Lecture 31: DNA Replication

Question 1

Prokaryotic DNA replication steps

Answer 1

Occur simultaneously in vivo:
1. Initiation
2. Priming
3. Synthesis
4. Termination

Question 2

Prokaryotic DNA replication - initiation process

Answer 2

1. DnaA initiator protein recognizes 9 bp repeats in oriC (origin of replication)
2. Replication bubble opens; 3 AT-rich repeats adjacent to oriC are less stable. DnaA uses histone-like proteins.
3. DnaB/C complex expands replication fork bidirectionally (helicase)
4. DnaA complex displaced
5. ssDNA regions stabilized by ssbinding proteins (SSBs)

Question 3

DNA replication

Answer 3

Most accurate biological process; semi-conservative. Requires activated substrates (dNTPs)

Question 4

Prokaryotic DNA replication - priming requirement

Answer 4

No DNA polym. can init. new strand synthesis; req. unreacted 3’ OH group at end of base-paired primer strand.

Question 5

Prokaryotic DNA replication - initiation site

Answer 5

Circular bacterial chromosome has 1 DNA replication start site: oriC
Synthesis is bidirectional + continuous (new rounds start before 1st is over)
DnaA = initiator protein

Question 6

Prokaryotic RNA primer

Answer 6

5-10 NTs long, created 5’ to 3’, allows DNA poly. to start new strand synthesis.

Question 7

Primosome complex

Answer 7

DnaG (primase) is part of primosome complex; binds both strands of replication bubble to make short RNA primers

Question 8

Prokaryotic DNA polymerases involved in replication

Answer 8

1. DNA polymerase I: primer excision + repair
2. DNA polymerase III: replicative chain elongation + repair
   5 prokary. DNA polym. total

Question 9

DNA polymerase I activities

Answer 9

3 activities:
1. Polymerase
2. 3’ exonuclease (3’-5’ direction)
3. 5’ exonuclease (5’-3’ direction)

Question 10

DNA polymerase III features

Answer 10

• Very high rate of addition
• Very high processivity (adding multiple NTs before disengaging)
  Distinct but important qualities!

Question 11

DNA polymerase III structure

Answer 11

Holoenzyme w/ 10 subunits, 1 at each replication fork

Core enzyme (dimer):
- α = polymerase
- ε = 3’ exonuclease (proofreading)
- θ = req. piece
+ β subunit sliding clamp = increases processivity (low internal affinity for DNA w/ inside neg. residues)
+ τ maintains holoenzyme dimerization

Clamp loader (γ) complex:
- γ, δ, δ’, ψ, χ

Question 12

Leading and lagging strands

Answer 12

DNA synthesis only occurs 5’ to 3’:
Leading strand = made continuously
Lagging strand = made discontinuously, req. multiple RNA primers, forms Okazaki fragments

Question 13

DNA polymerase III proofreading

Answer 13

DNA polym. III recognizes mismatches; ε 3’ to 5’ exonuclease removes mismatch so DNA poly can try again

Question 14

Prokaryotic DNA replication - termination regions

Answer 14

• ter site is opposite oriC
• TUS binds repeats in funneling orientation
• TUS allows one-way DNA polym. procession, resulting in termination at ter site

Question 15

Replication fork features

Answer 15

• DnaA (initiator, dissociates)
• DnaG (primase)
• SSBs (stabilize ssDNA)
• 1 DNA polym. III at each end
• Topoisomerase to introduce supercoiling to newly synthesized DNA (+ refolds to supercoils)

Question 16

Major steps in termination

Answer 16

1. DNA polym. III reaches ter site and stops synthesis
2. Nick translation by DNA polym. I
3. Type II topoisomerase separates 2 daughter chromosomes

Question 17

Prokaryotic nick translation

Answer 17

Performed by DNA polym. I
1. 5’ to 3’ exonuclease hydrolyzes RNA primer
2. Simultaneous extension of 3’ end of Okazaki fragment
3. DNA ligase seals nick

Question 18

Prokaryote and eukaryote DNA replication similarities

Answer 18

1. Both semi-conservative
2. Both bi-directional
3. Both use short RNA primers required to initiate new strand synthesis
4. Continuous leading strand synth. + discontinuous lagging strand synth.

Question 19

Prokaryotic vs eukaryotic DNA replication enzymes

Answer 19

Prokaryotic = numbers/Roman numerals
Eukaryotic = Greek letters
Similar basic mechanisms, but eukary. more complex