PHAR lecture 5
Replication: Differences between
eukaryotes and prokaryotes
Replication in Eukaryotes
• Replication is intimately linked to cell
division
• Cell division in eukaryotes is known as the
cell cycle; this will be covered in a later
lecture.
• It takes some 18 – 24 h to complete.
• There are checkpoints along the process.
The cell cycle
The cell cycle consists of:
• G1 = growth and preparation of the
chromosomes for replication
• S = synthesis of DNA (and centrosomes)
• G2 = preparation for M
• M = mitosis
The cell cycle :
• When a cell is in any phase of the cell
cycle other than mitosis, it is often said to
be in interphase (G1, S or G2).
• If a cell is not dividing it is said to be in G0.
Terminally differentiated cells are in G0.
Checks before replication
• As the cell passes through each phase of
the cell cycle there are checkpoints. If
something goes wrong the cell division
process can be aborted.
• If the cell remains in this aborted or
arrested state for too long it will be
directed to commit suicide, a process
known as apoptosis
Check for replication
• There is a commitment point late in G1.
After that there is no turning back, the cell
is committed to cell division.
• This point is known as the restriction point.
• Extracellular factors; mitogens and protein
growth factors must be present. These
factors regulate proliferation
Check for replication
• DNA damage is checked for at G1 before
the cell enters S
• A check for the completion of S phase
(replication) is the presence of Okazaki
fragments.
• Spindles are checked before the cell
actually divides (M phase)
Cyclins
• At critical stages, G1, S and M, special
cytoplasmic proteins known as cyclins
increase in concentration then subside
once the cell has passed through that
stage.
Checks before replication
• There are also a group of enzymes known
as cyclin dependent kinases (cdks) which
phosphorylate protein targets involved in
the control of the cell cycle.
• The catch is that, although the Cdks are
present at fairly steady concentrations in
the cell independent of the cell cycle
stage, they only become activated when
they bind the appropriate cyclin.
Drives the cell
from G1 to S
Cyclin A
Cyclin E
Cyclin B
Binds to
Cdk4 & 6
Binds to
Cdk1
Cdk
levels
Concentration
Activity
Cyclin D
Binds to
Cdk2 & 1
Binds to
Cdk2
Drives the cell
from G2 to M
G1
S
G2
M
Similarities between prokaryotic
and eukaryotic replication
•
•
•
•
•
More similarities than differences
Bi-directional process
DNA polymerases work 5’ to 3’
Leading and lagging strands
Primers are required
Differences in Replication
• Linear chromosomes  Ends or telomeres
• More genetic material; eukaryotic cells have
on average 50 times more genetic material
• More packaging, nucleosomes and
nuclear scaffolds
• Different enzymes; they are slower!
Eukaryotic replicating Enzymes
• Despite the large increase in genetic
material eukaryotic DNA polymerases
work much slower NOT faster!!
• At the rate they work it would take 30 days
to copy the human genome if it was left to
2 replication forks!
• The unpacking and repacking with
histones could account for the slow pace.
Eukaryotic replicating Enzymes
• The average E. coli replication fork works
around the chromosome at a staggering
105 bases per minute.
• Our eukaryotic counterpart can only
manage somewhere between 500 and
5 000 bases per minute.
• The major enzymes are DNA polymerase
a, d and e.
Eukaryotic replicating Enzymes
• To get around these slack work habits we
have multiple initiation sites.
• These sites are scattered around the
genome 30 to 300 kb apart. Humans have
some 20,000 separate initiation sites.
• The whole replication process does not
happen simultaneously.
Eukaryotic replicating Enzymes
• Clusters of 20 to 80 sites are initiated at a
time
• Forks extend in both directions from each
site.
• Replication takes place throughout S
phase and takes several hours.
Initiating replication
• Each origin of replication must have a
protein complex bound to it; imaginatively
named the Origin Recognition Complex
(ORC).
• This complex will remain on the DNA
throughout replication.
Initiating replication
• Other protein factors will then bind to the
ORC and recruit proteins which coat the
DNA.
• This process is essential for replication.
• These accessory proteins or licensing
factors accumulate during G1 of the cell
cycle.
Initiating replication
• The initiation event must be licensed or
allowed.
• It must also be prevented from re-initiating
the process until that round of cell division
has finished.
• Licensing factors have a role in allowing
the initiation and preventing re-initiation.
The start!
The parent DNA about to embark on replication
Multiple initiation sites form along the DNA, ORC
+other licensing factors.
The human genome will have some 20,000 such sites
often activated in clusters. These multiple origins of
replication separated by only 30-300 kb and clustered in
groups of 20-80 in various regions of the DNA.
The ends!
The parent DNA about to embark on replication
Let’s focus on the end
working from the last
initiation site.
The ends!
5’
3’
5’
3’
RNA primer
Newly synthesised DNA
strand, the arrow head is the
3’ OH
The ends!
5’
3’
5’
3’
RNA primer removed
Newly synthesised DNA
strand, the arrow head is the
3’ OH
The ends!
5’
3’
3’
3’
3’
No 3’ end to fill in from
Gaps filled in from the 3’ end
Newly synthesised DNA
strand, the arrow head is the
3’ OH
5’
The ends!
5’
3’
5’
3’
Known as the
end problem
RNA primers removed
Newly synthesised DNA
strand, the arrow head is the
3’ OH
The ends!
5’
3’
3’
5’
Overhangs at the 3’ end of the parent strand
5’
3’
3’
5’
Newly synthesised
DNA strand
Parent
DNA
strand
The ends!
