna lol aplha (primase) get replaced by in dna pol switching

Why

A

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
Q

What is the role of the sliding clamp protein

How does this help

A

Keeps the polymerase close to the dna template

This increases the processivity of the dna pol

35
Q

What is the origin of replication

A

Sites on the dna that unwind to intimate synthesis

36
Q

What is the replicator

A

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
Q

What are initiator proteins

A

A bunch of sequence specific proteins that come and bind to the dna to start replication

38
Q

What is the replicon

A

The new strand of the dna that has been replicated from a origin of replication

39
Q

What is the ORC

A

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
Q

What elements on the replicator are recognized by the ORC

A

The B1 and A elements

41
Q

What does the B2 element of the replicator do

A

Recruits proteins for dna unwinding (like helicase)

42
Q

What needs to happen for the ORC to interact with the DNA strand

A

Atp hydrolysis

43
Q

Does ORC binding always mean the strand will separate

A

No

44
Q

How many replicator sites are there on the dna

What does this mean

A

Multiple

Many replications of the dna can occur at the same time

45
Q

Explain how replicator inactivate and activate during dna replication

A

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
Q

What happens to each replicator after the dna has been replicated at that site

A

They become inactive to prevent multiple replications

47
Q

What is the role of G1 and S in the initiation of replication

A

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
Q

What type of selection is it in the G1 and S to make specific things happens as different times

A

A temporal selection

49
Q

What is helicase loading and when does it happen

A

The helicase gets added by the orc

Occurs in g1

50
Q

What are the steps in helicase loading during late g1

A

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
Q

On what way does the Mcm2-7 get added to the replicator

A

In a head to head fashion (two subunits)

52
Q

What actually triggers the unwind g of the dna after helicase is recurred

A

The hydrolysis of atp

So this hydrolysis doesn’t happen until s phase

53
Q

Once the Mcm2-7 helicase is loaded, what happens

This is all happening in s phase now

A

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
Q

What is the CMG complex

A

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
Q

After the CMG complex is formed what happens

A

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
Q

After helicase is unwinding dna what happens

A

The dna pol aplha (primase) and dna pol delta (lagging) are recruited

57
Q

Before helicase activate (in g1) what did the helicase look like

What about after (s phase)

A

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
Q

Since replication can happen only once during each cell cycle , what has to happen to the replicator site

A

The replicator has to be inactivated at the end of the replication

This is important to maintain the genomic stability

59
Q

What is required for loading of the helicase

What about to initiate dna replication

A

Low CDK activity, which is in g1 NO ACTIVATION of helicase

High activity in S phase, this helps activation of the helicase

60
Q

Ultimately, what controls the activation of the helicase and this activation of replication

A

The CDK activity

61
Q

After s phase what happens to the complex on the replicator

A

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
Q

Where is high and low cdk activity

A

In g1 low for loading

S g2 M high

63
Q

What is the replication of the circular DNA in prokaryotes

A

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
Q

What is the end replication problem of a linear chromosome

A

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
Q

What is the first possible solution to the end replication problem

A

Instead of using an rna primer, use a protien primer at the end of the lagging strand

66
Q

Explain how you can use a protien primer to fix the end replication problem

And what organism can use it

A

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
Q

What is the second solution to the end replication problem

What can make it happen

A

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
Q

What is telomerase

A

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
Q

What are the similarities in telomerase and dna polymerase

A

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
Q

What are the differences in telomerase and dna polymerase

A

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
Q

How much of the telomeric sequence does the rna subunit of telomerase encode

What does this mean

A

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
Q

What is the process of telomerase fixing the end replication problem

What usually uses this solution

A

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
Q

How does the extension of the telomere sequence help the end relocation problem

A

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
Q

What is good about the repeating telomere extension

A

The telomeric dna is non protein coding

Meaning the extra telomere sequence doesn’t interfere with cellular functions

75
Q

Since we don’t want the telomeres to replicate and extend infinitely how do we stop it

In s cerevisiae

A

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
Q

Since we don’t want the telomeres to replicate and extend infinitely how do we stop it

In humans

A

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
Q

What type of formation does the end of the telomeric sequence take and why and in what organisms

A

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 (6 decks)

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