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Molecular Biology > DNA replication 1 > Flashcards

DNA replication 1 Flashcards

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1
Q

At what point in the cell cycle does DNA replicate?

A

S (synthesis) phase

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2
Q

What is semi conservative replication?

A

When the two DNA strands separate and act as a template for the new strand. Each new duplex is made up of one parental strand and one new daughter strand.

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3
Q

What is conservative replication?

A

Where the two parental strands stay together and the daughter duplex is completely new.

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4
Q

What is dispersive replication?

A

Where the parental strands are fragmented and coexists with the new DNA.

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5
Q

In 1958 how did Meleson and Stahl try to prove semi conservative replication?

A

performed a CsCL (caesium chloride) equilibrium density-gradient centrifugation to distinguish between E. coli duplex DNA containing only 14N only 15N or a mixture of both.

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6
Q

Describe briefly the process they used to prove semi conservative replication.

A

Grew DNA on 15N for several generations so that there were only heavy duplexes (H/H). Then quickly changed the medium to 14N and test the DNA periodically.

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7
Q

What would be the expected out come of Meleson and Stahl’s experiment if it was conservative replication?

A

There would two bands one heavy - for the parental strands and one light for the daughter strands.

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8
Q

What would be the expected out come of Meleson and Stahl’s experiment if it was semi conservative replication?

A

For the first generation there would an intermediate strand - lying in the middle of the gradient as it is a mixture of heavy and light.
For the second generation there would be two strands one intermediate and one light.

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9
Q

What would be the expected out come of Meleson and Stahl’s experiment if it was dispersive replication?

A

For the first generation there would be an intermediate band between heavy and light.
For the second generation the band would shift slightly towards the light - 25%H and 75%L

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10
Q

Which form of replication did Meleson and Stahl prove to be correct?

A

Semi-Conservative

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11
Q

What do replicating bacterial chromosomes resemble?

A

Theta (greek letter) - there are know as replication eyes or bubbles.

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12
Q

Draw the structure of a bacterial chromosome and indicate the replication forks.

A

See picture on phone.

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13
Q

What does electron microscopy show about the replication forks?

A

It indicates that the replication forks are moving away from the central origin.

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14
Q

What is the enzyme for DNA replication in eukaryotes and prokaryotes?

A

DNA polymerase.

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15
Q

Describe briefly how DNA polymerase works.

A

Uses single stranded DNA as a template.
Adds free dNTPs to the free 3’ OH group of an already bound nucleotide, to synthesis the complimentary strand.
Nucleotides selected on ability to (Watson + Crick) base pair with template strand.
New DNA strand forms duplex with template strand.

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16
Q

Is synthesis slow or quick? Nucleotides per second?

A

Very rapid - 1000 nucleotides per second

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17
Q

How does DNA polymerase ensure accuracy?

A

Has proof reading activity.

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18
Q

What drives the addition of dNTPs to the 3’OH group?

A

The release of pyrophosphate (two phosphorus groups) and the subsequent hydrolysis of pyrophosphate into two inorganic phosphates (Pi).

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19
Q

What are the two major DNA polymerases?

A

DNA polymerase I and DNA polymerase III

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20
Q

Describe the activity of DNA polymerase III.

A

5’-3’ polymerase activity.

3’-5’ exonuclease activity (proof reading).

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21
Q

Describe the activity of DNA polymerase I.

A

5’-3’ polymerase activity.
3’-5’ exonuclease activity (proof reading).
5’-3’ exonuclease activity.

22
Q

What kind of strands does DNA replication require?

A

ss template strands - duplex must be separated.

23
Q

Why is it hard to separate DNA?

A

The DNA duplex is very stable.

24
Q

What is required to separate a DNA duplex in vitro? (in glass)

A

Very high temperatures.

25
Q

What is used to separate the duplex in vivo?

A

The enzyme DNA helicase.

26
Q

Describe DNA helicase in brief.

A

They are diverse group of enzymes, which harness the power of ATP hydrolysis to unwind short sections of AT rich parental duplex DNA.

