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Cellular and Molecular Biology > Lecture 8: DNA replication [G] > Flashcards

Lecture 8: DNA replication [G] Flashcards

Monday 14th October

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1
Q

What does the fact that bacterial DNA is usually a lot larger in size than the actual bacteria tell us?

A

That bacterial DNA is very compacted

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2
Q

When did Watson and Crick propose the DNA double helix structure?

A

In 1953. This revolutionarised our understanding of genetics. However, their hypothesis remained theoretical until tested and confirmed by subsequent experiments.

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3
Q

What ways is DNA compacted?

A
  • By looping, with loops being attached at the base in an unknown way.
  • By supercoiling
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4
Q

Is our DNA packed more tightly than E coli’s?

A

Yes

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5
Q

Did Watson and Crick test their double helix DNA model?

A

No, they left it to other people to test

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6
Q

What is the length of an E coli cell?

A

1 - 2 micrometres

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7
Q

How long is the DNA inside E-coli?

A

1.6 mm

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8
Q

How big is our haploid genome?

A

3.2 Gbp

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9
Q

How much DNA does a typical somatic cell have?

A

6.4 Gbp of DNA

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10
Q

What is the spacing between base pairs?

A

0.34 nm

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11
Q

What is the total length of DNA in our typical somatic cells?

A

6.4 x 10⁹ x 0.34 x 10⁻⁹ m
~ 2.2 m

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12
Q

Is our DNA packed more tightly than E coli’s?

A

Yes

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13
Q

How is eukaryotic DNA organised?[compaction]

A

1) DNA wraps around histone proteins to form nucleosomes (~11 nm diameter).

2) Nucleosomes coil into a smaller 30 nm fiber.

3) Fiber forms higher-order loops (~300 nm diameter).

4) Loops coil further to create chromosome arms (~700 nm diameter).

5) Final condensed chromosome width: ~1400 nm.

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14
Q

Is the error rate for DNA replication high or low?

A

Low

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15
Q

What is the error rate for DNA replication?

A

~1 in 109 base pairs in most organisms

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16
Q

How many mutations do we generate at each cell division?

A

About 6 mutations

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17
Q

What did Watson and Crick say on April 25, 1953?

A
  • “It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”
  • Can copy DNA by making a complementary copy to each of the strands.
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18
Q

Are the daughter strands in DNA replication identical?

A

Yes

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19
Q

What were the 2 predictions by Watson and Crick in regards to DNA replication?

A

1) DNA strands are held together by ‘Watson-Crick’ base- pairing, consistent with Chargaff’s rules. To make this work, the strands must be anti-parallel.

2) Each strand is therefore complementary to the other, so each can act as a template for DNA replication. DNA replication should therefore be semi-conservative

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20
Q

Describe semi-conservative replication

A

Each daughter DNA molecule contains one parental strand and one newly-replicated strand.
WATSON-CRICK model

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21
Q

Describe conservative replication

A

The entire parental DNA molecule remains intact, and a new, separate double-stranded DNA molecule is synthesized.

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22
Q

Describe dispersive replication

A

parent helix is broken into fragments, dispersed, copied and then assembled into two new helices with interspersed old and new DNA.

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23
Q

Who tested models of DNA replication in 1958?

A

Meselson and Stahl

24
Q

How did Meselson and Stahl investigate DNA replication?

A

Using the technique of Caesium chloride (CsCl) equilibrium density gradient centrifugation, a technique that separates molecules based on their densities.

25
Q
A
  • Take 2 strong glass tubes and add a solution of a very heavy salt, caesium chloride (CsCl).
  • Spin the test tubes very fast for a long time
  • This forms a CsCl gradient, where there’s a higher concentration of salt at the bottom of the tubes and vice versa.
  • Take samples and weigh equal volume samples.
26
Q

How did Meselson and stahl establish their conditions for the DNA replication experiment?

A
  • They grew E coli for ~14 generations in medium containing 14N NH4Cl. (light nitrogen)
  • They did the same in a medium of 15N NH4Cl. (heavy nitrogen)
  • They prepared DNA from both of these tubes, mixed them together, mixed them with CsCl.
  • They then made a balanced tube of the same weight and spun this at 140,000 x g
    for 20hrs.
  • They hoped that the 2 different DNA’s would have different densities because of the weight of the nitrogen.
  • Photographing under UV light allowed the different bands of DNA to be identified.
27
Q

Describe the results of Meselson and Stahl’s experiment (read)

A
  • Only three forms of DNA were seen: H, hybrid and L: after one generation, all the DNA was hybrid.
  • For later generations there was a gradual loss of hybrid 14N/15N DNA and replacement with light DNA.
  • Hence proving that DNA replication is semi conservative.
28
Q

What led Arthur Kornberg to believe that here would be an enzyme capable of replicating DNA: a DNA polymerase?

A

Beadle and Tatum’s ‘one gene, one enzyme’.

29
Q

How did Arthur Kornberg discover DNA polymerase?

