DNA Replication

(Lecture 3)
VI. Eukaryotic DNA Replication

A. Eukaryotic DNA Polymerases

DNA Polymerase α DNA Polymerase γ

-involved in initiation -the DNA-replicating enzyme
-synthesizes an RNA primer then adds dNTPs of mitochondria
-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 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)

1
 Additional Proteins Involved in Eukaryotic DNA Synthesis

PCNA (Proliferating Cell Nuclear Antigen)

-confers high processivity to DNA Polymerase δ
-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

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 δ binds to PCNA at the 3’ ends of the growing to carry out Polymerase
highly processive DNA synthesis Switching

2
RFC Mediates Polymerase 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 α 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 δ approaches the RNA primer of the downstream Okazaki fragment,
RNase H1 removes all but the last RNA nucleotide of the RNA primer
5) the FEN1/RTH1 exonuclease complex removes the last RNA nucleotide
6) DNA Pol δ fills in the gap as the RNA primer is being removed
7) DNA ligase joins the Okazaki fragment to the growing strand

3
 A Model for Eukaryotic DNA Synthesis

Leading

Pol α
RNA
DNA
DNA Pol α syn. 20-30-nt primers

RFC loads PCNA to the end of the primer

Lagging PCNA displaces DNA Pol α

DNA Pol δ binds to PCNA and
elongates the Okazaki fragment

RNase H1 nuclease removes all

but one nucleotide of the RNA
primer
FEN1/RTH1 exonuclease removes
the last ribonucleotide

DNA ligase joins the Okazaki

fragment to the growing strand

After maturation of Okazaki fragments…

4
B. SV40 As a Model System
-Simian Virus 40 causes cancer in rhesus monkeys
-Early polio vaccine, generated in monkey tissue culture cells, was contaminated with
SV40
-Can study DNA replication in cultures of monkey cells (in vivo)
-Also a model for studying eukaryotic DNA replication in vitro
1) it has a small circular dsDNA genome of about 5 kb
2) possesses an ori (64 bp-long)
3) dependent almost entirely on host-cell proteins
4) requires only 1 viral protein (Large Tumor Antigen; T Ag) for replication in vivo
and in vitro
5) can do reconstitution experiments with DNA replication proteins purified from
human tissue culture cells
-Causes cancer by disrupting the cell-cycle control of DNA replication

5
Model of in vitro (Fig. 12-12, Lodish)
replication of SV40
DNA by eukaryotic
systems

6
T Ag function is regulated by protein phosphorylation
1) Phosphorylating of Thr124 activates T antigen to bind the SV40 ori and initiate
replication
2) Dephosphorylation at different amino acid phosphorylation sites on T antigen also
activate it.

E. coli vs. Human DNA Replication Enzymes

E. coli Human
SSB RPA

β2 Sliding Clamp PCNA

γ Complex Clamp Loader RFC (5 subunits)

Pol III Pol α, then Pol δ

Primase (DnaG) Primase activity of Pol α

Pol I 5’-3’ exo RNase H1 & FEN1

DNA Ligase (NAD) DNA Ligase (ATP)

Dna B Helicase Helicase [T-Ag (SV40)]

7
C. Cell Cycle Control of DNA Replication

The Eukaryotic Cell Cycle

(Fig. 30.19, G&G)

Eukaryotes have Multiple Origins of Replication

-Depending on the organism there is a replication origin or “replicator” every 1-300 kbp
of DNA to make sure that DNA replication occurs continuously throughout the
chromosome.
-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”

8
 Model for Initiation of the DNA Replication Cycle in Eukaryotes (Yeast)

ORC=origin recognition complex

-multi-subunit protein
-binds to replicators
-is bound to replicators throughout the
cell cycle

Cdc6p
-replication activator protein

Phosphorylation by
these proteins triggers
DNA replication

MCM
-is a “replication licensing factor (RLF)
-licenses or permits replication to occur
-mini-chromosome maintenance
(required for the maintenance of
plasmids in yeast)
(Fig. 30.20, G&G)

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

9
D. Telomeres

The ends of eukaryotic chromosomes are called telomeres (chromosomes are linear dsDNA molecules)
Telomeres are needed for chromosomal integrity and stability (protect ends from degradation).

Tandem telomere repeats (TTTAGG)

The ends of the lagging strands cannot be copied completely

Scenario #1: Not enough room for at the 5’ end of the lagging strand to lay down a new primer.
5’ 3’

3’ 5’

Scenario #2: After maturation of Okazaki fragments, there is a primer gap (as depicted above).

10
 Telomerase prevents progressive shortening of lagging strands during eukaryotic DNA replication

Telomerase is a
ribonucleoprotein
complex consisting In this example, the
of proteins and an telomerase of
RNA that acts as a Oxytricha adds
template for telomere TTTTGGGG
addition to repeats to the ends
chromosome ends of its chromosomes

(Fig. 12-13, Lodish)

11
Telomerase
-maintains telomere length by restoring telomeres to the 3’-ends of chromosomes.
-a ribonucleoprotein complex
-consistists of a 126 kDal RNA-dependent DNA polymerase, other proteins and a 450-nt RNA
-the telomerase polymerase is a “reverse transcriptase”
-the template sequence comes from the telomerase RNA and is AAAACCCC
-uses the 3’-end of the DNA as a primer and adds successive repeats to it (TTTTGGGG for
Oxytricia; TTTAGG for humans).
-ciliated protozoans such Oxytricha and Tetrahymena are good models for studying telomerase
activity because they have thousands of “minichromosomes.” Oxytricha has 107 gene-sized
chromosomes in its macronucleus.

Facts about Telomeres

Somatic cells lack telomerase activity because the TRT gene is switched off
Therefore, the telomeres get shorter with each cell division. (About 50 bases are lost from each telomere
every time a normal cell divides.)
Mammalian cells in culture will divide only ~ 50X
“Telomere theory of aging”—cells senesce and die when the telomeres are gone.
Evidence?: Over-expression of telomerase activity extends the life span of cells.
Reactivation of Telomerase activity in cancer cells

How to Clone a Telomere?

5’ 3’

3’ 5’
Bal31 Nuclease

EcoR I Digestion

E E E E E E E

Ligate EcoR I digested DNA into a blunt-EcoRI

and EcoR I-blunt vector (directional cloning)

Blunt E E Blunt

12
 Telomerase expressing cells

FISH (fluorescence in situ hybridization

1) green-tagged probe for telomere DNA
sequences
2) pink-tagged probe for centromeric
DNA sequences
3) purple (DAPI dye) stain for all DNA

Almost senescent cells

13
Artificial Chromosomes Can Be Used to Clone up to 1 X 106 bp Fragments

14
Reverse Transcriptase (R.T.)

In Retroviruses, R.T, is Needed to Convert The Retrovirus ssRNA Genome into dsDNA

The
Retrovirus
Life Cycle

(Fig. 6-22, Lodish)

Reverse Transcriptase as three activities:

1. RNA-directed DNA polymerase activity
2. RNase H activity
3. DNA-directed DNA polymerase activity

RNA

RNA-directed DNA polymerase

RNA
DNA

RNase H

DNA +
DNA-directed DNA pol

DNA
DNA

Avian Myeloblastosis Virus R.T. = 1 subunit of 71 kdal

HIV-1 R.T. = 2 subunits of 66 & 51 kdal (a heterodimer)

15