Blok 9

Eukaryotic DNA Replication

Eukaryotic DNA Polymerases

DNA Polymerase
involved in initiation synthesizes an RNA primer then adds dNTPs a complex of four subunits 50-kD and 60-kD are primase subunits;180-kD subunit DNA polymerase synthesizes 8-10 nt RNA primers, then adds DNA to the RNA primers low processivity of DNA synthesis (200 nt) has no 3 -5 exonuclease activity (proofreading), yet has high fidelity

DNA Polymerase
the principal DNA polymerase in eukaryotic DNA replication has 3-5 exonuclease activity consists of a 125 kdal and a ~50 kdal subunit the 50 kd subunit interacts with PCNA (Proliferating Cell Nuclear Antigen) is highly processive when in association with PCNA

DNA Polymerase -required for replication, but its role is unclear -may substitute for DNA polymerase in lagging strand synthesis
DNA Polymerase -role in DNA repair (doesnt participate in replication)

Additional Proteins Involved in Eukaryotic DNA Synthesis

PCNA (Proliferating Cell Nuclear Antigen)

confers high processivity to DNA Polymerase the eukaryotic counterpart of the 2 Sliding Clamp of E. coli PCNA also encircles the double helix, but is a homotrimer of 37 kD subunits

RPA (Replication Protein A)

-ssDNA-binding protein that facilitates the unwinding of the helix to create two replication forks -the eukaryotic counterpart of the SSB protein of E. coli

RFC (Replication Factor C)

the eukaryotic counterpart of the complex Clamp Loader of E. coli

E. coli vs. Human DNA Replication Enzymes

E. coli SSB 2 Sliding Clamp Complex Clamp Loader Pol III Primase (DnaG) Pol I 5-3 exo DNA Ligase (NAD) Dna B Helicase Human RPA PCNA RFC (5 subunits) Pol , then Pol Primase activity of Pol RNase H1 & FEN1 DNA Ligase (ATP) Helicase [T-Ag (SV40)]

A Model for Eukaryotic DNA Synthesis

Leading strand synthesis

1. starts with the primase activity of DNA Pol to lay down a primer 2. lays down an RNA primer, then the DNA pol component of Pol adds a stretch of DNA 3. RFC assembles PCNA at the end of the primer 4. PCNA displaces DNA Pol . 5. DNA polymerase d binds to PCNA at the 3 ends of the growing to carry out highly processive DNA synthesis

RFC Mediates Polymerase Switching

1. Assembly of PCNA 2. Removes DNA Pol 3. Addition of DNA Pol

PCNA

Pol

RPA
RFC

Lagging strand synthesis

1. 2. 3. 4. 5. 6. starts off the same way as leading strand synthesis RNA primers synthesized by DNA polymerase a every 50 nt and consist of 10-nt RNA + 10-20-nt DNA polymerase switching as before to extend the RNA-DNA primers to generate Okazaki fragments when the DNA Pol d approaches the RNA primer of the downstream Okazaki fragment, RNase H1 removes all but the last RNA nucleotide of the RNA primer the FEN1/RTH1 exonuclease complex removes the last RNA nucleotide DNA Pol d fills in the gap as the RNA primer is being removed DNA ligase joins the Okazaki fragment to the growing strand

7. 8.