MB - DNA Replication I

Question 1

What do you need to replicate the entire chromsome? (6)

Answer 1

1. Helicase
2. Topoisomerases
3. Primase
4. SSB proteins
5. DNA polymerase I
6. DNA ligase

Question 2

What are the 5 different polymerases involved in DNA Replication?

Answer 2

Pol I: DNA repair and replication

Pol II: DNA repair

Pol III: Principal DNA replication
enzyme

Pol IV: DNA repair

Pol V: DNA repair

Question 3

What are the main differences between pol I and pol III? (5)

Answer 3

Pol I:

• 1 gene
• 109 kDa
• 400 copies per cell
• 10 nt/s
• > 100 hours per genome

Pol III:

• 22 genes
• 106 kDa
• 10 copies per cell
• 1600 nt/s
• 40 minutes per genome

Question 4

How can we determine which genes (proteins) are important?

Answer 4

Temperature-sensitive mutants allow proteins to be switched on or off by changing the temperature
e.g. protein works at 20, but not 37°C

Question 5

What is the difference between quick stop mutants and slow stop mutants?

Answer 5

Quick stop mutants: Replication immediately stops

Slow stop mutants: Current round of replication finishes, but a new one can’t start

Question 6

What are problems with DNA replication? (4)

Answer 6

1. Strands being coiled (topology)
2. Circular DNA molecules (topology)
3. Antiparallel strands (polarity & topology)
4. Mutations/errors (fidelity)

Question 7

How are DNA strands unwound?

Answer 7

They are plectonemically coiled

• Helicases separate and unwind the duplex using ATP hydrolysis

Question 8

How do helicases unwind DNA? (3)

Answer 8

1. Conformational changes in the helicase pull on the DNA strand, separating it from its partner
2. Helicases move towards the 3’ end of the strand they are clamped to (one on each strand)
3. Helicases separate and unwind the two DNA strands, creating a replication bubble

Question 9

What causes supercoiling?

Answer 9

Unwinding part of the duplex creates strain elsewhere (results in supercoiling)

Question 10

What equation describes DNA topology?

Answer 10

Lk = T + W

Lk = Linking number (fixed in circDNA)

T = Twist (number of duplex turns)

W - Writhe (number of duplex self-crossings

Question 11

What is the relaxed form called (W=0)?

Answer 11

Lk0

Question 12

How can you determine +ve supercoiling and -ve supercoiling?

Answer 12

If Lk > Lk0 there is +ve supercoiling

If Lk < Lk0 there is -ve supercoiling

Question 13

What is the formula for Superhelical density?

Answer 13

σ = (Lk-Lk0)/Lk0

In relaxed DNA, σ = 0

Question 14

Where is -ve supercoiling found?

Answer 14

Purified cellular DNA is negatively supercoiled

• σ = -0.06 (eukaryotes and prokaryotes)

Question 15

What problem occurs that results in +ve supercoiling formation? (3)

Answer 15

1) Helicase-based unwinding → overwinding elsewhere

2) Overwinding will resist replication fork movement

3) Lk can’t change to relieve the stress without breaking the phosphodiester bond

• Positive supercoils form