DNA replication

Watson and Crick duplex structure of DNA immediately suggested how genetic
material was replicated from one generation to the next.

The realization that bacterial genomes and eukaryotic chromosomes consist of single DNA
millimeters to centimeters in length raised a series of structural and biochemical questions
about DNA replication.

Questions to be answered by the course:

How does the replication begin; how does it progress along the chromosome?
What mechanism ensure that only one round of replication occures before cell division?
Which enzymes take part in DNA synthesis, and what are their functions?

Importantly – as DNA serves as genetic link between the generations, the base sequence
must not only be copied correctly during replication, but also maintained throughout
lifespan. To keep DNA sequences accurate cells possess enzymes that catalyze DNA
repair.

DNA recombination provides a mechanism for generating new genetic diversity.

1
 Watson and Crick’s model of replication

The base-pairing principal inherent

for Watson-Crick model suggests
that the two new strands are copied
from two old strands.

…two chains unwind and separate.

Each chain then acts as a template for
the formation onto itself of a new
companion chain…
Upon the replication of double-helix
each of the two daughter molecules
will have one old strand (parental) and
one newly made strand.
Such a process is called
semiconservative replication.

Alternative models or replication

To explain the phenomenon of heredity, biological information

must be accurately copied and transmitted from each cell to all of
its progeny. Three ways for DNA molecules to replicate may be
considered, each obeying the rules of complementary base pairing.
• Conservative replication would leave intact the original DNA
molecule and generate a completely new molecule.

•Dispersive replication would produce two DNA molecules with

sections of both old and new DNA interspersed along each strand.

•Semiconservative replication would produce molecules with

both old and new DNA, but each molecule would be composed of
one old strand and one new one.

2
 Three models of DNA replication.

The Meselson-Stahl Experiment

Bacteria in Predictions
medium Conservative Semiconservative Dispersive
with 15N

Centrifuge
DNA sample
after 20 min

Second replication
Bacteria
transferred
to medium
with 14N Centrifuge
DNA sample
after 40 min
centrifugation CsCl gradient

3
 Summary of Meselson-Stahl experiment

The replication in bacteria is semiconservative

The conservative model could be ruled out after the first round of
replication, since the only one intermediate bend was present

Dispersive model was ruled out by two major observations:

• when the hybrid molecule was heat denatured after the first
round of replication, the density of the single strands corresponded
to either 15N- or 14N-profile but not an intermediate;
• the second round of replication resulted in the presence of two
bends in semiconservative (intermediate and 14N forms) and would
result in one shifted bend in dispersive model.

Semidiscontinuous replication
Both strands can not replicate continuously – as polymerase goes
only 5’ to 3’.

Leading strand- continuously; lagging strand – discontinuously.

Discontinuity comes from its direction – opposite to the
direction of fork moving.

Okazaki’s model of semidiscontinuous replication made two

predictions:
1. Because at least half of newly synthesized DNA appears in
pieces, one ought to be able to label them before them are
stitched together allowing short pulces of radioactive DNA
precursor.
2. 2. If one eliminates enzyme DNA ligase is responsible for
stitching these short pieces ought to be detectable.

4
 Experiment:

•T4 phage – simple

•Shorter and shorter pulses of H3 labeled thymidine (2 sec).
•Measured sizes of DNA that was synthesized

•Already at 2 seconds DNA was visible in the gradient – short

pieces about 1000-2000nt.

•Increasing pulse time –labeled DNA appeared much nearer to

the bottom of the tube – result of attaching the small, newly
formed pieces of labeled DNA to much larger, preformed
pieces that were made before the labeling began. They did not
show up before ligase joined small labeled pieces to them.

•Small pieces of DNA that are initial products of replication are

known as Okazaki fragments.

5
 Most DNA replication is bidirectional

Starts at the
origin -defined
sequence of base
pairs Some linear
DNA viruses
Each region
served by one
DNA origin is Certain plasmids
called a replicon

Most
common for
eukaryotes
and
prokaryotes

Bidirectional DNA replication

6
 Bidirectional DNA replication in eukaryotes

Mechanisms of the strand growth

Mechanism one: one strand derives from the origin and the other
strand derives from another origin.Only one strand of the duplex
grows at each growing point. In this mechanism, which operates in
linear DNA viruses such as adenovirus, the ends of the DNA
molecules serve as fixed sites for the initiation and termination of
replication.

