Chapter 2

DNA Replication

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The Gene is the Fundamental Unit of

Heredity

The unit of heredity is known as a gene. Each gene is

responsible for asingle inherited property or characteristic of
the organism.

Inheritance of GeneticInformation
Since each cell needs a complete set of genes, it is necessary
for theoriginal cell to duplicate its genes before dividing.
Because the genes aremade of DNA and make up the
chromosomes, this means that each chromosome must
beaccurately copied. Upon cell division, both daughter
cellswillreceive identical sets of chromosomes, each with a
complete set of genes.

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Replication of DNA
Replication of the DNAin molecular terms
means that the DNA of the original, or mother,
is duplicated to give two identical copies.
This process is known as replication.
Upon cell division each of the descendants
gets one completecopy of the DNA. The
original genes of the mother cell are on a
doublestranded DNA molecule so the first
step in replication is to separate the
twostrands of the DNA double helix.
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Experiments in the 1950s showed

that DNA is the hereditary
material
Scientists raced to determine the
structure of DNA
1953 - Watson and Crick proposed
that DNA is a double helix

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2
bonds

3 bonds
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The next step is to

build acomplementary strand on
each of the two
original strands.
SinceA only pairs
with T, and since G
only pairs withC,
the sequence of
each strand
dictates
thesequence of its
complementary
strand.
5

A=T,

C = G.

We nowhave two double stranded DNA

molecules, bothwith sequences identical to the
original one. One of these daughter molecules
has the original leftstrand and the other
daughter has the originalright strand.
This is known assemiconservative
replication of the progeny, conserveshalf of
the original DNA molecule.
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Semiconservative Replication

1
2
3
4
Parental
strands

Parental
strands
separate

Parental strand
receives new strand of
DNA (1 set - one old
and one new strand)

Strand 1 is identical to strand 3

Starnd 2 is identical to strand 4
Strand 1 is complementary to strand
2
Starnd 3 is complementary to strand
4
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n

Each parent strand

remains intact

Every DNA molecule is

half old and half new

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How Does a Double Helix Separate into Strands?

Because the two strands forming a DNA molecule
areheld together byhydrogen bonding and
twisted around eachother to form a double
helix,they cannot simply be pulled apart.
Worse still,the DNA inside a cell is
alsosupercoiled to pack it into a small space.
Before separating thestrands , both the supercoils
and the double helixmust be unwound.

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How Does a Double Helix Separate into Strands?

This is done in two stages :
1. First the supercoils are unwound by anenzyme
known as DNA gyrase (DNA topoisomerase).
The gyrase cutsboth strands of double stranded
DNA to give a double stranded break.
However, it keeps hold of all of the cut ends.The
two halves of the gyrase then rotaterelative to
each other and the ends arerejoined. This
untwists the supercoils. Eachrotation costs the
cell a small amount of energy.
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Action of DNA Gyrase

Double stranded breaks done by DNA gyrase will relieve

supercoiling but will not pull the strands apart because the strands
are still held by the hydrogen bonds between the bases. Once
untwisted the ends are rejoined.
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2. Once the supercoils have beenuntwisted, the

double helix is unwound by the enzymeDNA
helicase. Helicase does not break the DNA
chain,it simply disrupts the hydrogen
bondsholding the base pairs together.

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How are the Parental Strands of DNA Kept Apart?

The two separated strands of the parental DNA

molecule are complementary to eachother.
Consequently all of their respectivebases are
capable of pairing off and binding to each other.
In order to manufacturethenew strands, the two
original strands, despite their desire to cling
together,must somehow be kept apart.
This is done by means of a special
"divorce"protein which binds to the unpaired
single stranded DNA and prevents thetwo parental
strands from getting back together. This is known
asSingle Strand Binding protein (SSB)
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Single strand binding protein

5
helicase
3
Single-strand
binding
protein

Direction
of
Replicatio
n

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Making a New Strand of DNA

The critical issue in replication is the base pairing of A
withT and of G with C. Each of theseparated parental
strands ofDNA serves as atemplate strand for the
synthesisof anewcomplementary strand.
Theincoming nucleotides for the newstrand recognize
their partners bybase pairing and so are lined upon
the template strand. Actually, things are a bitmore
complicated.

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Base Pairing during

Replication
Each old strand
serves as the
template for
complementary
new strand

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Althoughhydrogen bonding alone wouldmatch

bases correctly (99%) of the time this is not
good enough.
The enzyme that linksthe nucleotides known as
DNA polymerase III or pol III can also sense if
the base are correctly paired. If not, the
mismatched base pair is rejected.

