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DNA replication
Three models of DNA replication.
1
Most DNA replication is bidirectional
Starts at the
origin -defined
sequence of
base pairs
Each region
served by one
DNA origin is
called a replicon
Some linear
DNA viruses
Certain plasmids
Most
common for
eukaryotes
and
prokaryotes
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Bidirectional DNA replication
Bidirectional DNA replication in eukaryotes
3
Such studies have
revealed clusters
of active replicons
Replicating mammalian cells were exposed first to high then to
low concentration of H3Thymidine – DENA will be heavily
labeled near the origin and lightly later.
Most DNA replication is bidirectional
Prokaryotic chromosomes have a
single origin of replication with
two replication forks
Much larger eukaryotic
chromosomes have many
origins of replication
Each region served by one DNA
origin is called replicon.
First evidence of bidirectional
fork growth came from fiber
autoradiography of labeled
DNA molecules from
mammalian cultured cells. Such
studies revealed clusters of
active replicons, each of which
contain 2 growing forks moving
away from a central origin.
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Number of growing forks and their rate of movement
In E. coli cells
it takes 42 minutes to replicate the single circular chromosome
that has 4 639 221 bp and is about 4.1mm in length.
Since the chromosome is duplicated from one origin by two
growing forks, we can calculate that the rate of the fork movement
is about 1000bp/second/fork.
Number of growing forks and their rate of movement
The rate of fork movement in human cells, based on fiberlabeling experiments, is only about 100bp/second/fork.
The entire human genome of 3 x 109 bp replicates in about 8
hours, suggesting that human genome might have about 1000
forks. However, fiber autoradiography and electron microscopy
indicate that growing forks are spaced closer than 3 x 106 apart.
A most likely estimate is that human genome contains 10 000100 000 replicons, each of which is actively replicating for only
part of the 8 hours required for replication of the entire genome.
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Replication bubbles
DNA Replication Origin
Replication origin = site on the DNA double helix where replication
is initiated.
•site where the double helix first opens ---> replication bubble.
•consist of specific nucleotide sequences recognized by initiator
proteins.
oA-T rich (easier to separate)
o100 bp (base pairs) in length
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Number of replication origins
•Prokaryotes
o1 replication origin per chromosome
oreplication rate = 500 nucleotides per sec.
•Eukaryotes
omultiple replication sites on each chromosome.
oreplication rate = 50 nucleotides per sec.
In eukaryotes replication origins are activated in clusters of 20
to 80 adjacent origins = replication units.
The pattern of replication is controlled, temporally and
spatially.
DNA Replication Origin of E.coli
E. coli replication origin oriC is an ≈240bp DNA segment present at the start
site for the replication of the E. coli chromosomal DNA.
Plasmids or any other circular DNAs containing oriC are capable of
independent and controlled replication in E. coli cells.
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DNA Replication Origin of E.coli
1
13
17
5’ GATCTNTT TATTT
29
GATCTNTT TATTT
CTAGANAAATAAA
3’ CTAGANAAATAAA
58
32
44
GATCTNTT TATTT
CTAGANAAATAAA
66
TGTGGATAA
ACACCTATT
166
174
TTATACACA
AATATGTGT
201
209
240
TTTGGATAA
AAACCTATT
248
TTATCCACA
AATAGGTGT
3’
5’
Consensus sequence of the minimal bacterial replication origin based on analyses of
genomes from six bacterial species
13 bp repetitive sequences are rich in A and T. The 9bp sequences exist in both
orientations. These sequences are referred as 13-mers ans 9-mers.
DNA Replication Origin of E.coli
Cell wall
Plasma membrane
origins
The origins of the replicated chromosomes have independent points of attachment to the
membrane and thus move further apart as new membrane and cell wall forms midway
along the length of the cell.
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Yeast autonomously replicating sequences
Each yeast chromosome has multiple origins of replication: about
400 origins exist on 17 chromosomes of S. cerevisiae.
Each yeast origin, called autonomously replicating sequence
(ARS), confers on a plasmid the ability to replicate in yeast and is
a required element for YACs.
Detailed mutational analysis of one ≈180 bp ARS called ARS1
revealed only one element, a 15-bp segment, designated element A,
stretching from position 114 to 128.
Three other short segments – elements B1, B2 and B3 – increase
the efficiency of ARS functioning. Comparison of the sequences
required for functioning of many different DNA segments that act
as ARSs led to recognition of an 11-bp consensus sequence:
(5’) A/T-T-T-T-A-T-A/G-T-T-T-A/T (3’)
Element A in ARS1 is identical in 10 out of 11 positions of the
consensus sequence, and element B2 – in 9 of 11.
DNA footprinting revealed that 6 different proteins called the
ORC (origin recognition complex) binds specifically to the
element A in ARS1 in an ATP-dependant manner.
