Week 5 - DNA Replication

Question 1

stages of replication

Answer 1

1. initiation
2. replication
3. termination

Question 2

DNA unwinding element (DUE)

Answer 2

13 bp sequences (x3) that unwind the DNA

Question 3

oriC

Answer 3

245 bp long where replication is initiated

Question 4

initiation at oriC

Answer 4

• DUE unwinds dsDNA
• 4-5 DNA-A proteins bind to the 9 bp repeat in oriC
• complex made when DNA wraps around the DNA-A protein
• complex induces supercoiling (local positive writhe)
• negative supercoiling induced to compensate for positive writhe which causes denaturation of AT-rich bps in oriC
• DNA-B binds to each strand to prepare for replication

Question 5

DNA-A protein

Answer 5

• binds to 9 bp sequences (x5) at oriC

- binding ability is regulated as it requires DNA to be fully methylated

Question 6

DNA-B protein

Answer 6

helicase that binds to each exposed strand

Question 7

DNA-C protein

Answer 7

in complex with DNA-B before it binds to strands

Question 8

DNA-B mechanism

Answer 8

hydrolyzes ATP and uses energy gained to drive conformation change that enables the winding of helix

Question 9

DNA adenine methylation (Dam) methylase

Answer 9

methylates N6 adenine in palindromic sequence (GATC)

Question 10

hemi-methylated

Answer 10

after replication, strands are hemi-methylated, therefore new strands cannot bind DNA-A, which are protected from Dam methylase until cell is ready to replicate

Question 11

Dam methylase protection

Answer 11

oriC is “hidden” and sequestered from Dam methylase until replication is needed

Question 12

DNA gyrase

Answer 12

relieves torsional strain from unwinding

Question 13

DNA polymerase III

Answer 13

• fast polymerization rate
• high processivity
• multiple subunits
• alpha
• epsilon
• theta
• tau
• gamma
• beta

Question 14

alpha subunit

Answer 14

• polymerization activity

- part of the core polymerase

Question 15

epsilon subunit

Answer 15

• 3’-5’ exonuclease activity (proof-reading)

- part of the core polymerase

Question 16

theta subunit

Answer 16

• stabilizes epsilon subunit

- part of the core polymerase

Question 17

tau subunit

Answer 17

• core enzyme dimerization

- part of the clamp-loading complex

Question 18

gamma subunit

Answer 18

• clamp loader

- part of the clamp-loading complex

Question 19

beta subunit

Answer 19

keeps enzyme on the template for optimal processivity

Question 20

lagging strand synthesis

Answer 20

• DNA-B has primase which will add primer to the lagging strand which is replaced with DNA by DNA polymerase 1
• new primase binds to DNA-B to synthesize new primer as the Okazaki fragment before nears completion
• a new clamp is loaded onto the new template primer by the clamp loader
• core subunits (alpha, epsilon, theta) are transfered to new template primer and the old beta clamp is discarded

Question 21

DNA polymerase 1

Answer 21

• catalyzes strand replacement
• 5’-3’ exonuclease activity
• binds at the nick and cuts off primer, replacing it with DNA (dNTPs)

Question 22

DNA ligase

Answer 22

creates phosphodiester bonds between neighbouring residues (DNA) to seal nicks
- 3’OH will attack the 5’ phosphate group

Question 23

DNA ligase mechanism

Answer 23

1. DNA ligase adenylated (AMP attached to it)
2. 5’ phosphate group creates an phosphoanhydride bonds when it is activated through AMP transfer (AMP is a good leaving group)
3. nucleophilic attack of 3’ hydroxyl seals the nick when it creates a phosphodiester bond