Lecture 20 - DNA replication

Question 1

How is a new nucleotide added?

Answer 1

Comes in on a dNTP (deoxynucleoside triphosphate), 3’-hydroxyl performs nucleophilic attack on alpha phosphate, breaks bond between alpha phosphate and oxygen. Means that DNA is always synthesized 5’-3’

Question 2

Describe the replication fork

Answer 2

Starts at origin of replication (defined DNA sequence), creates 2 replication forks (DNA synthesis is bidirectional. Leading strand synthesized continuously, lagging strand synthesized by Okazaki fragments.

Question 3

Describe DNA polymerase 1 and 3

Answer 3

DNA pol 1 –> 3’-5’ exo activity, 5’-3’ exo activity, removes RNA primer (nick translation), proof reading
DNA pol 1 –> 3’-5’ exo activity, high processivity, elongation of chain and proof-reading structure

Question 4

Describe the replication enzyme complex

Answer 4

Helicase - unwinds DNA
Topoisomerase - reduces strain
ss Binding proteins - prevent DNA from rewinding, protect from nucleases
Primase - RNA polymerase that produces RNA primer
DNA pol 3 - proof reading, chain elongation
DNA pol 1 - proof reading, removal of RNA primer (nick translation)
Ligase - joins Okazaki fragments by creating phosphodiester bonds

Question 5

How does DNA polymerase ensure fidelity?

Answer 5

On the minor groove, H-bond acceptors are all in the same location regardless of base, bind to H-bond donors in polymerase. If wrong base pairing, H-bonds won’t form

Question 6

What is the function of the beta clamp?

Answer 6

Ensures high processivity, pair of beta subunits clamp between template and daughter strand