A

Kornberg took Escherichia coli protein extracts, and worked out the in vitro conditions that were required for this extract to make new DNA:

1) A template DNA to copy

2) deoxyucleotide triphosphates (dATP, dTTP, dCTP and dGTP) (building blocks of DNA)

3) the co-factor Mg2+

4) an energy source, ATP

5) … and a small piece of DNA (a PRIMER) with a free 3’ OH

30
Q

Kornberg’s experiment

A
  • Primer bound to template DNA
  • Added 3 non-radioactive nucleotides and a radioactive nucleotide (dTTP*).
  • Added E Coli protein extract.
  • After the reaction, the reaction mixture was treated with acid: unincorporated dNTPs remain soluble, but polynucleotides precipitate.
  • 32P was found in the pellet – so therefore there was new DNA present.
  • Radioactive labelling found that new strands are extended in the 5’ → 3’ direction by a polymerase (pol 1)
31
Q

What are the functions of pol 1

A
  • Could extend DNA strand from 5’ to 3’
  • 3’ → 5’ exonuclease activity
  • 5’ → 3’ exonuclease activity
  • This suggests that Pol I has editing/proof-reading functions and can correct mistakes, such as incorrectly paired nucleotides.
32
Q

Did Kornberg’s experiments confirm key predictions of the Watson-Crick model?

A

Yes:

  • 1) DNA strands are held together by ‘Watson-Crick’ base- pairing, consistent with Chargaff’s rules. To make this work, the strands must be anti-parallel.
  • 2) Each strand is therefore complementary to the other, so each can act as a template for DNA replication. DNA replication should therefore be semi-conservative
33
Q

Pol III is essential for DNA replication in E. coli.

A

Pol I is NOT the enzyme that is used for replication of E. coli DNA.

34
Q

What is DNA polymerise often envisaged as?

A

a three-fingered hand. Kornberg proposed that the polymerase enzyme works like a “hand,” where the “palm” holds the DNA and catalyzes the addition of new nucleotides. The enzyme “closes” around the incoming nucleotide to facilitate the chemical reaction.

35
Q

There is a conformational change in DNA polymerase (its ‘fingers’ tighten) only around the correct nucleotide

A
36
Q

Does polymerase edit mistakes in DNA?

A

Yes

37
Q

is is true that Pol III lacks 5’ → 3’ exonuclease activity?

A

Yes

38
Q

How kornberg tested strand polarity

A
  • Starting nucleotide radioactive
  • After the action of DNAse, the neighbouring nucleotide is radioactive.
  • How often was nearest neighbour a T?
  • GpA (0.065) = TpC (0.061)
  • These proportions can only match if the strands are anti-parallel
39
Q

How did kornberg test Watson-Crick base pairing?

A

Using a template strand with a known sequence.

Providing dNTPs with specific radioactive labels.

Allowing DNA synthesis to proceed and analyzing the composition of the newly synthesized DNA.

Observing that complementary base pairing (A-T, G-C) followed Watson-Crick rules.

He showed that the proportions of incorporated nucleotides matched the base composition of the template strand.

40
Q

How does editing work with DNA polymerase?

A

① The wrong nucleotide can be incorporated at low frequency

② This leaves an unpaired 3’-OH end that blocks further elongation by DNA polymerase

③ DNA polymerase 3’ → 5’ exonuclease activity removes the mismatch to leave a base-paired 3’-OH end

DNA replication can now resume

41
Q

What are the 2 active sites for polymerisation for?

A

one for polymerisation, and the other for editing. The editing site has the 3’-5’ exonuclease activity.

42
Q

Is it true that new DNA is made in the 5’ → 3’ direction?

A

Yes

43
Q
A

① Replication requires priming

② The primers are extended, growing in the 5’ → 3’ direction

③ Because new DNA is built 5’ → 3’ this strand needs new priming and extension … lagging strand

④ … whereas this strand supports continual synthesis (LEADING strand
)

44
Q

Is it true that DNA polymerase has a proof-reading 3’ → 5’ exonuclease activity?

A

Yes

45
Q

Who showed that DNA polymerase III is primarily responsible for large-scale DNA replication in cells?

A

Thomas Kornberg. ( Pol I was mainly involved in smaller, more specific repair and replication tasks.)

46
Q

Are there 2 different active sites for polymerisation and for editing errors?

A

Yes

47
Q

What did Arthur Kornberg use radioactive nucleotides to do?

A

To trace the incorporation of individual nucleotides into newly synthesised DNA. This allowed him to track the process of DNA synthesis

48
Q

How does DNA polymerase replicate DNA on the leading strand?

A

In a continuous manner, towards the replication fork

49
Q

How does DNA polymerase replicate DNA on the lagging strand?

A

In short strands, called Ozaki fragments, away from the replication fork. This requires repeated priming.

50
Q

Which ion is critical to the role of DNA polymerase?

A

Mg2+

51
Q

What did Kornberg realise was needed to initiate DNA replication?

A

A primer

52
Q

What are the challenges faced with lagging strand synthesis?

A
  • As the fork progresses, single-stranded DNA is exposed.
  • New primers must be laid down periodically for continued synthesis.
53
Q

How did Kornberg validate Watson Crick base pairing?

A
  • Kornberg used radiolabeled nucleotides to confirm complementary pairing.
  • Found consistent proportions of A-T and G-C pairing across strands.
54
Q

How did Kornberg validate Watson Crick antiparallel strands?

A

Base pairing and nucleotide ratios validated DNA’s antiparallel orientation.

55
Q

Summarise the key features of DNA replication

A
  • Semi-conservative.
  • Antiparallel in strand orientation.
  • Directional: 5’ → 3’ synthesis.
  • Semi-continuous: Leading strand is continuous; lagging strand is fragmented.
  • Proofreading minimizes errors to ~1 in 10⁹ bases.
  • Requires multiple enzymes and priming for lagging strand synthesis.

Cellular and Molecular Biology (33 decks)

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