BM: DNA Replication

Question 1

How does DNA replicate?

What does this mean?

Answer 1

Semi-conservative replication.

• Half the strands in each new DNA molecule are from the original DNA molecule.*
• Means there is genetic continuity between generations of cells.*

Question 2

What are the four requirements needed for semi-conservative replication to take place?

Answer 2

• The four types of (free) nucleotides, with bases A, T, G & C, must be present.
• Both strands of DNA must act as a template for these nucleotides to attach onto.
• Enzyme DNA polymerase must be present.
• Chemical source of energy is required to drive the process.

Question 3

Explain the process of semi-conservative replication:

Answer 3

• Enzyme helicase breaks hydrogen bonds between base pairs.
• Helix unwinds to form two single strands.
• Exposed polynucleotide strands act as templates.
• Free floating nucleotides are attracted to their complementary exposed bases on each original strand - due to complementary base pairing.
• Condensation reactions join the nucleotides of the new strands together - catalysed by the enzyme DNA polymerase.
• Hydrogen bonds form between the bases on the orignal and the new strand.
• Each new DNA molecule contains one strand from original DNA molecule and one new strand.

Question 4

What two enzymes are involved in semi-conservative replication?

What is the function of each?

Answer 4

Helicase = breaks hydrogen bonds between base pairs.

Polymerase = catalyses condensation reactions between nucleotides of new strand.

Question 5

How does DNA polymerase move across the DNA molecule?

Answer 5

It moves in opposite ways along antiparallel DNA strands.

Question 6

Describe the arrangement of the DNA strands in a DNA molecule:

Answer 6

The strands are antiparallel.

Question 7

What does ‘antiparallel’ mean in terms of DNA strands?

Answer 7

They run in opposite directions.

Question 8

How are the two ends of a DNA polynucleotide strand different?

Answer 8

One end is 5’ and one end is 3’

Question 9

What is the active site of DNA polymerase complementary to?

What does this mean the enzyme can do?

Answer 9

The 3’ end of the newly forming DNA strand.

This means the enzyme can only add nucleotides to the new strand at the 3’ end.

Question 10

If DNA polymerase can only add nucleotides to the new strand at the 3’ end, how does this affect the direction in which the new strand is made?

How does the enzyme move across the template strand?

Answer 10

It means the new strand is made in a 5’ to 3’ direction.

The enzyme moves down the template strand in a 3’ to 5’ direction.

So you end up with antiparallel strands.

Question 11

Who came up with the theory of semi-conservative replication?

Answer 11

Watson and Crick

Question 12

Who validated the theory of semi-conservative replication through experiments?

Answer 12

Meselson and Stahl

Question 13

Before any theory was validated, what did scientists think about DNA replication?

Answer 13

They were unsure whether it was conservative or semi-conservative.

Question 14

If DNA replication was conservative, what would this mean?

Answer 14

The original DNA strand would stay together and the new DNA molecules would contain the two new strands.

Question 15

What did Meselson and Stahl experiment with?

Answer 15

Two isotopes of nitrogen (as DNA contains nitrogen) - heavy nitrogen (15N) and light nitrogen (14N).

Question 16

Describe Meselson and Stahl’s experiment:

Answer 16

• Two samples of bacteria were grown - one in nutrient broth containing light nitrogen and one in heavy nitrogen.
  
  • Bacteria reproduced and took up nitrogen to help make nucleotides, so it gradually became part of DNA
• Sample of DNA taken from each batch and spun in a centrifuge.
  
  • DNA from heavy nitrogen settled lower down centrifuge than DNA from light nitrogen bacteria, as it is heavier.
• Then, bacteria growin in heavy N were put in broth containing light nitrogen.
• Left for one round of DNA replication and spun in centrifuge.

Question 17

What were the results from Meselson and Stahl’s experiment?

• What would the results have been if DNA replication was conservative?*
• What would the results have been if DNA replication was semi-conservative?*

Answer 17

Conservative = original heavy DNA, which would still be together, would settle at the bottom and the light DNA would settle at the top.

Semi-conservative = new bacterial DNA would contain one strand of old DNA containing heavy N and one strand of new DNA containing light N. So, DNA would settle out between original settlement of light DNA and heavy DNA.

As it turned out, DNA settled in middle showing DNA contained mixture of heavy and light nitrogen - bacterial DNA replicated semi-conservatively in light nitrogen.

Question 18

Once Meselson and Stahl confirmed replication was semi-conservative in bacteria, what happened?

Answer 18

Other scientists carried out experiments to show that it was the universal method for DNA replication.