Mechanism two entails one origin –one growing fork (the point
where DNA replication occurs), which moves along the DNA in
one direction with both strands of DNA being copied. Certain
bacterial plasmids replicate in such way.

7
 Mechanisms of the strand growth
A third mechanism is that synthesis might start at a single origin
and proceed both directions , so that both strands are copied at each
growing fork.
The available evidence suggests that the third alternative is most
generally used in prokaryotic and eukaryotic cells – replication
proceeds bidirectionally from given starting site with both strands
being copied at each fork.
In circular DNA molecules present in bacteria, plasmids and some
viruses one origin often suffices – two resulting growing forks
merge on the opposite site of the circle to complete replication.
However, long linear chromosomes of eukaryotes contain multiple
origins;
origins the two rowing forks from particular origin continue to
advance untill they meet advancing forks growing from
neighboring origins.

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.

8
 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.

Demonstration of bidirectional chain growth from a single origin in

viral DNA
EM - replication bubbles
The replication viral DNA from
origin EcoRI SV40-infected cells was cut by
restriction site
EcoRI, which recognizes single site
Circular viral chromosome and examined by electron
EcoRI
microscopy.
Series of ever large bubbles whose
centers maps to the same site.
Time of replication

Replication bubble
The EM pictures showed a
collection of increasingly growing
replication bubbles, the centers of
which are a constant distance from
each end of the cut molecules, thus
indicating that chain growth
occurs in two directions from a
common origin.

9
 Number of growing forks and their rate of movement

9In E. coli cells

9it takes 42 minutes to replicate the single circular chromosome
that has 4 639 221 bp and is about 4.1mm in length.

9Since 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

9The rate of fork movement in human cells, based on fiber-

labeling experiments, is only about 100bp/second/fork.

9The entire human genome of 3 x 109 bp replicates in about 8

hours, suggesting that human genome might have about 1000
forks.

9However, fiber autoradiography and electron microscopy

indicate that growing forks are spaced closer than 3 x 106 apart.

9A most likely estimate is that human genome contains 10 000-

100 000 replicons, each of which is actively replicating for only
part of the 8 hours required for replication of the entire genome.

10
 DNA replication begins at specific chromosomal sites

DNA replication as many other processes is controlled by initiation

step.

Replication of DNA begins at a defined sequence of base pairs near

the center of the replication bubbles, called replication origin.

A replication origin is a stretch of DNA that is necessary and

sufficient for replication of a circular DNA molecule, usually a
plasmid or virus, in an appropriate host cell.

In yeast this definition has been refined to include sequences that

direct replication once per S phase – the period of the cell cycle when
chromosomal duplication takes place.

Replication bubbles

11
 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

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.

12
 DNA Replication Origin of E.coli
9E. coli replication origin oriC is an ≈240bp DNA segment present at the start
site for the replication of the E. coli chromosomal DNA.
9Plasmids or any other circular DNAs containing oriC are capable of
independent and controlled replication in E. coli cells.

9Comparison of oriC with the origins of five other bacterial species including
the distant species Virbio harveyi revealed that all contain repetitive 9bp and
AT-rich 13 bp sequences, called 9-mers (dnaA boxes) and 13-mers.

9These are binding sites for DnaA protein that initiates replication.

9In addition, the E. coli genome contains a segment of DNA with a relatively
high A+T content adjacent to the oriC. This sequence appears to facilitate local
melting of DNA segments onto which the replication machinery is loaded.
9After E. coli replication has initiated, replication origins in the two daughter
DNA duplexes become linked to specific proteins on the plasma membrane. As
the cell wall divides and forms this linkage assures that one of the daughter
DNA duplexes is delivered to each daughter cell.

DNA Replication Origin of E.coli

1 13 17 29 32 44
5’ GATCTNTT TATTT GATCTNTT TATTT GATCTNTT TATTT
3’ CTAGANAAATAAA CTAGANAAATAAA CTAGANAAATAAA
58 66
TGTGGATAA
ACACCTATT

166 174 201 209 240 248

TTATACACA TTTGGATAA TTATCCACA 3’
AATATGTGT AAACCTATT AATAGGTGT 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.

13
 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.

DNA Replication Origin of E.coli, oriC and comparisons

with other origin sequences in other bacteria.

14
 Yeast autonomously replicating sequences

¾Each yeast chromosome has multiple origins of replication: about

400 origins exist
¾on 17 chromosomes of S. cerevisiae.

