DNA Synthesis

Question 1

Describe replication origins.

Answer 1

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 2

What are the differences between the bacterial and mammalian cell cycle?

Answer 2

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 3

What are the mutliple different polymerases?

Answer 3

In bacteria, there is I, II and III repair (there are others that also repair).

In eukaryotic cells, there is:
- α replication
- β replication
- γ mitochondrion
- δ replication (causes elongation)
- ε replication (causes elongation)
(there are others also possibly involved in repair).

Question 4

What are some DNA Polymerase key properties?

Answer 4

• 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 (becuase 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 5

Describe DNA replication.

Answer 5

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 6

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

Answer 6

• HELICASE: Separates the base pairs producing single strands
• TOPOISOMERASE: goes in front of helicase and gets rid of the coils in DNA (as becomes supercoiled due to helicase)
• PRIMASE: lays down some RNA (produces 3’ –OH), so that DNA polymerase knows where to start replicating
• DNA BINDING PROTEINS: stabilise the single-stranded DNA, and stop it being re-annealed with the other parent strand
• REPLICATIVE DNA POLYMERASE: copies the parental strand
• REPAIR DNA POLYMERASE: repairs the fragments and removes the RNA so it will be a full DNA strand
• DNA LIGASE: splices the fragments together

Question 7

Why is the error rate of DNA Polymerase so low?

Answer 7

• due to base pairing and proofreading/editing function of the enzyme
• due to the mismatch repair system, which corrects most of the polymerase errors.

Question 8

Mention some DNA replication inhibitors.

Answer 8

ANTIBACTERIAL: 
- Ciprofloxacin 
- Levofloxacin 
- Novabiocin 
(Gyrase inhibitors)  

ANTITUMOUR: 
- Etoposide
- Doxorubicin
- Mitoxantrone
(Topo II inhibitors) 

ANTIVIRAL:
- AZT
(Reverse transcriptase)