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Molecular Biology > Mod 2- DNA Replication > Flashcards

Mod 2- DNA Replication Flashcards

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1
Q

What are the different types of DNA replication?

A

Semiconservative

Conservative

Dispersive replication

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2
Q

What does semiconservative replication entail?

A

The two DNA strands separate and DNA is copied using the parent strand as a template

Ends up with 2 identical strands same as parent DNA- each daughter strand has half the information from the parent.

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3
Q

Why is DNA replication essential?

A

To pass on equal amounts of DNA when cells divide

Defects can lead to diseases like cancer.

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4
Q

What role do topoisomerases play in DNA replication?

A

They solve the problem of DNA double helix unwinding during replication

Allow for efficient and accurate replication.

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5
Q

What was the initial belief about semiconservative replication?

A

It was thought to be impossible due to DNA being plectonemic (Strands can’t be separated without unwinding the double-helix structure)

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6
Q

What did the Meselson-Stahl experiment demonstrate?

A

That DNA is replicated by the semiconservative process in living cells

Conducted in 1959 using heavy nitrogen.

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7
Q

What is the purpose of density gradient centrifugation in the Meselson-Stahl experiment?

A

To distinguish between 15N-DNA and 14N-DNA by buoyant density

Cesium chloride (CsCl) was used.

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8
Q

What are DNA supercoils, and why must they be managed?

A

Produced if strands are separated; they need to be removed or prevented

Managed by DNA topoisomerases.

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9
Q

What is the function of Type I DNA topoisomerase?

A

Separates the strands without unwinding the DNA double helix

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10
Q

What is the G segment in Type II topoisomerase action?

A

The DNA strand that gets completely cut to allow another strand to pass through.

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11
Q

What is the T segment in Type II topoisomerase action?

A

The DNA strand that gets passed through

Essential for managing DNA replication.

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12
Q

Fill in the blank: DNA replication is essential for _______.

A

[reproduction and continued life]

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13
Q

True or False: Conservative replication was the first hypothesis believed to be true about DNA replication.

A

True

Scientists initially thought semiconservative replication was impossible.

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14
Q

What is the method of DNA replication that ensures copies are made exactly without errors?

A

semiconservative

This method allows each new DNA molecule to contain one original strand and one newly synthesized strand.

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15
Q

What are the cellular enzymes that synthesize DNA strands from nucleotides called?

A

DNA polymerases

DNA polymerases play a crucial role in DNA replication and repair.

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16
Q

What is the direction of DNA synthesis during replication?

A

5’ to 3’

DNA synthesis always occurs in this direction, requiring a primer to initiate.

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17
Q

What is the role of a primer in DNA synthesis?

A

To initiate synthesis of the new strand

Without a primer, DNA synthesis cannot occur.

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18
Q

What enzyme is responsible for making the RNA primer in bacteria?

A

primase

The primer is typically 4-15 nucleotides in length.

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19
Q

What is the term for the short sections of DNA synthesized on the lagging strand?

A

Okazaki fragments

These fragments are later joined together to complete the lagging strand.

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20
Q

What type of activity allows DNA polymerases to correct errors during DNA synthesis?

A

exonuclease activity

This includes 3’ to 5’ proofreading, where the polymerase removes incorrectly inserted nucleotides.

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21
Q

What protects the separated single strands of DNA during replication?

A

single-strand binding proteins (SSBs)

These proteins prevent the strands from reattaching or being degraded by nucleases.

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22
Q

In eukaryotes, which DNA polymerase first extends the RNA primer?

A

DNA pol alpha

DNA pol alpha adds about 20 nucleotides before DNA pol delta synthesizes the rest.

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23
Q

True or False: DNA replication can occur in both directions at the replication fork.

A

False

DNA replication can only occur in one direction, but the two strands are oriented oppositely.

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24
Q

What happens to the RNA primers after Okazaki fragments are synthesized?

A

They are removed and the fragments are joined together

This process is essential for completing the lagging strand synthesis.

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25
Q

What is the purpose of DNA topoisomerases during DNA replication?

A

To relieve the stress of DNA double-helix unwinding

They nick or cut the DNA to facilitate this process.

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26
Q

What is the first step in synthesizing a new strand of DNA at the replication fork?

A

The creation of a primer

This primer is necessary for initiating DNA synthesis.

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27
Q

What is the enzyme involved in joining Okazaki fragments in eukaryotes?

A

FEN1

FEN1 is an endonuclease that plays a critical role in this process.

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28
Q

What are the two types of strands during DNA replication?

A

Leading strand and lagging strand

29
Q

What is the end replication problem?

A

DNA molecules gradually getting shorter with each round of replication

30
Q

What is the role of telomerase?

A

Prevents the ends of chromosomes from being shortened

It extends the parent DNA by adding a sequence (TTAGGG) several times

31
Q

When does DNA replication take place?

A

During the S phase of the cell cycle

32
Q

What is the leading strand?

A

The strand that is copied by continuous DNA synthesis

Copying of this strand requires a primer

33
Q

What is the primer made of in bacteria?

A

RNA

34
Q

What enzyme synthesizes the primer in bacteria?

A

Primase

(On leading strand)

35
Q

How long is the RNA primer in bacteria?

A

4-15 nucleotides in length

36
Q

Which DNA polymerase extends the RNA primer in eukaryotes?

A

DNA pol alpha

Adds about 20 nucleotides

Has no exonuclease activity

37
Q

What is the function of DNA pol delta in eukaryotes?

A

Makes the rest of the new strand after DNA pol alpha

Has 3’—>5’ exonuclease proofreading activity

38
Q

How is the lagging strand copied?

