Download MBLG1001 Lecture 9 The Flow of Genetic Information Replication

The Flow of Genetic
Information
replication
MBLG1001 Lecture 9
DNA
DNA
Transcription
RNA
Replication
Chapter 7 Malacinski
Chapter 5 Clark
Translation
Folding, modification,
translocation
Protein
Functional protein
Figure 28.1
Watson and Crick’s
famous paper, in its
entirety. (Reprinted
with permission from
Watson,J.D., and
Crick,F.H.C., Molecular
structure of nucleic acid,
1953. Nature 171:737738. Copyright 1953
Macmillan Publishers
Ltd.)
Replication
• Is the process :
–Conservative,
–Semi-conservative OR
–Dispersive?
The Messelson Stahl Experiment
The Messelson Stahl Experiment
• Cells were grown up on the “heavy”
isotope of N, 15N, abbreviated as H
(15NH4Cl)
• then the medium was changed to one
containing normal 14N (light or L) as sole
nitrogen source.
• DNA was isolated at various time points
after the media change and applied to a
CsCl density gradient.
• This technique separates by buoyant
density
• DNA containing 2 light strands (L:L) will
sediment at a different density to a hybrid
Heavy:Light (H:L) nucleic acid or the
Heavy:Heavy (H:H) form of DNA.
• Old parent DNA will be heavy (H)
• Newly synthesised DNA will be light (L).
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The Messelson Stahl Experiment
LL
HL
HH
Concentrated CsCl
solutions, when
centrifuged really
fast form a gradient.
Compounds
separate by their
buoyant density in
such a gradient.
If replication was semi
conservative…
• In the first generation after medium
change the DNA would be composed of
solely H:L
• In the next generation you would expect
H:L and L:L in a ratio of 1:1.
• In the following generation the H:L and
L:L would have a ratio of 1:3. In the next
generation it would be 1:7.
If replication was conservative…
• The DNA one cell division after medium
change would be composed of H:H and
L:L in equal proportions.
• In the second generation there should be
3 L:L to 1 H:H.
• The third generation..7 L:L and 1 H:H
If replication was dispersive…
• Hybrid H:L DNA would result but if the
individual strands were analysed under
denaturing conditions (in CsCl with NaOH
to keep the strands apart) they would also
have an intermediate density.
• The individual DNA strands would always
be completely H or L in the other models.
E. coli Replication:
the quintessential example
• E. coli can, under optimal growth
conditions double cell numbers every 20
min. Clark p125
• It has 1 large circular chromosome; 4.6
million bp
• The replication fork moves at a constant
1000 NMPs/sec.
• There are 2 forks which move in opposite
directions
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E. coli Replication:
the quintessential example
The replication forks
oriC
• At this rate it takes 40 min to copy the whole E.
coli genome (4.6 million bases pairs) and
another 20 min to separate the cellular
components.
• To double in less than 60 min means the cell
must initiate the next round of replication before
the previous one had finished.
• To scale up this process it is a 400 k trip made
by 2 machines in 40 min with an error made
every 170 k.
E. coli’s problems:
• Replication is bi-directional. The theta
model.
• Bacterial DNA is a closed circle so it will
get tangled when it is unwound.
• Enzymes are needed to copy the DNA
• The strands must be pulled apart and
unwound.
The discovery of DNA polymerases
What enzymes are involved in
copying DNA?
• As soon as the structure of DNA was
elucidated the hunt was on for the
enzymes which copy it.
• These enzymes are known as
polymerases
• Over the past 50 years many such
enzymes have been found. Some even
copy an RNA.
Arthur Kornberg
• The first DNA polymerase (DNA pol I) was
isolated in 1956, only 3 years after the
structure of DNA was published.
• Arthur Kornberg isolated DNA pol I and
won the 1959 Nobel prize for his efforts.
• At the time it was thought to be the main
replicative enzyme.
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Problems with DNA pol I
• It didn’t work fast enough to copy the
whole genome.
• John Cairns and Paula DeLucia isolated
mutants of E. coli which had ~1% of the
DNA pol I activity but still divided at normal
rates.
• Moral of the story: get the prize before
anyone can prove you wrong.
DNA polymerase III
Search for DNA copying enzymes
• Since then another 4 polymerases have been
identified in E. coli: DNA pol II, III, IV and V
• These have been isolated and purified from
polA- mutants.
• DNA pols II, IV and V are repair enzymes and
DNA pol III was the Big one!
• There have been to date over 15 eukaryotic
DNA pols isolated and purified and some
interesting viral versions.
The father and son act!
• It wasn’t until 1970 that DNA pol III was
isolated by Arthur’s son, Thomas.
• This is a truly large enzyme with ~10
subunits.
• It has a circular donut-like pair of subunits
which clamp the enzyme to the DNA. This
gives it its processivity (ability to remain
tightly associated with the template
through many nucleotide additions)
The main players in E coli
Replication.
• DNA polymerase I has :
– 5’ to 3’ exonuclease
– 3’ to 5’ exonuclease (proof reading)
– 5’ to 3’ polymerase (new strand)
• DNA polymerase III has;
– 3’ to 5’ exonuclease (proof reading)
– 5’ to 3’ polymerase (new strand)
Properties of DNA polymerases
Clark p187, Malacinski p 128
• 5’ to 3’ polymerase activity; All polymerases
(DNA and RNA) synthesise the new strand of
nucleic acid in a 5’ to 3’ direction.