27
Q

How does DNA helicase separate duplex DNA?

A

It binds to ssDNA moves along the strand until in encounters dsDNA whereby it prises the helix apart using ATP hydrolysis.

28
Q

How quickly does DNA helicase prise the duplex DNA apart? bp/second

A

1000 base pairs per second.

29
Q

What prevents the separated strands of DNA from re-annealing?

A

Single stranded DNA binding protein.

30
Q

What is SSB (single stranded binding) protein called in eukaryotes?

A

RPA - replication protein A

31
Q

How does SSB (RPA) prevent re-annealing of the two DNA strands

A

SSB coats ssDNA and must be stripped away for replication to occur.

32
Q

SSB also prevents secondary structures forming in the ssDNA how does it do this?

A

By binding not only to the ssDNA but also to the adjacent SSB protein - this is cooperative binding and straightens out the DNA strand.

33
Q

What is the replication fork?

A

The point at which the two strands of DNA are separated to allow replication of each strand.

34
Q

What is the issue when trying to replicate DNA?

A

DNA is a double helix, where the strands wrap around each other. This means for the replication fork to advance the helix would have to rotate rapidly.

35
Q

What happens when Circular DNA or Eukaryotic chromosomes unwind?

A

They are not free to unwind - positive supercoils form ahead of the helicase.

36
Q

What does the formation of supercoils ahead of helicase cause?

A

Intolerable strain on the DNA - becomes too hard to unwind and must be relaxed.

37
Q

How could the DNA be relaxed?

A

If there was some form swivel that allowed the strands to rotate. (suggested by John Cairns 1963)

38
Q

What would allow DNA to rotate (swivel)?

A

A nick in one of the strands phosphodiester bonds the other could swivel round.

39
Q

What enzyme produces this nick in one the DNA strands?

A

Topoisomerase.

40
Q

Do bacteria, Archaea and eukaryotes all produce topoisomerase enzymes?

A

Yes.

41
Q

How many classes of topoisomerase are there?

A

2 classes - topoisomerase I and topoisomerase II.

42
Q

Explain the difference between topoisomerase I and topoisomerase II?

A

topoisomerase I - makes ss breaks.

topoisomerase II - makes staggered ds breaks.

43
Q

Which residue is the active site of topoisomerase I?

A

Tyrosine.

44
Q

Explain how topoisomerase I causes a break in the ssDNA?

A

It covalently attaches to the DNA’s 5’ phosphate group resulting in the breakage of the phosphodiester linkage in the DNA strand. The two strands can now rotate relative to each other and this relieves the accumulated strain.

45
Q

How is the DNA strand reformed by topoisomerase I?

A

The original phosphodiester bond energy is stored in the phospho-tyrosine linkage making the reaction reversible. The phosphodiester bond spontaneously reforms, this regenerates the DNA helix and topoisomerase I.

46
Q

What is the main problem with DNA polymerase in DNA replication?

A

It cannot initiate synthesis it can only add nucleotides to the 3’ end.

47
Q

Which enzyme initiates synthesis of a new DNA strand?

A

DNA primase.

48
Q

How does DNA primase initiate replication?

A

It catalyses synthesis of a short RNA primer at the origin of replication, then stops. (Unlike DNA polymerase, it can start a new polynucleotide chain by joining two ribonucleotides together).

49
Q

What are DNA helicase and DNA primase together called?

A

Primosome.

50
Q

What is the τ subunit for in DNA polymerase III?

A

Dimerisation of the core polymerases.

51
Q

Why is DNA polymerase III more processive than DNA polymerase I?

A

The high processivity is provided by the β subunits of DNA polymerase III, which encircle the DNA and act like a clamp that keeps the DNA polymerase III complex tethered to the DNA template during elongation.

52
Q

How do eukaryotes overcome the problem of replicating such a large genome in a reasonable time frame?

A

By having multiple origins of replication, spaced about 30,000 base pairs apart.

Molecular Biology (15 decks)

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