This complex also binds to other ARSs. The ORC remains
bound to an ARS throughout the cell cycle and during
replication becomes associated with other proteins –this triggers
DNA synthesis.
Yeast mutants defective in any of the proteins of ORC are
defective in DNA replication.
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SV 40 origin of replication
A 65-bp region in the SV40 chromosome is sufficient to promote
DNA replication both in animal cells and in vitro.
Researchers have used mammalian proteins and plasmids
carrying the SV40 origin to study the molecular mechanisms of
DNA replication.
Common features of replication origins
Although the
specific nucleotide sequences of replication origins from E.coli,
yeast, and SV40 are very different, they share several properties:
Replication origins are unique DNA segments that contain
multiple short repeated sequences.
These short repeat units are recognized by multimeric originbinding proteins. These proteins play a key role in assembling
DNA polymerases and other replication enzymes on the sites
where replication begins.
Origin regions usually contain an AT-rich stretch. Origin-binding
proteins control initiation of DNA replication by directing the
assembly of replication machinery to specific sites on the
chromosome.
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General features of chromosomal replication
1. The general features of chromosomal replication seem to
apply with little modification to all types of cells.
2. DNA replication is semiconservative.
3. Once replication has started it continues until the entire
genome has been duplicated.
4. It starts at origin. An origin “fires” ones and only ones
during the cell cycle.
5. Replication is bidirectional.
6. At the place of the replication start (origin) helix
unwinds and creates two replicational forks.
The DNA replication machinery
DNA polymerases are unable to melt duplex DNA (I.e. break
certain hydrogen bonds) in order to separate strands that are to be
copied
All DNA polymerases so far discovered can only elongate a preexisting DNA or RNA strand, the primer; they can not initiate
chains.
The two strands in the DNA duplex are opposite (5’→3 and 3’
→5’) in chemical polarity, but all DNA polymerases catalyze
nucleotide addition at the 3’hydroxyl end of a growing chain –
only 5’→3 direction.
11
DnaA protein initiates replication in E.coli
Genetic studies suggested that initiation of replication at oriC in
E.coli is dependent upon protein coded by dnaA gene. DnaA
protein binds with oriC.
Although DnaA can bind to duplex E.coli origin DNA in the
relaxed-circle form, it can initiate replication only when the DNA is
negatively supercoiled.
Supercoiling is controlled by enzymes called topoisomerases.
Binding of DnaA to oriC 9-mers facilitates melting of duplex DNA,
which occurs at oriC 13-mers. This process requires ATP and
yields so called open complex.
12
DnaA protein initiates replication in E.coli
13
DnaB is a helicase that melts duplex DNA
Helicases constitute a class of
enzymes that can move along a
DNA duplex utilizing the energy of
ATP hydrolysis to separate the
strands.
SSB protein - binds ssDNA
Helicases exhibit directionality with
respect to unwinding reaction.
DnaB moves along the single strand
of DNA to which it binds in the
direction of it’s free 3’ end – it
unwinds DNA 5’→3’ direction.
E. coli primase catalyzes formation of RNA primers for for DNA synthesis
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E. coli primase catalyzes formation of RNA primers for for DNA synthesis
The primers used during DNA replication in eukaryotes and
prokaryotes are short RNA molecules whose synthesis is catalyzed
by the RNA polymerase primase.
Primase is usually recruited to a segment of single-stranded DNA
by first binding to DnaB hexamer already attached at that site. The
term primosome is now generally used to denote a complex
between primase and helicase, sometimes with other proteins.
In initiation of E. coli DNA replication, a primosome is formed
by binding of primases to DnaB in prepriming complex.
After bound primases synthesize short primer RNAs
complementary to both strands of duplex DNA , they dissociate
from the single stranded template.
E. coli primase catalyzes formation of RNA primers for for DNA synthesis
15
Replication, Okazaki fragments
16
Ligation reaction:
Polymerases
DNA polymerases are important enzymes involved in DNA
replication.
Three polymerases have been purified from E.coli.
In addition to important role in filling the gaps between
Okazaki fragments, DNA polymerase I is the most important
enzyme for gap filling during DNA repair.
DNA polymerase II functions in the inducible SOS response;
this polymerase fills the gap and appears to facilitate DNA
synthesis directed by damaged templates.
DNA polymerase III catalyzes chain elongation at the
growing fork of E. coli.
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DNA polymerase I
1957 – Arthur Kornberg isolated an enzyme (DNA polymerase I)
from E. coli that was able to direct DNA synthesis in vitro.
Major requirements for in vitro DNA synthesis were:
1. All four deoxyribonucleoside triphosphates (dATP, dCTP,
dGTP, dTTP = dNTP).
