Eukaryotic DNA Replication
Chromosomes
are densely
packed in
mitosis
The accuracy of DNA replication is seen
in the quality of the product
Fertilised Egg
Product
Characteristics of ARSs
• Budding yeast replication origins map within such ARS elements on both
chromosomal and plasmid DNA.
• ARS elements comprise a short 11 bp A element or ‘ARS consensus
sequence’: 5’-(A/T)TTTA(T/C)(A/G)TTT(A/T)-3’,
plus flanking regions of 100 - 200 bp (‘B’ elements) that enhance origin function.
B3
B2
B1
ACS
Which proteins bind to and define
eukaryotic replication origins?
Replication origins in metazoans (somatic cells)
The structure of replication origins in higher eukaryotes
is unclear.
•
• Small extrachromosomal DNA sequences replicate
poorly, even when carrying >10 kb genomic DNA known
to act as origins when in the chromosome.
• Replication initiates at specific regions at a
characteristic time in S phase. Both place and timing
may change with cell type.
• Replication forks can potentially initiate at a number of
different sites throughout an “initiation zone” that may
extend over >10 kb.
The ‘Origin Number’ Paradox
E. coli:
Genome, 4 Mb = 4 x 106 bp
Fork rate approx. 800 bp / sec
Replication time approx. 40 minutes = 2,400 secs
Amount replicated by 2 forks in 40 mins = 2 x 2400 x 800 = 3,840,000 bp (~4 Mb)
Eukaryotes
Genome 20 Mb (yeast) up to 6,000 Mb (human)
Fork rate 10 bp / sec (frog) - 50 bp / sec (mammal)
Amount replicated by 2 forks in 8 hr (human cells) = 2 x 50 x 28,800 = 2,880,000
(~ 3 Mb, a 2,000-fold deficit)
46 chromosomes (human cells) - with one origin per chromosome, at least 92
replication forks gives approx. 140 Mb replicated in 8 hours (still a 40-fold deficit)
The solution:
- eukaryotes replicate their chromosomes from multiple replication origins
Electron micrograph showing an approx. 300 kb stretch of replicating
chromosomal DNA from the yeast S. cerevisiae. Replication forks
are indicated by an arrow. (Petes, Newlon, Byers, & Fangman 1974;
Cold Spring Harb Symp Quant Biol. 38:9-16 ).
The study of replication origins using
DNA fibre autoradiography
Protocol:
a) Pulse label proliferating cells with 3H-thymidine for 5 min (pulse I)
b) Dilute label to 1/5 activity for further 5 min (pulse II)
c) Isolate DNA and spread on a photographic plate
d) expose for 6 months
e) develop and examine grains under microscope
light labelling heavy labelling
Interpretation:
Before pulse I:
End of pulse I:
End of pulse II:
Chromosome regions replicate at different times
Protocol:
BrdU
a) Pulse cells, at different times, with
BrdU for 1 hr.
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
b) “Chase”, collect chromosomes.
c) Stain with anti-BrdU antibodies.
BrdU
BrdU
BrdU
BrdU
Duration
(hours)
G1
~8
~2 ~1
BrdU
S
G2 M
2 hr
BrdU
5 hr
9 hr
= BrdU pulse
= chase
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
late S
2 hr chase
mid S
5 hr chase
early S
9 hr chase
Organization of replication during S phase
Typical somatic cell
template DNA
early-firing origins
late-firing origins
duplicated DNA
The global pattern of origin usage can also change: eg early embryonic
versus somatic cells:
Drosophila somatic cell (transcriptionally active)
S phase = 10 hours (600 mins); mean origin spacing = >40kb
Early Drosophila embryo (transcriptionally quiescent)
S phase = 3.4 mins; mean origin spacing = 7.9kb
Early Drosophila embryo
near-synchronous
initiation
Why so many origins?
To allow sections of the genome to replicate faster?
To allow different sections of the genome to replicate at different times?
To prevent problems if origins do not initiate with 100% probability?
Excess origins are used to lower the probability of a lethal ‘double stall’?
stalled fork
replication completed by
other fork of pair
double stall: no way of replicating intervening DNA
Facts I
• Rate of progression of replication forks is fairly constant for a given organism
• Forks generally stop only when they encounter an oppositely moving fork
• Chromosome replication is regulated mainly through control of the initiation of
new replication forks
For example:-by regulating the number and spacing of origins that fire eg. during
development
-by regulating the time during S phase at which different origins are activated
Facts II
• In somatic mammalian cells, most inter-origin distances (replicon sizes) are
between 30 - 300 kb (ie would take 5 - 50 min to replicate completely).
