Module 2 - Lecture 2 - DNA Replication II

- DNA replication occurs by the semiconservative method

- DNA topoisomerases nick/cut the DNA to relieve the stress of DNA unwinding
- Helicases break the base pairs
- The replication fork progresses along the DNA molecule
o Where the DNA is being copied and being made
- Strand are SEPERATED
o Via helicase
- Then the strands unwind to relieve the stress via DNA topoisomerase to get rid to the
supercoils

How are the new strands of DNA synthesized at the replication fork?
- The process is called template dependent DNA synthesis
- The template is DNA, so this is DNA-dependent DNA synthesis

DNA-dependent DNA synthesis is carried out by an enzyme called a DNA-dependent DNA

polymerase
- DNA synthesis is always in the 5´→3´direction
- This requires a primer to initiate synthesis of the new strand
- Without a primer there is no DNA synthesis
 - Most DNA polymerases can also degrade DNA
o this is called the exonuclease activity
o there are two possible types of exonuclease activity
- 3´→5´exonuclease activity
o the polymerase can remove nucleotides it has just inserted
o this is called proofreading – allows errors to be corrected

- 5´→3´exonuclease activity
o the polymerase can remove DNA already attached to the template

DNA synthesis
- you lose two phosphates to join the
nucleotides
o pyrophosphates
Replication fork
- First, the separated single strands must be protected
o if not, they may just re-attach to one another
o or they might be attacked by nucleases
- The strands are protected by single-strand binding proteins or SSBs
o Example in eukaryotes Replication protein A

- One strand can be copied by continuous DNA

synthesis
- This is called the leading strand
- Copying of this strand requires a primer

- The primer is made of RNA

o in bacteria the primer is made by the primase
enzyme
o the primer is 4-15 nucleotides in length
o once the primer has been made, DNA pol III makes
the new strand

- In eukaryotes the process is slightly different

o The RNA primer is first extended by DNA
pol alpha
o DNA pol alpha adds about 20 nucleotides
o then DNA pol delta makes the rest of the
new strand
Lagging strand
- DNA synthesis is always in the 5´→3´direction
o the lagging strand must therefore be copied from a primer
placed at the replication fork.

- What happens when the replication fork moves?

o The leading strand is extended by more DNA synthesis
o But the lagging strand must be made in sections
o The sections are called Okazaki fragments
 200-300 nucleotides long
o These must be joined together, and RNA primers removed to
complete lagging strand synthesis

Joining two Okazaki fragments in Bacteria

- DNA polymerase 3 stops when it reaches the RNA
primer
o No exonuclease activity
o It gets blocked
- Switch to DNA polymerase 1
o Has 5’ to 3’ exonuclease activity
o Removes primer
- DNA ligase joins the two
- Fragments

Joining two Okazaki fragments in Eukaryotes

- DNA polymerase delta and helicase push the primer

aside
- You get a flap
- FEN1 enzyme cuts the flap at the branch point
o It is a endonuclease
 Endonuclease cuts within a molecules
 Exonuclease cuts are the ends
- Ligase joins the nucleotides together where there is a
missing phosphodiester bond