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Base-pairing of
DNA

ne
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w

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DNA polymerase III

phosphate

T
G
A
A

TEMPLATE
STRAND

3OH

GROWING
STRAND

OH

5
OH
3 DNA POLYMERASE III MAKING DNA
DNA Polymerase III Pol (III) enzyme that
makes most of the DNA when chromosome are
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replicated

A Closer Look at
Strand Assembly

Energy for
strand
assembly is
provided by
removal of two
phosphate
groups from
free nucleotides

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DNA polymerase III

The nucleotides are thenjoined together by the
enzyme.This DNA polymerase has twosubunits.
i. One of these is thesynthetic subunit and
isresponsible for manufacturingnew DNA.
ii. The other subunitis shaped like a doughnut
and slides up and down like a curtain ring on
thetemplatestrand of DNA. This "sliding
clamp" subunitbinds the synthetic subunit to
the DNA.

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Dna Polymerase III The Sliding Clamp

Pol III- synthetic
subunit
Pol III- sliding clamp
subunit

Newly synthesized DNA

SSBs
Original strand of
DNA

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Sliding clamp subunit

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Synthesis Always Goes from 5' to 3

As you know, nucleotides have three components:
a. a phosphate group
b. sugar and
c. the base.
In DNA the sugar is deoxyribose, and is joined
tothebase at position 1' and to the
phosphategroup at position 5'.
(Thecarbon atoms of the deoxyribose sugarare
numbered with prime marks todistinguish them
from those of the basewhich have plain numbers)

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5
Phosphate-O-CH2

BASE
1

4
H
H
3
Next nucleotide is
joined here

OH
H
2

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H2O
O
OH

P
O

BASE

H2C 5

5
4

H
3

H
OH
2

3
OH

1
2

H
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Direction of DNA Synthesis

When anew nucleotide is added it is joined, via its own
phosphate group on position 5', to the 3' position as
indicated by the arrow.
New DNA strandsalways start at the 5 end and grow in
the 3 direction. In fact, all nucleicacids, whether DNA or
RNA, are always made in the 5 to 3 direction.C
However, DNA is normally double stranded, and it happens
that thetwo strands run in opposite directions, that is, if
one goes 5' to 3' then itscomplementary partner will run
from 3' to 5'. The strands are saidto be anti-parallel.

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Double stranded DNA is antiparallel

American James Watson joined with
Francis H. C. Crick in England to
work on structure of DNA. Watson
and Crick received the Nobel Prize in
1962 for their model of DNA.

Using information generated by

Chargaff and Franklin, Watson and
Crick built a model of DNA as double
helix; sugar-phosphate molecules on
outside, paired bases on inside.
Complementary base pairing is
the paired relationship between
purines and pyrimidines in DNA,
such that A is hydrogen-bonded to T
and G is hydrogen-bonded to C.

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Double stranded DNA is antiparallel

GG TT GG AA AA TT GG CC GG
CC

AA

CC

TT

TT

AA

CC

GG CC
5

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The Replication Fork Is Where the Action Is!

The replication fork is the total structure in the region
where the DNAmolecule is being duplicated.
It includes the swivel where the DNA is beingtwistedby
DNA gyrase, the helicase following right behind, and
thestretches of single stranded DNA held apart by the
single strand bindingprotein.
It also hastwo molecules of DNA polymerase III which
are busymaking two new strands of DNA. Since DNA is
always made in the 5' to 3' direction, and since the
twostrands of double helical DNA are antiparallel, this
means that duringDNA replication the two new strands
must be synthesized in oppositedirections.
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SSB

Leading
strand

DNA helicase
5

3
5

Lagging
strand

3
5

3
5

The Replication
Fork

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The Replication
Fork

a, Nucleoside triphosphates serve as

a substrate for DNA polymerase,
according to the mechanism shown
on the top strand. Each nucleoside
triphosphate is made up of three
phosphates (represented here by
yellow spheres), a deoxyribose sugar
(beige rectangle) and one of four
bases (differently coloured
cylinders). The three phosphates are
joined to each other by high-energy
bonds, and the cleavage of these
bonds during the polymerization
reaction releases the free energy
needed to drive the incorporation of
each nucleotide into the growing
DNA chain. The reaction shown on
the bottom strand, which would
cause DNA chain growth in the 3 to
5 chemical direction, does not occur
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nature.
n

The DNA replication fork the

explanation
b, DNA polymerases catalyse chain
growth only in the 5 to 3 chemical
direction, but both new daughter
strands grow at the fork, so a
dilemma of the 1960s was how the
bottom strand in this diagram was
synthesized. The asymmetric nature
of the replication fork was recognized
by the early 1970s: the leading
strand grows continuously, whereas
the lagging strand is synthesized by
a DNA polymerase through the
backstitching mechanism illustrated.
Thus, both strands are produced by
DNA synthesis in the 5 to 3
direction.