¾Each yeast origin, called autonomously replicating sequence

(ARS), confers on a plasmid the ability to replicate in yeast and is a
required element for YACs.

¾Detailed 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.

¾Three 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’)

¾Element A in ARS1 is identical in 10 out of 11 positions of the

consensus sequence, and element B2 – in 9 of 11.
¾DNA 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.
¾This 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.
¾Yeast mutants defective in any of the proteins of ORC are
defective in DNA replication.

15
 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:

9Replication origins are unique DNA segments that contain

multiple short repeated sequences.

9These short repeat units are recognized by multimeric origin-

binding proteins. These proteins play a key role in assembling DNA
polymerases and other replication enzymes on the sites where
replication begins.

9Origin 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.

16
 General features of chromosomal replication - conclusions

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

¾DNA polymerases are unable to melt duplex DNA (I.e. break

certain hydrogen bonds) in order to separate strands that are to be
copied
¾All DNA polymerases so far discovered can only elongate a pre-
existing DNA or RNA strand, the primer; they can not initiate
chains.
¾The 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.

17
 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.
The reason – negative supercoiles are tightly wound and are easier
to melt locally (thus providing a single-stranded template region)
than DNA molecules w/o supercoiles.

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.

18
 DnaA protein initiates replication in E.coli

DnaA binds oriC.

Genetic studies of recombinant E. coli pointed that DnaA binds oriC,

forming initial complex, and melts DNA at 9-mers and 13-mers.

19
Further melting of the two strands in E.coli chromosome to generate
unpaired template strands is mediated by the protein product of the
dnaB locus - a helicase that is essential for DNA replication.

One molecule of DnaB,

DnaB, a hexamer of identical subunits,
subunits clamps
around each of the two single strands in the open complex formed
between the DnaA and oriC.
This binding requires ATP and the protein product of the dnaC
locus.

20
The function of DnaC is to deliver DnaB to the template. One
DnaB hexamer clamps around each single strand of DNA at oriC,
forming the prepriming complex. DnaB is a helicase,
helicase and the two
molecules then proceed to unwind the DNA in opposite directions
away from the origin.

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.
DnaB, like many other proteins that act on
DNA, is processive. Because it forms the
clamp around ssDNA DnaB does not fall
off until it reaches the end of the strand or
is “unloaded” by other protein.
Other kinds of helicases unwind in
opposite direction, moving along the
strand to which they are bound toward the
free 5’ end.

21
E. coli primase catalyzes formation of RNA primers for for DNA synthesis

E. coli primase catalyzes formation of RNA primers for for DNA synthesis

Primase

Catalyzes the formation of an RNA strand, complementary and

antiparallel to a single DNA strand:

oRNA strand grows 5'--> 3'

ocomplementary to the DNA, read 3'-->5'

Process:

•Primer --> a short length of RNA-DNA duplex (about 10

nucleotides in length)

9DNA polymerase attaches to the duplex

9DNA polymerase forms a new DNA strand, starting at the 3'-end
of the RNA strand.

22
E. coli primase catalyzes formation of RNA primers for for DNA synthesis

9The primers used during DNA replication in eukaryotes and

prokaryotes are short RNA molecules whose synthesis is catalyzed
by the RNA polymerase primase.

9Primase 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.

9In initiation of E. coli DNA replication, a primosome is formed

by binding of primases to DnaB in prepriming complex.

9After 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

23
 DNA replication is
Replication, Okazaki fragments continuous on the
leading strand (1
primer); and
discontinuous on the
lagging strand – many
primers.
When newly formed
fragment approaches
the 5’ end of the other
one DNA polymerase
I takes over. It has
exonuclease activity –
removes RNA primer
and fills the gap by
adding
deoxynucleotides.
Steps in the discontinuous synthesis of the lagging strand:
strand this process
requires multiple primers, two DNA polymerases, a ligase that joins the 3’
hydroxyl end of one Okazaki fragment with the 5’ phosphate of the adjacent
fragment.

24
 Ligation reaction:

During this reaction ligase transiently attaches covalently to the 5’

phosphate on one stand, thus activating the phosphate group.
E. coli DNA ligase uses NAD+ as a cofactor, generating NMN and
AMP. Bacteriophage T4 ligase, commonly used in DNA cloning,
uses ATP, generating PPi and AMP.

Polymerases
9DNA polymerases are important enzymes involved in DNA
replication.

9Three polymerases have been purified from E.coli.

9In 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.

