1. Origin of Replication Primase is a Specialized RNA Polymerase
Primase does not need a primer, because like other RNA polymerases, they synthesize RNA directly from the template so a primer is not needed

2. DNA Replication: Primase
How does Primase “know” where to lay down the RNA primer?
Primase is a specialized RNA polymerase that makes SHORT RNA primers on a single stranded DNA template (RNA primers are 5-10 bp long).
Primase does not require a “promoter” sequence like RNA polymerase
Primase activity is greatly increased when it associates with helicase and single stranded DNA. This ensures that Primase is recruited to the replication fork.

3. Synthesizing the lagging strand

4. Synthesizing the lagging strand

5. Replication Fork Events

6. Single Stranded Binding Proteins

7. DNA Sliding Clamp Sliding clamp’s purpose is to Increase Processivity
Prokaryotes – 2 Subunits
Eukaryotes – 3 Subunits
Clamp Loader Complex

8. DNA Sliding Clamp
DNA polymerase needs help to stay associated with the template strand
DNA polymerases are associated with DNA sliding clamps proteins. They look like a doughnut.
DNA polymerase still disengages from the DNA-- BUT it does not diffuse away. The sliding clamp protein “holds” it in place and ensure that the DNA polymerase binds to the same primer:template junction
Primer:template junction

9. DNA Sliding Clamp
The DNA polymerase has a higher affinity to a single stranded: double stranded junction DNA. If double stranded DNA only is in the active site of DNA poly the affinity for the sliding clamp is reduced. Thus DNA poly disengages and diffuses away.
junction
Okazaki Fragment

10. DNA Sliding Clamps
E. coli
Eukaryotic
Viral
DNA sliding clamps are HIGHLY conserved—They all have similar diameters

11. The replisome
Method of Coordinating Leading and Lagging Strands

12. Replication in Eukaryotes

13. Removal of RNA Primer
RNA primer bound to DNA creating a RNA-DNA Hybrid
RNase H can only degrade a RNA-DNA Hybrid
After the RNA is degraded a new primer:template junction is formed
DNA polymerase can attach (exposed 3’OH group) and synthesize new DNA.
5’ exonuclease activity of DNA polymerase removes the last RNA nucleotide.
Rnase H: H stands for hybrid

14. DNA Ligase Mechanism

15. Coolest DNA replication video ever!

16. Too much 5’ exonuclease activity– it would just keep on chopping!
Different Types of DNA Polymerases
E. coli
Pol III
Chromosome replication
High processivity
Pol I
RNA primer removal
5’ exonuclease activity
Low processivity
Why is this a good thing that PolI has low processivity?
Too much 5’ exonuclease activity– it would just keep on chopping!

17. Different Types of DNA Polymerases
Eukaryotes
~ 15 different types per cell and three are required during DNA replication

18. Different Types of DNA Polymerases
The newly unwound strands are asymmetric
Pol ε (epsilon) associates with the Leading Strand
Pol ε requires the presence of the helicase to securely attached to the DNA
Pol δ (Delta) associates with Lagging Strand
Pol δ binds more tightly to the DNA Clamp
More DNA clamps on the lagging strand?
Helicase
DNA Clamp
Pol ε
Pol δ
Credit: Image courtesy of Rockefeller University, O’Donnell Lab, 2014