• Humans are more complicated than yeast and have multiple origins
• for picture- green spots are potential origins and origin of choice is random
• Replicons are organized as functional domains that contain several potential DNA replication origins (on average five), already licensed. A single origin is activated per replicon during the S phase. 
• The origin choice within each replicon can occur stochastically or can be influenced by specific cell fates or transcriptional activity. Replicon clusters include several consecutive replicons that are activated simultaneously (Berezney et al. 2000). 
• Replicons could be organized in chromatin loops where activation of one origin silences the other origins within the same replicon    

Shuttle mechanism of editing in DNA polymerases

• Polymerase binds to primer, then adds a nucleotide and then exonuclease checks
• The 3′ end of the primer strand in polymerizing mode is duplex and lies near three catalytically important carboxylates in the polymerase active site. The 3′ end of the primer strand in editing mode is single-stranded and lies in the 3′-5′-exonuclease domain active site (6). B, the shuttling model for polymerase editing proposes that the equilibrium between the 3′ end of the primer strand being bound as a single strand in the exonuclease active site (right) and bound as duplex at the polymerase active site (left) is shifted toward the editing mode by mismatched base pairs, which destabilize duplex DNA and retard addition of the next nucleotide. The shuttling of the 3′ end between the two active sites is fast compared with the rate of next nucleotide addition.    

 G1 phase- when helicast is loaded on DNA (1): starts with double hexemer and single hexamer goes on each strand. common in all organims 

 What the ORC does is mark or tag the origin and cells the cell where to load the helicase – load it as a double hexamer (MCM2-7) – different than viral SV40 which has the helicase built on the DNA. Euks

(2) and gate opens - this is common in all organisms

(3) and then DNA is twisted because of steric exclsion

(4) DNA begins to ooze out- now they start moving and they move and as they move they un-wind the DNA – steric exclusion. As they move, the dimer will hit the steric – fork. This provides the structure to open it. These keep moving a long – opening the DNA and it gets replicated by the polymerases.

Replication machinery – like a ribosome

Lagging strand – been taken and twisted around so that the direction of replication is the same on the two strands. The two replication polymerases are going in the same direction = loop!

MCM Helicase- hexameric helicase (6 proteins) initial step – got to take the dsDNA and open it up to single strand.

3 DNA polymerases: Alpha, Epsilon, Delta

epsilon does leading strand and alpha and delta make loop and piece it together 

All DNA polymerases require a primer: dsDNA and a single strand DNA it won’t do anything without a small fragment of DNA annealed to the large DNA with a 3’ hydroxyl – polymerase looks for the 3’ hydroxyl and gets on that and polymerizes from there.

Replicative Helicases 

spins DNA as fast as Jet engine to unwind DNA 

one strand continuously copies other is done in loops because it is reversed (3' to 5')

SV40 T-antigen: has 6 of the same proteins (hexamer - paralogs) hole in the center where DNA does in the hole and that’s how we open it up.

Methanon-cockus (first crystalize helicase)– Archaea organism, very small genome (1 mega bases, 25% the amount of DNA in ecoli) you can sequence their DNA and you find out there replication machinary is eukaryotic like – but they look and smell like bacteria. Have circular genomes like bacteria, with eukaryotic-like simple replication machinary. Has 1 protein that forms a homo-hexar.

Blue = basic amino acids; basic AA bind to the DNA molecule that is negatively charged through the phosphates and eventually will twist that DNA and open it up and allow replication.

SV40 is better model for studing eukaryotes because more similar due to 6 protein than archaea

Taken the lagging strand and twisted it around so that 2 polymerase delta molecules are in the same direction (dimeric) and they both now replicate towards the fork.

Processesivity –polymerase gets on DNA and replicates it until it is finished.

Polymerase Chain Nucleoanagen – PCNA- holds polymerase on DNA ring shaped, and is necessary for completion of replication in timely manner

Alpha and primase- primase is unsual and a RNA polymerase (RNA polymerases don’t require a primer). Primase makes a little piece of RNA (10-20 bp) and at the end of the RNA is a 3’hydroxyl – alpha is part of same complex, gets on 3’ hydroxl and makes a piece of DNA 40-50 bp long- to finish okazaki fragment we need polymerase delta or epslion. Three make a 200 bp piece of DNA.

Delta can replicate both leading and lagging strands.

Virus have small chromosomes – amazing molecule that is needed because the viruses have such small circular chromosomes.

SV40 has 2 deltas and humans need epsilon because of more complicated genome

T-antigen Acts like ORC – recognizes DNA sequence of the origin of the virus; acts as the helicases (MCM protein) and it is a hexameric protein like cellular but homo-hexamer (same protein with 6 molecules), and it brings in the cellular DNA polymerases.; acts as a transcription factor too.

SV40 to human cell: squiggle is the RNA primer, put in by primase, dna pol alpha replicated a small piece of dna after that and delta finished it out.

Epsilon does the leading strand and delta is reserved for lagging strand.