A

From a primer placed at the replication fork

39
Q

In what direction is DNA synthesis always carried out?

A

5’ → 3’ direction

40
Q

What are the sections of the lagging strand called?

A

Okazaki fragments

41
Q

What must be done to complete lagging strand synthesis?

A

Join Okazaki fragments and remove RNA primers

42
Q

What enzyme is involved in joining Okazaki fragments in eukaryotes?

A

FEN1 (an endonuclease)

43
Q

What is the sequence added by telomerase?

A

TTAGGG

This is unique to each organism but is this sequence in humans

44
Q

What type of complex is telomerase?

A

RNA/protein complex

45
Q

Why is telomerase particularly important in Tetrahymena thermophila?

A

It has a large number of chromosomes (40,000)

46
Q

What is senescence in cell culture?

A

Cells can divide about 50 times before dying

47
Q

What happens to chromosome ends in senescent cells?

A

They become shortened

48
Q

During which phase of the cell cycle does DNA replication occur?

A

S phase (synthesis phase)

Lasts about an hour/ couple of hours

49
Q

What can be visualized to detect sites of DNA replication?

A

Modified nucleotides incorporated into DNA and detected with fluorescently labelled antibodies to visualise sites of DNA replication

50
Q

What are the two types of DNA that are replicated during S phase?

A
  • Euchromatin = open, active DNA where genes get transcribed and are used
  • Heterochromatin = condensed, inactive DNA where genes aren’t expressed or being used
51
Q

How does Type I DNA topoisomerase work?

A
  • Nicks the DNA once
  • Cuts it at one strand
  • Enzyme pulls strand through
  • Joins again by ligase
  • So less helical (one fewer ‘twist’)
52
Q

What is the mechanism for Type II topoisomerase?

A

Major enzyme used

  • Double strand is cut
  • Pass second segment of the DNA molecule through the gap (another double strand)
  • Rejoin the two cut strands
53
Q

How are new strands of DNA synthesised at the replication fork?

A

By template-dependent DNA synthesis (ensures identical copy of DNA)

The template is DNA so this is DNA-dependent DNA synthesis

54
Q

What is exonuclease activity?

A

Where DNA polymerase degrades DNA

Acts on ends

55
Q

What are the 2 possible types of exonuclease activity?

A

3’—>5’ exonuclease activity
- going in opposite direction to synthesis
- the polymerase can remove nucleotides it has just inserted
- this is called proofreading = allows errors to be corrected (removes any incorrect bases that have been inserted = important to accuracy)

5’—>3’ exonuclease activity
- the polymerase can remove DNA already attached to the template

56
Q

Does DNA pol Alpha have exonuclease activity?

A

No

It’s function is priming during replication

57
Q

Does DNA pol Delta have exonuclease activity?

A

Yes

ONLY 3’—>5’ exonuclease activity (proofreading)

This is the main replicative enzyme

58
Q

What happens at the replication fork?

A

Replication occurs

Separated single strands must be protected, if not
- they may just reattach to one another
- they might be attacked by nucleases

The strands are protected by single-strand binding proteins (SSBs)

59
Q

What happens once the primer is made in prokaryotes?

A

DNA pol III makes the new strand

DNA pol III has a 3’—>5’ exonuclease proofreading activity

60
Q

What happens when the replication fork moves?

A

The leading strand is extended by more DNA synthesis (continued synthesis)

The lagging strand has to be made in sections = requires multiple primers (ensures 5’ to 3’ direction)

61
Q

Describe the roles and structure of bacterial DNA polymerases

A

DNA pol I
- has 1 subunit
- Has 3’ to 5’ AND 5’ to 3’ exonuclease activity
- Function = DNA repair and replication

DNA pol III
- has at least 10 subunits
- has 3’ to 5’ exonuclease activity ONLY
- Function = main replicative enzyme

62
Q

Describe the roles and structure of Eukaryotic DNA polymerases

A

DNA pol Alpha
- has 4 subunits
- NO exonuclease activity
- Function = priming during replication

DNA pol Delta
- has 2 or 3 subunits
- has 3’ to 5’ exonuclease activity ONLY
- Function = main replicative enzyme

63
Q

How are Okazaki fragments in eukaryotes joined?

A
  • DNA pol delta and helicase push aside the primer
  • forms a ‘flap’
  • FEN1 (flap endonuclease) cuts in the middle of the molecule at the branch point
  • leaving a missing phosphodiester bond
  • DNA ligase links the two DNA fragments
64
Q

How are Okazaki fragments in bacteria joined?

A
  • DNA pol III stops when it reaches the RNA primer (as no exonuclease activity)
  • DNA pol I (has 5’—>3’ exonuclease activity) continues synthesis until it gets rid of all the RNA
  • DNA ligase links the two DNA fragments
65
Q

Why can the final Okazaki fragment not be made in eukaryotes?

A

Because the priming site would be after the end of the parent molecule which would lead to DNA molecules gradually getting shorter

66
Q

How does telomerase act as an extension at the end of a human chromosome?

A

Telomerase is an RNA/protein complex

It acts as a template

A few thousand telomerase repeats at the end of human chromosomes TTAGGG acts as a big buffer

(RNA sequence is AAUCCC)

67
Q

Do all cells have telomerase?

A

No, only stem cells have telomerase

(Turns off telomerase activity once cells differentiate)

68
Q

What do the patterns of DNA replication observed during S phase show?

A

Patterns reflect the different types of DNA being replicated at different times of the cell cycle

Molecular Biology (10 decks)

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