• Some adjacent origins (“origin clusters”, typically 2 - 5 origins) initiate
synchronously
• Different origins / origin clusters initiate at different times during S phase
Typical mammalian cell replicates 6,000 Mb in 8 hr = 6 x 109 ÷ 28,800
bp/sec ie. ~200,000 bp/sec
For fork rate of 50 bp / sec = 200,000 ÷ 50
~ 4,000 forks active at any given time in S phase
Restoration of chromatin after
replication
The principle chromatin
assembly reactions during
DNA replication. Reaction
(a): parental nucleosomes
are partially disrupted during
DNA replication and the
histones are directly
transferred to the replicated
DNA, reassembling into
nucleosomes. Reaction (b):
the assembly of new
nucleosomes from newly
synthesized and soluble
histones is mediated by a
chromatin assembly factor
Initiation of SV40
replication
SV40 T antigen binds and
distorts the viral origin.
RP-A (‘replication protein
A’) binds to the singlestranded DNA.
DNA polymerase a -primase
puts down an RNA primer and
extends it with DNA.
RF-C displaces pol aprimase and loads PCNA to
establish the leading strand.
Trykkfeil: Cdt1, ikke Ctd1
Mcm2-7
Mcm2-7 (mini-chromosome maintenance) proteins were originally
identified in yeast because as mutants affecting replication origin usage.
Fractionation showed them to be a key component of Licensing Factor.
Highly conserved throughout eukaryotes; archaea also possess an
Mcm2-7 homologue
They are loaded onto DNA in anaphase and are removed from
chromatin during S phase.
They form a hexameric ring, capable of encircling double-stranded DNA.
Recap
 Whether a cell has only one chromosome (as in prokaryotes) or has
many chromosomes (as in eukaryotes), the entire genome must be
replicated precisely once of every cell division.
 Two general principles: (1) Initiation of DNA replication commits the
cell to a further division. Replication is controlled at the stage of
initiation. Once replication has started, it continues until the entire
genome has been duplicated. (2) If replication proceeds, the
consequent division cannot be permitted to occur until the
replication event has been completed.
Replicon
the unit of DNA in which an individual act of replication occurs.
Origin
site at which replication is initiated.
Terminus
site at which replication stops.
A genome in a prokaryotic cell constitutes a single
replicon; thus the units of replication and segregation
coincide.
A plasmid is an autonomous circular DNA genome that
constitutes a separate replicon; may show single copy
control or under multicopy control. Any DNA molecule
that contains an origin can be replicated autonomously in
the cell.
Each eukaryotic chromosome contains a large number of
replicons; each must be activated no more than once in
each cell cycle.
The DNA of mitochondria and chloroplasts may be
regulated more like plasmids that exist in multiple copies
per bacterium.
Replicons Can Be Linear or Circular
Key Concepts
A replicated region appears as an eye within nonreplicated
DNA.
A replication fork is initiated at the origin and then moves
sequentially along DNA.
Replication is unidirectional when a single replication fork is
created at an origin.
Replication is bidirectional when an origin creates two
replication forks that move in opposite directions.
Figure 15.1. Replicated DNA is seen as a replication eye
flanked by nonreplicated DNA.
Figure 15.2. Replicons may be
unidirectional or bidirectional,
depending on whether one or
two replication forks are formed
at the origin.
Figure 15.3. A replicatin eye forms a θ structure in circular DNA.
Origins Can Be Mapped by Autoradiography and Electrophoresis
Key Concepts
 Replication fork movement can be detected by autoradiography using
radioactive pulses.
 Replication forks create Y-shaped structures that change the
electrophoretic migration of DNA fragments.
Figure 15.5. Different densities of radioactive labeling can be used to
distinguish unidirectional and bidirectional replication.
Figure 15.6. The position of the origin and the number of replicating forks
determine the shape of a replicating restriction fragment, which can be
followed by its electrophoretic path (solid line). The dashed line shows
the path for a linear DNA.
15.4 Does Methylation at the Origin Regulate Initiation?
Key Concepts
 oriC contains eleven GATC/CTAG repeats that are methylated on
adenine on both strands.
 Replication generates hemimethylated DNA, which cannot initiate
replication.
 There is a 13-minute delay before the GATC/CTAG repeats are
remethylated.
 What feature of a bacterial (or plasmid) origin ensures that it is
used to initiate replication only once per cycle?