9DNA polymerase II functions in the inducible SOS response;

this polymerase fills the gap and appears to facilitate DNA
synthesis directed by damaged templates.

9DNA polymerase III catalyzes chain elongation at the

growing fork of E. coli.

25
 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 (UV-
sensitive).

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

26
 Role of polymerases in vivo

Polymerase I :
-removes the RNA primer;
-fills the gaps that naturally occur as primers are removed;
-has proofreading function.
Polymerase II:
-is involved in UV-damaged DNA repair;
-has proofreading function.
Polymerase III:
-is the most replication relevant polymerase;
-has proofreading function.

Properties of Three Bacterial DNA Polymerases

I II III
Initiation of chain synthesis - - -
5’-3’ polymerization + + +
3’-5’ exonuclease activity + + +
5’-3’ exonuclease activity + - -
Molecules of polymerase/cell 400 ? 15
Synthesis from
Intact DNA - - -
Primed single strands + - -
Primed single strands plus SSB
Protein + - +
In vitro chain elongation rate 600 ? 30000
Mutation lethal? + - +

27
 DNA Polymerase III Holoenzyme

9 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.

9 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.

9 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.

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.

28
 DNA Polymerase III Holoenzyme

Out of the six remaining subunits 5 (γ,δ, δ1,χ and ψ) form so-
called 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.

Subunits of DNA Polymerase III Holoenzyme

Subunit Function Groupings

α 5’-3’ polymerization “Core” enzyme:
ε 3’-5’ exonuclease Elongates polynucleotide
chain and proofreads
θ ??
γ
δ Loads enzyme on
δ’ template (Serves
γ complex
as clamp loader)
χ
ψ
β Sliding clamp structure
(Processivity Factor)
τ Holds together the two core
polymerases at the replication fork

29
 Subunits of DNA Polymerase III Holoenzyme

Space-filling model based on X-ray Schematic diagram of proposed

crystallographic studies of the
dimeric β subunit binding to DNA
duplex. Two β subunits (red and
yellow) form a donat-shapes clamp.
That remains tightly bound to a
closed circular DNA molecule bur
readily slides off.
association of the core polymerase
with the β subunit clamp at the
primer-template terminus. This
interaction keeps the core from
falling off the template and
positions is near the point of
nucleotide addition.

Leading and lagging strands are synthesized concurrently

Leading and
lagging strands are
linked together by
a τ subunit dimer.
Two molecules of
core polymerase
are bound at each
growing fork: one
at leading strand,
the other one at
lagging strand.

1) A single DnaB helicase moves along the lagging strand towards its 3’ end and
melts the duplex DNA at fork. 2) One core polymerase (core1) quickly adds
nucletides at 3’ end of the leading strand as its single-stranded template is
uncovered by the helicase action of DnaB. This leading strand polymerase,
together with its β subunit clamp remains bound to DNA, synthesizing leading
strand continuously.

30
 Leading and lagging strands are synthesized concurrently

3) Second core
polymerase (core2)
synthesise the
lagging strand
discontinuously as
an Okazaki
fragment. The two
core polymerases
are linked by a
dimeric τ protein.

4) As each segment of the ss template for the lagging strand is uncovered, it

becomes coated with the SSB protein and forms a loop. Once synthesis of an
Okazaki fragment is completed, the lagging strand polymerase dissociates form
DNA but core remains bound to the τ dimer. The released polymerase
subsequently rebinds with the assistance of the another β clamp in the region of
the other Okazaki fragment.

Leading and lagging strands are synthesized concurrently

9Two molecules of core polymerase are bound at each growing

fork: one at leading strand, the other one at lagging strand.

9The core polymerase synthesizing the leading strand moves,

together with its β subunit clamp, along its template in the direction
of movement of the fork, elongating the leading strand.

9It follows closely the movement of DnaB protein that melts the
duplex DNA of the fork.

9Since the core polymerase remains attached to the duplex DNA

the leading strand is synthesized continuously.

31
 Leading and lagging strands are synthesized concurrently

9The other core-polymerase molecule, which elongates the lagging

strand, moves with its its β subunit clamp in the direction opposite to
the fork movement.

9As elongation of the lagging strand proceeds, the size of the DNA
loop between the fork and this core polymerase increases.
Eventually core polymerase synthesizing the lagging strand will
complete an Okazaki fragment , then it dissociates from the DNA
template but the τ-subunit dimer remains to link it to the fork
proteins.

