Blok 9

Eukaryotic DNA Replication

Eukaryotic DNA Polymerases

DNA Polymerase α

• involved in initiation • synthesizes an RNA primer then adds dNTPs • 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 δ

• 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 ε -required for replication, but its role is unclear -may substitute for DNA polymerase δ in lagging strand synthesis

•DNA Polymerase β -role in DNA repair (doesn’t participate in replication)

Additional Proteins Involved in Eukaryotic DNA Synthesis

PCNA (Proliferating Cell Nuclear Antigen)

• The sliding clamp in eukaryotes is assembled from a specific subunit of DNA polymerase delta

• Three PCNA molecules are tightly associated to form a closed ring encircling duplex DNA.

• confers high processivity to DNA Polymerase δ

PCNA (Proliferating Cell Nuclear Antigen)

• the eukaryotic counterpart of the β2 Sliding Clamp of E. coli

• a sliding clamp (A separate ring-shaped protein complex) , is a protein fold that serves as a processivity-promoting factor in DNA replication.

• In Bacteria, the sliding clamp is a composed of two identical β subunits of DNA polymerase III and is referred to as β2 sliding clamp.

RFC (Replication Factor C)

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

• the sliding clamp, attaches the polymerase to the DNA with the help of a molecular machine, the clamp loader, whose action depends on ATP.

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

E. coli vs. Human DNA Replication Enzymes

• E. coli

• SSB

• β2 Sliding Clamp

• γ 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 α, then Pol δ

• Primase activity of Pol α

• 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 α to lay down a primer

2. lays down an RNA primer, then the DNA pol component of Pol α adds a stretch of DNA

3. RFC assembles PCNA at the end of the primer

4. PCNA displaces DNA Pol α.

5. DNA polymerase d binds to PCNA at the 3’ ends of the growing to carry out highly processive DNA synthesis

RFC Mediates Polymerase α Switching

1. Assembly of PCNA

2. Removes DNA Pol α

3. Addition of DNA Pol δ

PCNA Pol δ

RPA

RFC

Lagging strand synthesis

1. starts off the same way as leading strand synthesis 2. RNA primers synthesized by DNA polymerase a every 50 nt and

consist of 10-nt RNA + 10-20-nt DNA 3. polymerase switching as before to extend the RNA-DNA primers

to generate Okazaki fragments 4. when the DNA Pol d approaches the RNA primer of the

downstream Okazaki fragment, 5. RNase H1 removes all but the last RNA nucleotide of the RNA

primer 6. the FEN1/RTH1 exonuclease complex removes the last RNA

nucleotide 7. DNA Pol d fills in the gap as the RNA primer is being removed 8. DNA ligase joins the Okazaki fragment to the growing strand

Eukaryotes have Multiple Origins of

Replication

• Depending on the organism there is a replication origin or “replicator” every 1-300 kbp of DNA

• In lower eukaryotes such as yeast replicator sequences are small (100-200 bp)

• In mammalian chromosomes the zones where initiation of replication occurs can span 500-50,000 bp.

• The human genome has 6 billion bp. Therefore, the average human chromosome has several hundred units of replication or “replicons”

Initiation of the DNA Replication Cycle in Eukaryotes (Yeast)

Cyclins • -get their name because they are synthesized at one phase of the cell cycle and

degraded in another ORC=origin recognition complex • -multi-subunit protein • -binds to replicators • -is bound to replicators throughout the cell cycle • Cdc6p • -replication activator protein MCM • -is a “replication licensing factor (RLF) • -licenses or permits replication to occur • -mini-chromosome maintenance • (required for the maintenance of • plasmids in yeast) Phosphorylation by these proteins triggers DNA replication