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Completing the Lagging Strand

Although the leading strand justkeeps getting longer
and longer, the lagging strand is handicapped.
After thereplication fork has passed by, the Iagging
strand is left as a series of shortpieces with gaps in
between. Thesenewly made pieces of DNA are
known as Okazaki fragments after their discoverer
and must be joined together to give a complete
strand of DNA.

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Replication Fork Revisited

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OKAZAKI
FRAGMENT

GAP

Gap in
lagging
strand

Pol I Fills
Gap with
nucleotid
es

Direction
Direction
of
of
synthesis
synthesis

Origin
al DNA
strand

DNA
Pol 1
5

Newly added
3 nucleotides

5
New 53 bond
Nick Remains

One nick
remains

3
DNA
ligase

Ligase
seals
nick

3
5

5
3

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Thisisaccomplished by two enzymes workingin

succession: DNA polymerase I and DNA ligase.
DNA polymerase I fills inthe gaps and DNA ligase
joins the gaps.
DNA polymerase I was discovered before DNA
polymerase III,hence the numbering.
Both DNApolymerase I and DNA ligase have
important uses in genetic engineering.

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Starting a New Strand

Up to now we have assumed that we have strands

of DNA with freeends that can be elongated by
DNA polymerase. But how do we get a newstrand
started?
Although the leading strand only needs to be
started once, the lagging strand is made in short
sections and we need to start again everytime
we make a new Okazaki fragment.
Curiously, DNA polymerase cannot start a new
strand by itself, it canonly elongate!

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New strands are started with short stretch, not

of DNA itself,self, but of RNA!
These short RNA piecesknown as primers
and the enzyme that starts synthesis of new
chains bymaking the RNA primers is called
primase.
So every time a new fragmentof DNA is made,
primase sneaks in and lays down a short RNA
primer toget things going. Only then can DNA
polymerase get to work elongatingthe strand.
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Recoiling the DNA into a Helix

As the two new strands of DNA are synthesized,

two double DNA molecules are produced, each
with one old and one new strand.
Once the replication fork has moved past, the
double stranded DNAmolecule automatically
rewinds into a helix.

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Enzymes involved in DNA Replication- details

Two DNA polymerase molecules are active at the fork at
any one time. One moves continuously to produce the new
daughter DNA molecule on the leading strand, whereas the
other produces a long series of short Okazaki DNA
fragments on the lagging strand.
Both polymerases are anchored to their template by
polymerase accessory proteins, in the form of a sliding
clamp and a clamp loader.
A DNA helicase, powered by ATP hydrolysis, propels itself
rapidly along one of the template DNA strands (here the
lagging strand), forcing open the DNA helix ahead of the
replication fork.

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(DNA gyrase)

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Enzymes- details
The helicase exposes the bases of the DNA
helix for the leading-strand polymerase to copy.
DNA topoisomerase or DNA gyrase enzymes
facilitate DNA helix unwinding.
In addition to the template, DNA polymerases
need a pre-existing DNA or RNA chain end (a
primer) onto which to add each nucleotide.
For this reason, the lagging strand polymerase
requires the action of a DNA primase enzyme
before it can start each Okazaki fragment.
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More stuff on the enzymes for replication

The primase produces a very short RNA

molecule (an RNA primer) at the 5 end of each
Okazaki fragment onto which the DNA
polymerase adds nucleotides
Finally, the single-stranded regions of DNA at
the fork are covered by multiple copies of a
single-strand DNA-binding protein, which
hold the DNA template strands open with their
bases exposed.
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Leading strand
DNA pol III
Leading
strand

Lagging
strand

DNA
helicase

DNA
gyrase

DNA
helicase

Lagging strand
DNA pol III

In the folded fork structure shown in the inset, the

lagging-strand DNA polymerase remains tied to
the leading-strand DNA polymerase. This allows
the lagging-strand polymerase to remain at the
fork after it finishes the synthesis of each Okazaki
fragment.
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Binary fission in bacteria

Replication of chromosomal DNA in bacteria

starts at a specific chromosomal site called
the origin and proceeds bidirectionally until
the process is completed.
When bacteria divide by binary fission after
completing DNA replication, the replicated
chromosomes are partitioned into each of the
daughter cells.

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The origin regions specifically and

transiently associate with the cell
membrane after DNA replication has been
initiated, leading to a model whereby
membrane attachment directs separation of
daughter chromosomes (the replicon model).
These characteristics of DNA replication
during bacterial growth fulfill the
requirements of the genetic material to be
reproduced accurately and to be inherited by
each daughter cell at the time of cell
division.
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DNA Replication in Bacteria

ORIGI
N

REPLICATION
FORK
(bidirectional
replication)

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Bacterial
chromosome

Bacterium before DNA

replication

DNA replication
begins

E.coli
undergoing
binary fission

Parent DNA
molecule

DNA replication completed

DNA copy
Membrane growth moves
DNA molecules apart

New membrane and cell

wall deposited

Cytoplasm divided into two.

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