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

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.

Synthesizing the lagging strand

Synthesizing the lagging strand

Replication Fork Events

Single Stranded Binding Proteins

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

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

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

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

The replisome Method of Coordinating Leading and Lagging Strands

Replication in Eukaryotes

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

DNA Ligase Mechanism

Coolest DNA replication video ever! https://www.youtube.com/watch?v=4jtmOZaIvS0

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!

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

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