DNA Synthesis

Question 1

How does DNA replicate?

Answer 1

Replication is semi-conservative.

Question 2

What are replication origins?

Answer 2

DNA replication is initiated at specific sites on DNA called replication origins. These origins are recognised by an initiation complex.
DNA at the origin unwinds to form a replication bubble and allow access to the replication machinery.

Question 3

What are the differences between bacterial and mammalian cell cycle?

Answer 3

The bacterial cell cycle is about 20-30 minutes long, while the mammalian cell cycle is 16-24 hours long.
The bacterial cell cycle consists of M - S PHASES, while the mammalian cell cycle consists of the M - G1 - S - G2 PHASES.
Bacterial DNA only has a single replication origin, while mammalian DNA has multiple replication origins.

Question 4

What are the different bacterial DNA polymerases?

Answer 4

I – repair
II – repair
III – replication

(there are others that also repair)

Question 5

What are the different eukaryotic DNA polymerases?

Answer 5

Alpha – replication
Beta – repair
Gamma – mitochondrion
Delta – replication (causes elongation)
Epsilon – replication (causes elongation)

(others possibly involved in repair)

Question 6

What are the properties of DNA polymerase?

Answer 6

• it acts in the 5’ to the 3’ direction, which can important consequences
• it utilises AT and CG base pairing to synthesis new DNA strands
• it requires a DNA template, a DNA/RNA primer (because DNA Polymerase requires an -OH on the 3’ carbon to start adding nucleotides), the four dNTP building blocks and Mg2+ ion
(you can only add building block to the 3’ end)
• it also has a proof reading function

Question 7

Describe DNA replication.

Answer 7

The hydroxyl group reacts with (attacks) the first phosphate and causes it to release pyrophosphate (crudely, a diphosphate). This leaves the 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’ way, means one DNA strand must be made discontinuously, while the other is made continuously.

A replication fork will appear at the replication origin, and the parental strands will separate. It is important to remember they are antiparallel. 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 synthesise 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 while it moves forward. This is called the lagging strand, and forms okazaki fragments as replication has to be re-initiated again and again.

Question 8

What are the 7 enzymes involved in DNA synthesis and what do they do?

Answer 8

1. Helicase – Separates the base pairs producing single strands
2. Topoisomerase – Goes in front of helicase and gets rid of the coils in DNA (as becomes supercoiled due to helicase)
3. Primase – Lays down some RNA (produces 3’ –OH), so that DNA polymerase knows where to start replicating
4. DNA binding proteins – Stabilise the single stranded DNA, stop it being re-annealed with the other parent strand
5. Replicative DNA polymerase – Copies the parental strand
6. Repair DNA polymerase – Repair the fragments and takes out the RNA so it will be a full DNA strand
7. DNA ligase – Splices the fragments together

Question 9

Why is the error rate of DNA polymerase so low?

Answer 9

♣ DNA polymerase error rate is about 1 in 108, due to base pairing and proof reading/editing function of the enzyme
♣ The mismatch repair system, which corrects most of the polymerase errors.
This multi-enzyme system is highly conserved across species

Question 10

What happens if there are defects in DNA synthesis?

Answer 10

Inherited defects in mismatch repair genes are involved in (colon) cancer, in essence, failure to correct DNA synthesis may lead to cancer

Question 11

What are DNA replication inhibitors?

Answer 11

DNA replication inhibitors are important antibacterical, antiviral and antitumour agents.

Question 12

Examples of Antibacterial agents.

Answer 12

• Ciprofloxacin
• Levofloxacin
• Novabiocin (Gyrase inhibitors) – blocks replication

Question 13

Examples of antitumour agents.

Answer 13

• Etoposide
• Doxorubicin
• Mitoxantrone (Topo II inhibitors) – blocks replication

Question 14

Examples of antiviral agents

Answer 14

• AZT (Blocks reverse transcriptase) – stops cDNA from being formed from viral RNA. Therefore stops viral replication.