Leading and lagging strands are synthesized concurrently

9Simultaneously, primase binds to the site adjacent to the DnaB

helicase on the single-stranded segment of the lagging strand
template and initiates synthesis of another RNA primer.
9The resulting DNA primer complex attracts another β subunit
clamp to this segment of lagging strand template, followed by re-
binding of the core polymerase, which is still attached to the
complex. This polymerase then proceeds to elongate the RNA
primer into another Okazaki fragment.
As each Okazaki fragment nears completion, the RNA primer is
remover by the 5’→3’ exonuclease activity of DNA polymerase I.
9This enzyme also fills the gaps between the lagging strand
fragments, which are ligated together by DNA ligase.

32
 Leading and lagging strands are synthesized concurrently

9Although the two core polymerase molecules are linked by τ-

subunit dimer, they are oriented in opposite directions.

9Thus, the 3’ growing ends of both leading and lagging strands are
close together but offset from each other. For this reason the point
of the template from which the lagging strand is being copied is
displaced from the point in the template at which leading strand
copying is occurring.

9 Nonetheless, the two core polymerases can add

deoxyribonucleotides to the growing strands at the same time and
rate, so that leading and lagging strand synthesis occur
9s concurrently.

Leading and lagging strands are synthesized concurrently

9One τ-subunit also contacts the DnaB helicase at the fork. This
interaction strongly increases normally slow unwinding activity of
the helicase.

9 Thus, there is a physical and functional link between the two

major replication machines at the fork – the two core polymerases
and the primosome complex of DnaB and primase.

33
 Synthesis of leading and
lagging strands

Cycling of polIII complex

34
 Replication fork in E. coli

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.

35
 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 assoxiated
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 nucleotude – fits to
its repair enzyme role.
Pol γ if found only in mitochondria.

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

36
 Properties of mammalian DNA polymerases

Mammalian polymerases α β γ δ ε
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 - - + - -

Eukaryotic replication machinery is generally similar to that of E. coli

9Like DNA replication in E. coli, eukaryotic DNA replication

occurs bidirectionally from RNA primers made by a primase,
synthesis of the leading strand is continuous, while synthesis of the
lagging strand is discontinuous.

9In contrast to E. coli two distinct polymerases, α and either δ or ε,

function on the eukaryotic growing fork.

37
9SV40 DNA can replicate in mammalian cells.

9Replication 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.

9One 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

9The 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.

9The activity of Pol α is stimulated by replication factor C (RF-

C).

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9PCNA (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.

9Next 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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 PCNA and DNA

Eukaryotic replication machinery is generally similar to that of E. coli

9Thus 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.

9As 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.

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9Synthesis 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.

9Finally, in eukaryotes as in E. coli topoisomerases play an

important role in relieving torsional stress induced by growing
fork movement and separating the strands.

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

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 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.

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

9Unlike bacterial chromosomes, that are circular, eukaryotic

chromosomes are linear and carry specialized ends called
telomeres.

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 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.

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

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NOTE:

The C-rich telomere strands is synthesized by ordinary RNA-

primed DNA synthesis, like the lagging strand in conventional
DNA replication.

This mechanism ensures that chromosome ends can be rebuilt and

not shorten with each round of replication.

Telomeres and telomerase.

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 Forming of telomeres

Telomerase prevents progressive shortening of lagging strands during

eukaryotic DNA replication

9Telomeres consist of repetitive oligomeric sequences. The

need for a specialized region at the ends of chromosomes is
apparent – all known DNA polymerases elongate DNA chains
from the 3’ end, and all require DNA or RNA primer.

9As growing fork approaches the end of the linear chromosome,

synthesis of leading strand continues to the end of DNA template
strand; the resulting completely replicated daughter DNA double
helix then is released.

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 Telomerase prevents progressive shortening of lagging strands during
eukaryotic DNA replication

9However, because the lagging strand is copied discontinuously, it

can not be replicated in its entirety.
9When the final primer is removed there is no upstream strand onto
which DNA polymerase can build to fill the resulting gap.

9Without some special mechanism the daughter DNA strand

resulting from lagging strand synthesis would be shortened at each
cell division.

Telomerase prevents progressive shortening of lagging strands during

eukaryotic DNA replication

9The enzyme that prevents this progressive shortening of lagging

strand is a modified reverse transcriptase, called telomerase.