• All DNA polymerases need a primer; a
short fragment of single stranded nucleic acid
bound to the template which provides a 3’OH to
make the next addition.
• All DNA polymerases have a 3’ to 5’
exonuclease activity
4
5’ to 3’ polymerase activity
5’ to 3’ polymerase activity
Parent strand
Parent strand
5’P
3’OH
HO
dNTP
5’P
3’OH
5’P
5’P
HO
dNTP
• The correct dNTP which base pairs to the
template base is added by the polymerase
• The correct dNTP which base pairs to the
template base is added by the polymerase
5’ to 3’ polymerase activity
5’ to 3’ polymerase activity
Parent strand
Parent strand
5’P
3’OH
5’P
HO
dNTP
5’P
3’OH
3’OH
5’P
Newly synthesised strand
• The correct dNTP which base pairs to the
template base is added by the polymerase
• Eventually the whole strand is copied
OH
At the molecular level
-O
P
O
O
O
P
O-
P
O
O
O-
P
H
H
O
H
H
BASE
O
CH2
Cleaving these
phosphodiester bonds
provides the energy for
the polymerisation
-O
H
O
O
BASE
O
O
O-
Growing strand now
(n+1) residues long
O-
H
:OH
P
O
H
H
-O
P
O
H
O-
O
-O
BASE
O
Growing
strand (n)
residues
long
2
H
O
H
H
New incoming
nucleotide
triphosphate:
dNTP
O
-O
P
O-
O
O
P
O-
H
OH
H
Pyrophosphate
PPi
O-
+
O
H
P
BASE
O
O
O-
H
H
OH
H
H
H
5
OH
2
-O
P
Proof reading or editing
O
Growing strand now
(n+1) residues long
O-
Rapidly
breaks down
to 2
phosphates
BASE
O
-O
P
O
O
O-
H
H
O
H
H
O
-O
P
O-
O
O
P
O-
O-
+
O
H
P
BASE
O
O
O-
Pyrophosphate
PPi
H
H
OH
H
H
Clark p 113 Malcinski p 129
• DNA polymerases, and not RNA
polymerases, have an editing function.
• The 3’ to 5’ exonuclease is a slow acting
nuclease
• It cleaves the newly added nucleotide if it
does not base pair properly to the
template.
H
The Primer
Clark p115 Malacinski p 135
• All DNA polymerases need a primer, even
reverse transcriptase and Klenow.
• RNA polymerases do NOT need a primer.
They generate the primer for DNA
synthesis.
• The need for a 3’OH is exploited in drug
design and certain techniques e.g. DNA
sequencing.
Klenow enzyme or fragment.
• This enzyme comes from DNA pol I.
• If you digest DNA pol I for a short amount
of time with a protease (called limited
proteolysis) you get 2 fragments:
– A ~66 kD fragment with polymerase and 3’ to
5’ exonuclease activity.
– A~33 kD fragment with 5’ to 3’ exonuclease
activity.
A quick journey through the
other polymerases
Klenow enzyme or fragment.
• The larger fragment is the Klenow
enzyme. It is very useful as a DNA
polymerase.
• It requires a primer (needs a 3’OH to add
the next nucleotide to).
• It is very good a copying DNA.
• It can be used to synthesise a labeled
strand of DNA for experiments
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The history of DNA Polymerases
Reverse Transcriptase
• Produced by retroviruses e.g. HIV
• Uses an RNA template
• Produces a DNA copy, known as
complementary DNA or cDNA
• Works 5’ to 3’ and requires a primer.
• First isolated in 1970 by Howard Temin
and David Baltimore independently.
Figure 28.16
The structures of AZT (3′-azido-2′,3′dideoxythymidine). This nucleoside
was the first approved drug for
treatment of AIDS. AZT is
phosphorylated in vivo to give
AZTTP (AZT 5′-triphosphate), a
substrate analog that binds to HIV
reverse transcriptase, HIV reverse
transcriptase incorporates AZTTP
into growing DNA chains in place of
dTTP. Incorporated AZTMP blocks
further chain elongation because its
3′-azido group cannot form a
phosphodiester bond with an
incoming nucleotide. Host cell DNA
polymerases have little affinity for
AZTTP.
Taq Polymerase
• A thermal stable DNA polymerase isolated
form the bacterium, Thermus aquaticus
which lives in the hot springs of
Yellowstone National Park.
• Used in a reaction known as Polymerase
Chain Reaction (PCR).
• PCR is able to amplify sections of DNA by
copying it over and over.
Other DNA polymerases
So what do we know…
• Repair: DNA pol IV and pol V.
• Eukaryotic DNA polymerases: α, β, δ,
and ε with γ found in mitochondria.
• Then there are the eukaryotic repair
enzymes!!
• ALL WORK to make a new strand in the 5’
to 3’ orientation.
• there are a lot of DNA polymerases which
are capable of copying a strand of DNA,
provided they are supplied with
nucleotides, template and a primer.
• But how and when does replication occur?
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