DNA synthesis

Question 1

Describe how DNA replicates (4)

Answer 1

1. Each DNA molecule unwinds (untwisted a bit at a time) catalysed by DNA gyrase.
2. Each DNA molecule is unzipped, hydrogen bonds between complementary bases are broken producing to individual DNA strands with exposed nucleotide bases. This is catalysed by DNA helicase.
3. Free phosphorylated nucleotides are bonded to these exposed nucleotide bases, following the complementary base pairing rules (A-T and C-G). Catalysed by DNA polymerase.
4. Ligase enzymes then catalyse the formation of the phosphodiester bonds between the deoxyribose and phosphate, using the energy provided by the hydrolysis of activated nucleotides.

Question 2

Give an outline of how DNA starts to replicate (5)

Answer 2

1. DNA replication occurs at specific sites on the DNA molecule known as replication origins. Where it can be recognised by initiation complexes.
2. The DNA molecule opens up to form a replication bubble that allows access to the cellular machinery.
3. Bacteria’s cell cycle is between 20-30 minutes long. Consists of S and M phase. Replication occurs at one site only.
4. Mammalian cell cycle takes 16-24hrs and consists of M, G1, S, and G2. Replication occurs at multiple sites.
5. DNA bubbles will grow larger and larger until they coalesce and form two daughter chromosomes all the whole of the DNA has been copied.

Question 3

List the different types of DNA polymerases for eukaryotes and bacteria

Answer 3

Alpha	Replication 
Beta	Repair 
Gamma 	Mitochondrion
Delta 	Replication 
Epsilon 	Replication 

Polymerases 1 Repair
Polymerase 2 Repair
Polymerase 3 Replication
Others Repair

Question 4

Describe in detail how DNA replicates and the repair systems involved

Answer 4

• Acts in the 5’ to 3’ direction, which has important consequences
• Utilises AT and CG base pairing to synthesise new DNA strands
• It requires a DNA template, a DNA or RNA primer (because DNA polymerase requires an –OH on the 3’ carbon to start adding nucleotides) the four deoxyribonucleoside triphosphate (dNTP) building blocks and Mg2+ ions.
• It also has a proof-reading function
• You can only add building blocks at the 3’end.
• The dNTPs are building blocks for DNA.
• The hydroxyl groups reacts with the first phosphate and causes it to release pyrophosphate (a diphosphate.) This leaves one phosphate which then reacts with the hydroxyl forming the sugar phosphate backbone, while the bases pair.
• The significance that DNA polymerase can only act in the 5’-3’ means one DNA strand must be made discontinuously, while the other is made continuously.
• A replication fork will appear at the replication origin. The parental stands will separate. It is important to remember they are anti-parallel. On the 5’-3’ strand, DNA polymerase will cause a continuous strand to be made, called the leading strand, as the DNA opens up and the DNA polymerase can continue to synthesis the complementary strand.
• However, on the other strand, DNA polymerase will be working in the opposite direction, this means that behind it, the strand will be opening up as it moves forward. Causing it to be a lagging strand. This forms Okazaki fragments. As replication has to be re-initiated again and again.
• DNA polymerase also has a proof reading and mis-match repair system, to ensure that DNA replication proceeds without making mistakes. Humans must replicate 3 times 10 to the 9 base pairs.
• Error rate is extremely low because:
• DNA polymerase error rate is about 1 in 10 to the power of 8, due to base pairing/editing function of the enzyme.
• The mis-match repair system, which corrects most of the polymerases errors. This multi-enzyme system is highly conserved across species.

Question 5

Describe the different enzymes involved in DNA replication (7)

Answer 5

1. Helicase - Unzips the DNA molecule, breaking the hydrogen bonds between complementary bases, forming 2 single-stranded DNA molecules with exposed nucleotide bases
2. Topoisomerase 2 - Goes in front of DNA helicase removing coils from the DNA molecule as it becomes supercoiled to DNA.
3. Primase - Adds an RNA primer as DNA polymerase requires -OH on the 3’ to start adding nucleotides
4. DNA binding proteins - Stabilise the single-stranded DNA molecules and prevent them from being re-annealed back together.
5. Replication DNA polymerase - Replicates the parental DNA molecule.
6. Repair DNA polymerase - Repairs the fragments and removes the RNA molecule so it will be a full DNA strand.
7. Ligase - splices fragments together, catalyses the formation of the phosphodiester bond between the deoxyribose and phosphate groups.