2. Template DNA
DNA Polymerases II and III
1969 – Peter DeLucia and John Cairns discovered a mutant strain
of E. coli that was deficient in polymerase I activity.
Observation: the mutant strain duplicated its DNA and reproduced
itself but cells are highly deficient in DNA repair (UVsensitive).
Conclusions:
1. At least one more enzyme is able to replicate E. coli DNA.
2. DNA polymerase I may serve a secondary (at least for
replication) function which is associated with DNA fidelity.
Two other unique DNA polymerases have been isolated
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Properties of Three Bacterial DNA Polymerases
Initiation of chain synthesis
5’-3’ polymerization
3’-5’ exonuclease activity
5’-3’ exonuclease activity
Molecules of polymerase/cell
Synthesis from
Intact DNA
Primed single strands
Primed single strands plus SSB
Protein
In vitro chain elongation rate
Mutation lethal?
I
+
+
+
400
II
+
+
?
III
+
+
15
+
-
-
+
600
+
?
-
+
30000
+
DNA Polymerase III Holoenzyme
 The DNA polymerase III holoenzyme is a very large (>600
kDa), highly complexed protein composed of 10 different
polypeptides. The so called core polymerase is composed
of 3 subunits.
 The α subunit contains active site for nucleoride addition,
and the ε subunit is a 3’-5’ exonuclease that removes
incorrectly added (mispaired) nucleotides at the end of
growing chain. The function of θ is still unknown.
 The central role of the remaining subunits is to convert the
Polymerase III from distributive enzyme which falls the
template after forming short stretches of 10-50 nucleotides
to processive enzyme which can form stretches of up to 5 x
105 nucleotides before being released from the template.
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DNA Polymerase III Holoenzyme
The key to the processive activity of polymerase III is β subunit that forms a donut-shaped dimer around the DNA duplex and
then associates with and holds the catalytic core polymerase
near the 3’ terminus of growing strand.
Once associated with DNA , the β subunit functions like a “clamp”
which can slide freely along the DNA as the associated core
polymerase moves. In this way active sites of core polymerase
remain near the growing fork and the processivity of the enzyme
is maximized.
DNA Polymerase III Holoenzyme
Out of the six remaining subunits 5 (γ,δ, δ1,χ and ψ) form socalled γ complex that mediates two essential tasks:
1) Loading of β subunit clamp onto the duplex DNA-primer
substrate in a reaction that requires hydrolysis of ATP;
2) unloading of β subunit clamp after a strand of DNA has been
completed. Loading and unloading of the β subunit clamp
require opening of the clamp ring, but exactly how the γ
complex does it is still unknown.
The final τ subunit acts to dimerize two core polymerases and is
essential to coordinate the synthesis of leading and lagging
strands.
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Subunits of DNA Polymerase III Holoenzyme
Subunit
Function
α
5’-3’
ε
polymerization 3’θ
5’ exonuclease
γ
??
Loads enzyme on
δ
template (Serves
δ’
as clamp loader)
χ
ψ
β
τ
Groupings
“Core” enzyme:
Elongates
polynucleotide chain
and proofreads
γ
complex
Sliding clamp structure
(Processivity Factor)
Holds together the two core
polymerases at the replication fork
Subunits of DNA Polymerase III Holoenzyme
21
Leading and lagging strands are synthesized concurrently
Synthesis of leading and
lagging strands
22
Cycling of polIII complex
Replication fork in E. coli
23
Replication in eukaryotics is very similar to that in
prokaryotic cells.
Because eukaryotic cells have more DNA they also have more
origins of replication.
A mammalian cell for example has about 1 x 109 basepairs of
DNA.
There is an origin of replication about every 30,000 basepairs of
DNA, though the structure of these sites is not clearly understood.
The DNA synthesis is also much slower than in prokaryotic cells
because of the chromatin proteins, synthesis is about 100
nucleotides per second.
Mammalian DNA polymerases
Much less is known about mammalian proteins involved in DNA
replication.
It had been thought that polymerase α synthesizes the lagging
strand because of its low processivity.
Polymerase δ is much more processive than α, as it is associated
with the PCNA clamp.
PCNA is enriched in proliferating cells and enhances the
processivity of pol δ about 40 times.
Pol β is not processive at all – it can do just 1 nucleotide – fits to
its repair enzyme role.
Pol γ if found only in mitochondria.
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Probable roles of eukaryotic polymerases
Polymerase α - priming replication on both strands
Polymerase δ - elongation of both strands
Polymerase β - DNA repair
Polymerase ε - DNA repair
Polymerase γ - replication of mitochodrial DNA
Properties of mammalian DNA polymerases
Mammalian polymerases
5’-3’ polymerization
3’-5’ exonuclease proofreading
activity
Synthesis from
RNA primer
DNA primer
Associated DNA primase
Sensitive to aphidicolin (inhibitor of
cell DNA synthesis)
Cell location
Nuclei
Mitochondria
α
+
-
β
+
-
γ
+
+
δ
+
+
ε
+
+
+
+
+
+
-
+
-
+
+
+
+
+
+
-
+
-
+
+
-
+
-
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SV40 DNA can replicate in mammalian cells.