9It can elongate the lagging strand template from its 3' hydroxyl
end. This unusual enzyme contains a catalytic site that polymerizes
deoxyribonucleotides directed by an RNA template, and the RNA
template itself is brought to the site of calalysis as part of the
enzyme.

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9The repetitive sequence added by telomerase is determined by
the RNA associated with the enzyme, which varies between the
telomerases from different sources.

9Once the 3’ end of the lagging strand is sufficiently elongated,

synthesis of the lagging strand can take place, presumable from
additional primers.

9For cells in many organisms, telomere length is increased many

times over early in development.

Interestingly

‰Most human somatic cells replicate in the absence of the

telomerase activity and thus gradually consume the telomeric
repeats added earlier in development.

‰The progressive shortening of the chromosome ends and

eventual loss of genetic information that results has been linked to
cell death.

‰It has been suggested that life span is determined by the number
of telomeres with which the individual starts.

‰Indeed, the reverse relationship between the age and telomere

length has been observed.

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 Mechanism of action of telomerase

To summerize –eukaryotic and prokaryoric DNA replication scenarios

are very similar

When DNA replicates, many different

proteins work together to accomplish the
following steps:
1.The two parent strands are unwound
with the help of DNA helicases.

2 . Single stranded DNA binding

The major types of
proteins, which must
work together during the
replication of DNA
proteins attach to the unwound strands,
preventing them from winding back
together.
3 The strands are held in position,
binding easily to DNA polymerase,
which catalyzes the elongation of the
leading and lagging strands. (DNA
polymerase also checks the accuracy of
its own work!).

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 4. While the DNA polymerase on
the leading strand can operate in a
continuous fashion, RNA primer is
needed repeatedly on the lagging
strand to facilitate synthesis of
Okazaki fragments. DNA primase,
which is one of several
polypeptides bound together in a
group called primosomes, helps to
build the primer.
5. Finally, each new Okazaki
fragment is attached to the
completed portion of the lagging
strand in a reaction catalyzed by
DNA ligase.

Summary – DNA replication machinery

¾The enzymes and other protein factors that carry out DNA
replication in E. coli and in eukaryotic cells are analogous,
suggesting that the biochemical mechanism of DNA replication is
similar in all cells.

¾The enzymatic events at the growing fork are a consequence of

two properties of the DNA double helix and two of polymerases: the
double helix contains two antiparallel strands and the two strands
are interwound, so they can not simply be melted along the entire
length at once.
¾Dna polymerases require a nucleic acid primer –either a DNA or
an RNA molecule to begin synthesis and all DNA chain growth
occurs by nucleotide addition at the 3’ end.

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 Summary – DNA replication machinery

¾In all cells, one new DNA strand, the leading strand is synthesized
continuously in the direction of movement of the growing fork by
elongation from the 3’ end of the RNA primer base-paired to a
template strand.Synthesis of the other strand, the lagging strand,
occurs in the direction opposite to the overall direction of the
replication fork movement from a series of short RNA primers
formed on the second template strand. The resulting segments of
RNA plus DNA are called Okazaki fragments. After the primers are
removed and the gaps are filled, they are joined.

¾Initiation of DNA replication in E. coli occurs by binding of DnaA

to oriC, followed by attachment of DnaB, a helicase that melts DNA
at the fork. Association of primase with this complex forms a
primosome. After primer synthesis primase dissociates.

Summary – DNA replication machinery

¾E. coli polymerase III catalyzes nucleotide addition to both

leading and lagging strands. DNA polymerase I removes the RNA
primers from Okazaki fragments and fills the gaps on the lagging
strand. Finally, DNA ligase joins the Okazaki fragments.

¾Eukaryotic proteins that replicate SV40 DNA in vitro exhibit

similarities with E. coli replication proteins. A viral protein called T
antigen functions similarly to the DnaB helicase, and host-cell
PCNA is similar to the β-subunit clamp associated with E. coli
DNA polymerase III. However, two distinct mammalian
polymerases, α and δ or ε, function on eukaryotic growing fork.

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 Summary – DNA replication machinery

The processivity of DNA polymerase is essential for efficient

polymerization and is facilitated by their association with the β-
subunit clamp in E. coli and PCNA in eukaryotes.

Telomerase, a reverse transcriptase that contains an RNA template,

adds nucleotides to the 3’end of the lagging strand template and
thus prevents shortening of lagging strands during replication of
linear DNA molecules such as those of eukaryotic chromosomes.

Reading – chapters 20,21, excluding DNA repair

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