DNA Replication II - Lecture 5 Flashcards
What enzyme breaks the base pairing?
Helicase
How are new strands of DNA synthesised?
DNA-dependent DNA synthesis
What enzyme carries out DNA-dependent DNA synthesis?
○ DNA polymerase
○ Requires a primer
○ DNA synthesis is always in the 5’ -> 3’ direction
What two types of exonuclease activity can most DNA polymerases do?
○ 3’ -> 5’ exonuclease activity
○ 5’ -> 3’ exonuclease activity
What does 3’ -> 5’ exonuclease activity involve?
○ Removes nucleotides it has just inserted
○ Called proofreading - allows errors to be corrected
What does 5’ -> 3’ exonuclease activity involve?
Can remove DNA already attached to the template
What are the bacterial DNA polymerases and their function?
○ DNA polymerase I: DNA repair and replication
○ DNA polymerase III: Main replicating enzyme
What are the eukaryotic DNA polymerases and their function?
○ DNA polymerase α: Priming during replication
○ DNA polymerase δ: Main replicative enzyme
What polymerase is not capable of 3’ -> 5’ activity?
DNA polymerase α
Which polymerase is the only one capable of 5’ -> 3’ activity?
DNA polymerase I
What happens at the replication fork?
Separated single strands are protected by SSBs (single-strand binding proteins) e.g. replication protein A in eukaryote
What is the leading strand?
Strand copied by continuous DNA synthesis
What is the primer made of?
RNA
How is the primer made in bacteria?
○ Made by the primase enzyme
○ 4-15 nucleotides in length
○ Once the primer has been made, DNA pol III makes the new strand
How is the primer made in eukaryotes?
○ Made by primase enzyme
○ DNA pol alpha extends it by adding about 20 nucleotides
○ DNA pol delta makes the rest of the new strand
How is the lagging strand copied?
Copied from a primer placed at the replication fork
What are the sections of the lagging strand called?
Okazaki fragments
How were Okazaki fragments discovered?
○ Added radioactive nucleotides to bacteria for different amounts of time
○ Separated DNA by size using centrifugation
○ Small DNA fragments only seen at shorter timepoints
How are Okazaki fragments joined in bacteria?
○ DNA pol III stops when it reaches RNA primer
○ DNA pol I removes RNA primer and a bit of the next fragment
○ DNA pol I continues synthesis
○ DNA ligase links the two DNA fragments
How are Okazaki fragments joined in eukaryotes?
○ DNA pol delta and helicase push aside primer
○ FEN1 (endonuclease) cuts at the branch point
○ DNA ligase links the two DNA fragments