Replication is initiated by binding of a virus-encoded protein
called T-antigen to the SV 40 origin of replication. This
multifunctional complex binding melts DNA through its helicase
activity. Opening of the duplex at the SV40 origin also requires
ATP and replication protein A (RPA), a host cell single stranded
binding protein, with a function similar to that of SSB of E.coli.
One molecule of polimerase α (Pol α) tightly associates with
primase, then binds to each unwound template strand.
Eukaryotic replication machinery is generally similar to that of E. coli
The primases form RNA primers, which are elongated for a
short stretch by Pol α, forming first leading strands, which grow
from the origins in to different directions.
The activity of Pol α is stimulated by replication factor C (RFC).
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PCNA (proliferating cells nuclear antigen) then binds to the
primer-template 3’ termini, displacing Pol α from both leading
strand templates and thus interrupting leading strand synthesis.
Next Pol δ binds to PCNA at the 3’ ends of the growing strands.
The association of Pol δ with PCNA increases the processivity of
the polymerase so that it can continue the synthesis of the leading
strand without interruption.
PCNA and DNA
PCNA is a trimer that forms a “ clamp” around duplex DNA.
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Eukaryotic replication machinery is generally similar to that of E. coli
Thus the function of PCNA is highly analogous to that of the β
subunit clamp of the E.coli polymerase III, as both proteins form
rings around DNA. But, the amino acid sequences of them are
different and β subunit clamp is a dimer and PCNA is a trimer.
As melting of the duplex DNA, catalyzed by a hexameric form
of Tag progresses further away from the origin, the primase-Pol α
complex associates with melted template downstream from
leading-strand primers.
Synthesis of the lagging strand is then carried out by combined
action primase and Pol α, along with RFC, Pol δ and PCNA
while leading strand synthesis on the other side of the origin also
proceeds.
Finally, in eukaryotes as in E. coli topoisomerases play an
important role in relieving torsional stress induced by growing
fork movement and separating the strands.
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A model for eukaryotic
chromosome replication
•Unwinding at origin of
replication
•DNA pol α-prim
initiates DNA synthesis
•PCNA, RF-C, pol δ, ε
bind
•Polymerase switch on
lagging strand
Termination of DNA Replication
Several steps are involved in the termination of DNA replication:
1) Removal of RNA primers by DNA polymerase
•When DNA polymerase encounters an RNA primer in its path,
its proofreading mechanism recognizes that it is not DNA-DNA
duplex and:
oremoves the RNA primer, one ribonucleotide at a time
(exonuclease activity)
oinserts a deoxyribonucleotide, complementary to the base of the
template strand.
orepeats the process until all of the RNA is removed and replaced
with DNA double strand.
•This process occurs:
oon the lagging strand, at the beginning of Okazaki fragments.
oon the leading strand at the replication origin.
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2) Closing the DNA-DNA gaps
Problem: Removal of RNA primers by DNA polymerase leaves
gaps between the 5'-P end of one nucleotide and the 3'-OH end of
another.
Therefore: There is no high energy phosphate bond to supply
energy to close the gap with a phosphodiester bond.
Solution: DNA ligase
Unlike bacterial chromosomes, that are circular, eukaryotic
chromosomes are linear and carry specialized ends called
telomeres.
Termination in prokaryotes
Replication has a beginning and an end.
In bacterial replication two forks approach each other in the
terminus region – which contains 22-bp terminator sites that bind
specific proteins.
In E. coli the terminator sites are called TerA-TerF (E,D,A,C,B,F).
They are the binding sites for the Tus proteins (terminus
utilization substance).
Sequences must be disentangled.
30
Termination in eukaryotes
Telomeres – ends of eukaryotic chromosomes are composed of
GC-rich sequences.
The GC-rich strand of a telomere is added at the very 3’ end of
DNA strands, in a semiconservative replication, by an enzyme
telomerase.
The exact repeat of telomere is species specific.
In vertebrates, including humans, it is TTAGGG/AATCCC
Telomerase add many repeated sequences at the ends of
chromosome.
Telomerase contains short RNA that serves as a template for
telomere synthesis.
Priming can then occur within these telomeres to make a C rich
strand
Telomeres and telomerase.
31
Forming of telomeres
Mechanism of action of telomerase
32
Literature:
Weaver, Molecular Biology, 2005.
Brown, Genomes, 1999
Lodish et al. Molecular Cell Biology 2001 and 2006.
Lewin, Genes VII.
33