 Some sequences that are used for this purpose are included in the
origin. oriC contains eleven copies of the sequence GATC/CTAG,
which is a target for methylation at the N6 position of adenine by
the Dam methylase (Figure 15.7).
 If the plasmid is methylated it undergoes a single round of
replication, and then the hemimethylated products accumulate
(Figure 15.8). Hemimethylated origins cannot initiate again until the
Dam methylase has converted them into fully methylated origins.
Figure 15.7. Replication of methylated DNA gives hemimethylated DNA,
which maintains its state at GATC sites until the Dam methylase restores
the fully methylated condition.
Figure 15.8. Only fully methylated origins can initiate replication;
hemimethylated daughter origins cannot be used again until they have
been restored to the fully methylated state.
Origins May Be Sequestered after Replication
Key Concepts
 SeqA binds to hemimethylated DNA and is required for delaying
rereplication.
 SeqA may interact with DnaA.
 As the origins are hemimethylated they bind to the cell membrane
and may be unavailable to methylases.
 The nature of the connection between the origin and the membrane
is still unclear.
Figure 15.9. A membrane-bound inhibitor binds to hemimethylated DNA
at the origin and may function by preventing the binding of DnaA. It is
released when the DNA is remethylated.
Each Eukaryotic Chromosome Contains Many Replicons
Key Concepts
 Eukaryotic replicons are 40 to 100 kb in length.
 A chromosome is divided into many replicons.
 Individual replicons are activated at characteristic times during S
phase.
 Regional activation patterns suggest that replicons near one another
are activated at the same time.
 S phase usually lasts a few hours in a higher eukaryotic cell.
 Individual replicons in eukaryotic genomes are relatively small,
typically ~40 kb in yeast or fly and ~ 100 kb in animal cells. The rate
of replication is ~ 2000 bp/min, which is much slower than the
50,000 bp/min of bacterial replication fork movement.
 A mammalian genome could be replicated in ~1 hour if all replicons
functioned simultaneously. S phase actually lasts for >6 hours in a
typical somatic cell, implying that no more than 15% of the
replicons are likely to be active at any given moment.
 Visualization of replicating forks by labeling with DNA precursors
identifies 100 to 300 “foci” instead of uniform staining; each focus
shown in Figure 15.11 probably contains >300 replication forks.
Figure 15.11. Replication forks are organized into foci in the nucleus.
Cells were labeled with BrdU. The leftmost panel was stained with
propidium iodide to identify bulk DNA. The right panel was stained using
an antibody to BrdU to identify replicating DNA.
15.7 Replication Origins Can Be Isolated in Yeast
Key Concepts
Origins in S. cerevisiae are short A-T-rich sequences that
have an essential 11-bp sequence.
The ORC is a complex of six proteins that binds to an ARS.
 Any segment of DNA that has an origin should be able to replicate,
so although plasmids are rare in eukaryotes, it may be possible to
construct them by suitable manipulation in vivo. This has been
accomplished in yeast, although not in higher eukaryotes.
 The discovery of yeast origins resulted from the observation that
some yeast DNA fragments (when circularized) are able to
transform defective cells very efficiently. These fragments can
survive in the cell in the unintegrated (autonomous) state, that is,
as self-replicating plasmids.
 This segment is called as ARS (for autonomously replicating
sequence). ARS elements are derived from origins of replication.
 An ARS element consists of an A-T-rich region.
 Figure 15.12: shows a systematic mutational analysis along the
length of an origin.
 Origin function is abolished completely by mutations in a 14-bp
“core” region, called the A domain, which contains an 11-bp
consensus sequence consisting of A-T base pairs.
 This consensus sequence (called ACS for ARS Consensus Sequence)
is the only homology between known ARS elements.
 Mutations in three adjacent elements, numbered B1 to B3, reduce
origin function. An origin can function effectively with any two of
the B elements, so long as a functional A element is present.
 The ORC (origin recognition complex) is a complex of six proteins
with a mass of ~400 kD. ORC binds to the A and B1 elements.
 There are about 400 origins in the yeast genome, meaning that the
average length of a replicon is ~ 35,000 bp.
Figure 15.12. An ARS extends for ~50 bp and includes a consensus
sequence (A) and additional elements (B1-B3).
Licensing Factor Controls Eukaryotic Replication
Key Concepts
Licensing factor is necessary for initiation of replication at
each origin.
It is present in the nucleus prior to replication, but is
inactivated or destroyed by replication.
