DNA replication

Question 1

What is the stronger base pairing? Why?

Answer 1

C to G as they have three hydrogen bonds compared to two between A and T

Question 2

What are the steps of DNA replication?

Answer 2

1. The DNA unwinds
2. Hydrogen bonds between the strands are broken via DNA helicase, forming a replication fork
3. DNA polymerase only works in a 5’ to 3’ direction (this means that it binds to the 3’ and works from there). This is fine for the leading strand, which can be continuously synthesised in this direction [note that this direction applies for the new strand being formed, so the strand on which this leading strand is constructed is the other direction].
   The other strand is known as the lagging strand and is fragments that are then added together by other enzymes

Question 3

When denaturing a DNA strand (i.e., breaking apart the hydrogen bonds), what controls the melting temperature?

Answer 3

• The nucleotide composition
• Higher temperatures are required to split apart C - G bonds than A - T bonds are they have three hydrogen bonds vs only two

Question 4

Why is the DNA replicated referred to as semi-conservative and how was this found?

Answer 4

One parent strand is conserved in each new double strand produced.
This was found by Meselson and Stahl using different isotopes of nitrogen. After using 15N for generations, e.coli were introduce to a new solution containing only the lighter 14N. This meant that all the new strands of DNA were formed using this 14N and the presence of both isotopes in new strands signalled the semi-conservative replication.

Question 5

What does DNA polymerase do?

Answer 5

Uses the template strand to synthesise a new complementary DNA strand in the 5’ to 3’ direction.

Question 6

Are the phosphate groups monophosphates or triphosphates?

Answer 6

• They are added as nucleoside triphosphates but are found in the chain as nucleoside monophosphates (lose it as they are added)

Question 7

What were the steps in understanding DNA replication?

Answer 7

1. Understood to be a double helix by Franklin, Watson and Crick
2. Discovered DNA denaturation by nearly boiling the DNA or subjecting it to extreme pHs - however, the reformation of a double helix was not yet understood
3. The semi-conservative nature of DNA replication was identified Meselson and Stahl
4. In-site studies identified the function of DNA polyerase (removed “background noise” by adding only specific components at a time)