5’
3’
3’
TTAGGG
AAUCCCAAU
5’
3’
5’
Telomerase, containing an RNA component.
This enzyme was discovered by an
Australian scientist, Elizabeth Blackburn.
The ends!
5’
3’
3’
TTAGGGTTA
AAUCCCAAU
5’
3’
5’
The ends!
5’
3’
3’
TTAGGGTTA
AAUCCCAAU
5’
3’
5’
The ends!
5’
3’
3’
TTAGGGTTAGGGTTA
AAUCCCAAU
5’
3’
5’
The ends!
TTAGGGTTAGGGTTA
AAUCCCAAU
5’
The ends!
TTAGGGTTAGGGTTAGGGTTA
AAUCCCAAU
5’
The ends!
TTAGGGTTAGGGTTAGGGTTA 3’
5’
The 5’ strand then extends by lagging strand
mechanisms. We still have an overhang on the 3’
end which often tucks in and caps the end.
Special capping proteins bind to the ends to
protect the ends from nucleases.
Telomerases
• Telomerase activity is high in germ-line
cells; the zygote starts with full length
telomeres.
• Somatic cells do not usually have any
telomerase activity
• Apart from germ-line cells, the highly
proliferative stem cells are the only other
normal cells to have high telomerase
activity
Telomerases
If most somatic cells do not have telomerase
activity then:
• Every time the somatic cell undergoes
mitosis the telomere shortens
• Eventually the telomere gets too short and
the cell is in danger of eroding coding
genes
• This is known as the Hayflick limit
Telomerases
• Human cells start with ~10,000 base pairs
on their telomeres at birth.
• This enables some cells to survive for an
entire life time of replication erosion
without suffering cell death.
• Telomeres could be a limiting factor in
determining an organism’s life span.
Telomerases and Immortality
• This explains why normal eukaryotic cells only
undergo a certain number of cell divisions in
culture before they die.
• Immortal cells however, such as cancer cells
continue to divide
• Whereas normal cells typically don’t have
telomerase activity, cancer cells invariably do!
They are selected for it! Otherwise they would die
out very quickly with the rapid cell division.
Immortal cells.
• The most famous immortal cells would
have to be HeLa cells.
• HeLa cells are derived from Henrietta
Lacks who died in 1951 of cervical
cancer.
• Her cervical cells have survived her by
~50 years.
• They continue to be used as the model
cell line.
Telomeres and aging
• This programmed number of cell divisions
means our normal cells progress to
terminal differentiation and turn over.
• This shortening of the telomeres is thought
to be responsible in the end for the
organism’s aging.
• Aging may be a protective mechanism
against uncontrolled proliferation or
cancer.
Telomeres and aging
• When they cloned Dolly the sheep (1996 –
2003) they inserted old DNA into Dolly’s
nucleus.
• The process involved removing the
genome in the zygote (fertilised egg) and
replacing it with a different genome.
• Dolly literally was a clone of the genome
donor.
Dolly the sheep
Dolly the sheep
1996: Birth
Birth of Bonnie, Dolly’s lamb
Dolly’s death 2003
Sheep have a life expectancy of 10 to 20 years,
averaging 12 years. Poor Dolly lasted 6 years.
Telomeres and aging
• Why did Dolly die early? Because the DNA
inserted had shorter telomeres and was
already well on the way to getting old.
• She was euthanased in 2003 at 6 suffering
from a lung infection and arthritis;
characteristics of older sheep.
• Likewise sufferers of Progeria, the aging
disease are born with short telomeres.
Progeria
• Progeria is the premature aging disease
• It is described as a sporadic autosomal
dominant mutation
• There are 2 types; Werners syndrome
(adult-onset progeria, average life span 47
years) and Hutchinson-Gilford progeria
(juvenile-onset progeria, average life span
13 years)
Progeria
• Hutchinson-Gilford progeria is the result of
a T  C substitution in the gene LMNA
• This gene codes for a protein lamin A
which is normally found in the nucleus.
• The mutation causes a splice problem 
defective laminA (50 aa shorter) and the
mutant protein (progerin) stays attached to
the nuclear membrane
Progeria
• From the point of view of telomerases….
• Some of the cells from Hutchinson-Gilford
patients are prone to early cell senescence.
• “Hutchinson-Gilford children show what appears to be
early aging of their skin, bones, joints, and
cardiovascular system, but not of their immune or central
nervous systems.”
• Skin fibroblasts from Hutchinson-Gilford patients
have shorter than normal telomeres and
consequently undergo early cell senescence.
Progeria
• At birth, the mean telomere length of these
children in certain cells is equivalent to
that of a normal eighty-five-year-old.
• Introduction of telomerase to these cells in
culture restores their telomere length and
immortalises them in culture.
Progeria
• To quote the web “Clinical interventional
studies using this strategy in humans are
pending”.
• Other cells unaffected by the disease have
normal length telomeres e.g. lymphocytes.
• They die of cardiovascular disease or
stroke BUT they do not suffer dementia or
suffer from increased infections.
This five year old boy has Hutchinson-Gilford progeria, a fatal, "premature
aging" disease in which children die of heart failure or stroke at an average age
of thirteen. Photo courtesy of The Progeria Research Foundation, Inc. and the
Barnett Family.
Telomerases as drug targets
Telomerase is active in between 80 and 90%
of all cancers
• Targeting the RNA component with
antisense oligodeoxynucleotides and
RNaseH
• Reverse transcriptase inhibitors; AZT
• Inhibitors of the catalytic protein subunit
• G-quadruplex stabilisers..the 3’ overhang is
G rich and tetra-stranded DNA is formed.