Initiation of another replication cycle becomes possible only
after licensing factor reenters the nucleus after mitosis.
 A eukaryotic genome is divided into multiple replicons, and the
origin in each replicon is activated once and only once in a single
division cycle.
 Figure 15.13: >1 replication cycle needs cytoplasmic factors.
 Figure 15.14: explains the control of reinitiation by proposing that
this protein is a licensing factor.
 It is present in the nucleus prior to replication. One round of
replication either inactivates or destroys the factor, and another
round cannot occur until further factor is provided. Factor in the
cytoplasm can gain access to the nuclear material only at the
subsequent mitosis when the nuclear envelope breaks down.
Figure 15.13. A nucleus injected into
a Xenopus egg can replicate only once
unless the nuclear membrane is
permeabilized to allow subsequent
replication cycles.
Figure 15.14. Licensing factor in the
nucleus is inactivated after replication.
A new supply of licensing factor can
enter only when the nuclear
membrane breaks down at mitosis.
Licensing Factor Consists of MCM Proteins
Key Concepts
The ORC is a protein complex that is associated with yeast
origins throughout the cell cycle.
Cdc6 protein is an unstable protein that is synthesized only
in G1.
Cdc6 binds to ORC and allows MCM proteins to bind.
When replication is initiated, Cdc6 and MCM proteins are
displaced. The degradation of Cdc6 prevents reinitiation.
Some MCM proteins are in the nucleus throughout the
cycle, but others may enter only after mitosis.
 The key event in controlling replication is the behavior of the ORC
complex at the origin. The origin (ARS) consists of the A consensus
sequence and three B elements. The ORC complex of six proteins
binds to the A and adjacent B1 element. The transcription factor
ABF1 binds to the B3 element; this assists initiation.
 Most origins are localized in regions between genes.
 Figure 15.15; summarizes the cycle of events at the origin.
 In yeast, Cdc6 is a highly unstable protein, with a half-life of <5
minutes. It is synthesized during G1 and typically binds to the ORC
between the exit from mitosis and late G1.
 In yeast the presence of Cdc6 at the origin allows MCM (minichromosome maintenance) proteins to bind to the complex. The
origin therefore enters S phase in the condition of a prereplication
complex, which contains ORC, Cdc6, and MCM proteins. When
initiation occurs, Cdc6 and MCM are displaced, returning the origin
to the state of the postreplication complex, which contains only
ORC.
Figure 15.15. Proteins at the
origin control susceptibility to
initiation.
D Loops Maintain Mitochondrial Origins
Key Concepts
Mitochondria use different origin sequences to initiate
replication of each DNA strand.
Replication of the H strand is initiated in a D loop.
Replication of the L strand is initiated when its origin is
exposed by the movement of the first replication fork.
 Initiation requires separating the DNA strands and commencing
bidirectional DNA synthesis. A different type of arrangement is
found in mitochondria.
 Replication starts at a specific origin in the circular duplex DNA.
Initially, though, only one of the two parental strands (the H strand
in mammalian mitochondrial DNA) is used as a template for
synthesis of a new strand. Synthesis proceeds for only a short
distance, displacing the original partner (L) strand, which remains
single-stranded, as illustrated in Figure 15.16. The condition of this
region gives rise to its name as the displacement loop, or D loop.
Figure 15.16. The D loop
maintains an opening in
mammalian mitochondrial DNA,
which has separate origins for
the replication of each strand.
The existence of D loops exposes a general principle: An
origin can be a sequence of DNA that serves to initiate
DNA synthesis using one strand as template.
The opening of the duplex does not necessarily lead to
the initiation of replication on the other strand. In the
case of mitochondrial DNA replication, the origins for
replicating the complementary strands lie at different
locations.
Origins that sponsor replication of only one strand are
also found in the rolling circle mode of replication.
A eukaryotic DNA replication fork
Teleomeres
What is a telomere?
• 5-8 bp G-rich tandem repeats
• Repetitive noncoding DNA
http://www.phoenixbiomolecular.com/regenerative_medicine.html
Why do we have them?
• Replication problem
– Lagging strand synthesis
• Unable to replicate the 3’
ends faithfully
• Loose chromosomal DNA
Evolutionary development of telomere
http://www.uic.edu/classes/bios/bios100/lecturesf04am/ReplicationFork.gif
Telomeres
Ends of linear chromosomes
Centromere
Telomere
Telomere
Repetitive DNA sequence
(TTAGGG in vertebrates)
Specialized proteins
Form a 'capped' end structure
Telomeres 'cap' chromosome ends
TELOMERE STRUCTURE
5’
3’
Telomeric
t loop
5'
3'
NUCLEAR
MATRIX
Telomeric
proteins:
TRF1
TRF2
TIN2
RAP1
TANKS 1,2
POT1
etc
Why are telomeres important?
