Blok 9

Eukaryotic DNA Replication

Eukaryotic DNA Polymerases
 DNA Polymerase
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
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)

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
 PCNA (Proliferating Cell Nuclear Antigen)

the eukaryotic counterpart of the 2 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 subunits of DNA polymerase III
and is referred to as 2 sliding clamp.
 RFC (Replication Factor C)

the eukaryotic counterpart of the complex

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