REPLICATION
Copying DNA
A complex reaction
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Uncoiling of parent molecule
Unzipping the two sister strands to
reveal the sequence of bases
Reading the sequence of bases
Choosing the complementary
nucleotide building blocks
Lining up the nucleotides and
bonding them together
Checking for errors
Recoiling the two DNA molecules.
© 2010 Paul Billiet ODWS
All controlled by
enzymes in
particular DNA
polymerase
Image Credit: DNA
polymerase III
A very rapid reaction
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The average length the DNA
molecule in a bacteriophage (a
large virus) is 34µm long
100 000 base pairs
10 000 turns (10 base pairs per
revolution)
Replication time 2 minutes
Replication speed 83
revolutions per second.
Phage particle releasing its DNA
Image Credit: http://www.biochem.wisc.edu/
Multiple replication forks
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Eukaryotes have much more DNA
They have their DNA divided up into many
molecules (chromosomes)
Replication in eukaryotes begins at many
points along each chromosome
This reduces the time taken.
© 2010 Paul Billiet ODWS
Where and when does replication
occur?
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In the nucleus of eukaryotes
During interphase
During S-phase
© 2010 Paul Billiet ODWS
The cell cycle
Cytokinesis division of the
cytoplasm
M
Some cells may stay in
this stage for over a year
G0
First growth phase.
Varies in length
G1
G2
Second growth
period
S
Copying of
chromosomes
= replication
G1 + S + G2 = INTERPHASE
© 2010 Paul Billiet ODWS
Meselson & Stahl’s experiment
Samples taken at
timed intervals
And DNA extracted
ultracentrifuge
Bacteria fed on
N-15 labelled
food for several
generations
© 2010 Paul Billiet ODWS
Bacteria
switched to
N-14 labelled
food
DNA settles a
level because of
its density
Meselson and Stahl’s results
DNA
Light
Medium
Heavy
0
0.3
0.7 1.0
1.1
1.5
1.9
GENERATIONS
© 2010 Paul Billiet ODWS
2.5
3.0
4.1 0 +1.9 0 + 4.1
Controls
Observations
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Initially all the DNA is “heavy”
Only one band appears
After one generation there is one band but it is
“medium”
After two generations there are two equal bands
“Medium” and “Light”
After three generations there are two bands
A strong light band and a weaker medium
This carries on, the light band getting stronger.
© 2010 Paul Billiet ODWS
Interpretation of the results
GENERATION
0
1
2
3
© 2010 Paul Billiet ODWS
Interpretation
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At each generation the DNA molecule splits
A new strand is fabricated alongside the old
one
The is semi-conservative replication.
© 2010 Paul Billiet ODWS
E.coli caught in the act!
Newly formed
daughter
strands
Initiation point
2 strands
of parental
DNA
1962 autoradiograph by John Cairns of a replicating
E. coli chromosome
Growing
point
Untwisting the helix & breaking
the hydrogen bonds
A=T
T=A
T=A
CG
GC
GC
CG
T=A
A=T
T=A
CG
CG
CG
A=T
T=A
T=T
A=T
A=T
A=T
© 2010 Paul Billiet ODWS
A=T
T=A
T=A
CG
GC
GC
CG
T=A
A=T
A
T
C
C
C
A
T
T
A
A
A
Helicase
G
G
G
T
A
T
T
T
T
A=T
T=A
T=A
CG
GC
GC
CG
T=A
A=T
A
T
Adding in the
nucleotides
C
C
C
G
G
G
T
A
A
T
T
A
Complementary
base pairing
© 2010 Paul Billiet ODWS
A
A T
T
A
T
DNA
Polymerase III
T
T
T
Deoxynucleoside
triphosphates
T
Two daughter strands
© 2010 Paul Billiet ODWS
A=T
T=A
T=A
CG
GC
GC
CG
T=A
A=T
T=A
CG
CG
CG
A=T
T=A
T=T
A=T
A=T
A=T
A=T
T=A
T=A
CG
GC
GC
CG
T=A
A=T
T=A
CG
CG
CG
A=T
T=A
T=T
A=T
A=T
A=T
Added complications
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DNA helicase III cannot start the process of
replication
A small primer of RNA is needed first
This requires another enzyme RNA primase.
© 2010 Paul Billiet ODWS
DNA
Polymerase III
A=T
T=A
T=A
CG
GC
GC
CG
T=A
A=T
A
T
G
C
G
C
G
C G
T
A G
A
T T
T
T AA
T
A T
T
A T
RNA
T
A T
primase
© 2010 Paul Billiet ODWS
Added complications
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DNA polymerase III can only add nucleotides
on one way (5’ to 3’)
BUT the DNA molecule is antiparallel
One strand can be replicated directly as it
unzips (the leading strand)
The other strand needs to wait until a certain
amount is unzipped (the lagging strand).
© 2010 Paul Billiet ODWS
5’
3’
Okazaki
fragments
3’
5’
5’
3’
© 2010 Paul Billiet ODWS
3’
5’
Leading strand
5’
Lagging strand
Added complications
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The lagging strand is replicated in fragments
about 1000 base pairs long
OKAZAKI fragments
Each fragment starts with an RNA primer.
© 2010 Paul Billiet ODWS
Added complications
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At the end the RNA primers are removed by
another enzyme, DNA polymerase I
Replaces the primers with DNA nucleotides
The ends of the Okazaki fragments are stuck
together using DNA ligase.
© 2010 Paul Billiet ODWS
Gaps need
connecting
DNA polymerase I
replaces the RNA
primers with DNA
© 2010 Paul Billiet ODWS
Ligase
connects the
fragments