Prevent chromosome fusions by NHEJ (non-homologous end joining)
NHEJ
FUSION
BRIDGE
Mitosis
BREAKAGE
Fusion-bridge-breakage cycles
Genomic instability
Cell death OR neoplastic transformation
The End Replication Problem:
Telomeres shorten with each S phase
5'
3'
3'
5'
5'
3'
5'
3'
5'
DNA replication is bidirectional
Polymerases move 5' to 3'
Requires a labile primer
OriEach round of DNA
replication leaves
50-200 bp DNA unreplicated
at the 3' end
Telomere: the end of eukaryotic
chromosome consisting of short tandem
repeats, actively extending the ends of
chromosomes
Synthesis of telomeric DNA.
Telomerase extends chromosomes
Telomerase
1) An enzyme to add repeating sequence of nucleotides to the 3’ end of a DNA
strand
2) consists of two molecules
a) each of human telomerase reverse transcriptase (TERT): 1131 amino acids
b) Telomerase RNA (hTR or TERC): RNA (template for DNA synthesis)
3)
Template: an enzyme-associated RNA (3'-CAAUCCCAAUC-5'., human, upto 450
bases)
4) Telomere: Repeated sequence of TTAGGG upto 5-15 kb
Telomerase
http://www.phoenixbiomolecular.com/regenerative_medicine.html
Is Telomerase activity linked to cell immortality?
Cell proliferation potential lower from older donors
•Cells from older donors have “used up” some of doublings
Cell proliferation potential greater in longlived species
Organism + Life Span:
-mouse about 3 years
-human about 100
-Galapagos tortoise about 150
Hayflick Limit:
-doublings about 20
-doublings about 40-60
-doublings about 140
Is Telomerase activity linked to cell immortality?
Telomerase: only in germ, stem and cancer (immortal) cells, but not
in normal cells
Telomere Length (humans,
kb)
Telomere Length and Cell Division Potential
20
10
Germ Cells (Telomerase Positive)
Normal
Somatic
Cells
(Telomerase
Negative)
+ Telomerase
Cellular (Replicative) Senescence
Number of Doublings
Cellular senescence
• Once the telomere shrinks to a certain extent,
the cell stops dividing.
– ~4kb in human cells triggers end to cell division.
• This leads to other changes called cellular
senescence:
– Cell morphology changes.
– Gene expression changes.
Yeast replicative lifespan regulated by telomere length
• Telomerase mutants have a short lifespan: when telomeres shorten to a critical
point, yeast cells stop dividing.
• Overexpression of telomerase:
• Longer telomeres.
• Increased replicative lifespan.
Telomeres in mice
• Lab strains of mice have very long telomeres.
• 30-40kb telomeres.
• Tert knock-out mice:
• Normal for four generations as their telomeres shorten,
• Premature aging phenotypes present in the 5th generation
Telomerase:
Biomedical uses
Expand cells for replacement therapies
(burns, joint replacements, etc)
Telomerase inhibitors to selectively kill cancer cells
The telomere hypothesis of aging
Telomeres shorten with each cell division
and therefore with age
TRUE
Short telomeres cause cell senescence and
senescent cells may contribute to aging
TRUE
HYPOTHESIS:
Telomere shortening causes aging and
telomerase will prevent aging
TRUE OR FALSE?
The telomere hypothesis of aging
Telomere length is not related to life span
(mice vs human; M musculus vs M spretus)
Telomeres contribute to aging ONLY if
senescent cells contribute to aging
Telomerase protects against replicative
senescence but not senescence induce by
other causes
Telomeres and Cellular Aging
• Cells removed from a newborn infant and placed in culture
will go on to divide almost 100 times. Well before the end,
however, their rate of mitosis declines (to less than once
every two weeks).
• Cells from an 80 year old would manage only a couple of
dozen mitoses before they ceased dividing and died out.
• Telomeres are important so their steady shrinking with
each mitosis might impose a finite life span on cells.
• This phenomenon is called replicative senescence
• Could shrinkage of telomeres be a clock that determines
the longevity of a cell lineage and thus is responsible for
replicative senescence?