Transcript Replication RNA Synthesis Decoding the Genetic Code Noel Murphy

Replication
RNA Synthesis
Decoding the Genetic Code
Noel Murphy
Reference Sources
Hartl & Jones, Genetics: Analysis of Genes and Genomes,
6th Edition
Chapter 6 – Replication
Chapter 10 – Transcription and the code
Klug & Cummings, Essentials of Genetics 5th Edition
Chapter 11 – Replication
Chapter 13 – Transcription and the code
Lectures http://www.tcd.ie/Genetics/staff/Noel_Murphy.htm
DNA is the Genetic Material
Therefore it must
(1) Replicate faithfully.
(2) Have the coding capacity to generate
proteins and other products for all
cellular functions.
• “A genetic material must carry out two jobs:
duplicate itself and control the development of the
rest of the cell in a specific way.”
• -Francis Crick
Replication
The Dawn of Molecular Biology
April 25, 1953
Watson and Crick: "It has not escaped our
notice that the specific (base) pairing we
have postulated immediately suggests a
possible copying mechanism for the
genetic material."
Models for DNA replication
1) Semiconservative model:
Daughter DNA molecules contain one parental
strand and one newly-replicated strand
2) Conservative model:
Parent strands transfer information to an
intermediate (?), then the intermediate gets copied.
The parent helix is conserved, the daughter
helix is completely new
3) Dispersive model:
Parent helix is broken into fragments, dispersed,
copied then assembled into two new helices.
New and old DNA are completely dispersed
MODELS OF DNA REPLICATION
(a) Hypothesis 1:
(b) Hypothesis 2:
(c) Hypothesis 3:
Semi-conservative
replication
Conservative replication
Dispersive replication
Intermediate molecule
Testing Models for DNA replication
Matthew Meselson and Franklin Stahl (1958)
Meselson and Stahl
Semi-conservative replication of DNA
Isotopes of nitrogen (non-radioactive) were used in this experiment
Generations
0
HH
Equilibrium Density
Gradient
Centrifugation
0.3
HH
Detection of
semiconservative
replication in E. coli
by density-gradient
centrifugation. The
position of a band of
DNA depends on its
content of 14N amd
15N. After 1.0
generation, all the
DNA molecules are
hybrids containing
equal amounts of 14N
and 15N
0.7
1.0
HL
1.1
HL
1.5
1.9
LL + HL
2.5
3.0
4.1
0 and 1.0
mixed
0 and 4.1
mixed
HL
LL
LL
LH
DNA replication
Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)
Replication as a process
• Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two molecules are made.
Each has one new and one old
DNA strand.
DNA Replication
• Since DNA replication is semiconservative, therefore
the helix must be unwound.
• John Cairns (1963) showed that initial unwinding is
localized to a region of the bacterial circular genome,
called an “origin” or “ori” for short.
Replication can be Uni- or Bidirectional
UNIDIRECTIONAL REPLICATION
Origin
BIDIRECTIONAL REPLICATION
3’
5’
5’
3’
5’
3’
Origin
3’
5’
John Cairns
Bacterial
culture
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in media with low
concentration of
3H- thymidine
Grow cells for several generations
Small amounts of 3H thymidine
are incorporated into new DNA
*T
*T
*T
All DNA is lightly
labeled with radioactivity
Grow for brief
period of time
Add a high
concentration
of 3H- thymidine
*T *T
*T
*T*T
*T
*T
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*T *T
*T
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*T*T *T *T*T *T
*T
*T
*T
*T
*T
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*T *T
Dense label at the replication fork
where new DNA is being made
Cairns then isolated the chromosomes by lysing the cells very very gently
and placed them on an electron micrograph (EM) grid which he exposed to
X-ray film for two months.
Evidence points to bidirectional replication
Label at both replication forks
Features of DNA Replication
• DNA replication is semiconservative
– Each strand of both replication forks is being
copied.
• DNA replication is bidirectional
– Bidirectional replication involves two
replication forks, which move in opposite
directions
Arthur Kornberg (1957)
Protein extracts from E. coli
+
Template DNA
Is new DNA synthesized??
- dNTPs (substrates) all 4 at once
- Mg2+ (cofactor)
- ATP (energy source)
- free 3’OH end (primer)
In vitro assay for DNA synthesis
Used the assay to purify a DNA polymerizing enzyme
DNA polymerase I
Kornberg also used the in vitro assay to characterize
the DNA polymerizing activity
- dNTPs are ONLY added to the 3’ end of newly
replicating DNA
5’
3’
5’
3’
5’
3’
5’
3’
3’ New progeny strand
3’
5’ Parental template strand
5’
3’
5’
3’
5’
-therefore DNA synthesis occurs only in the
5’ to 3’ direction
THIS LEADS TO A CONCEPTUAL PROBLEM
Consider one replication fork:
3’
3’
5’
Primer
Continuous replication
5’
Direction of
unwinding
3’
5’
3’
Discontinuous replication
5’
3’
5’
Evidence for the Semi-Discontinuous replication
model was provided by the Okazakis (1968)
Evidence for Semi-Discontinuous Replication
(pulse-chase experiment)
Bacterial
culture
Add 3H Thymidine Flood with non-radioactive T
Harvest the bacteria
at different times
For a SHORT time
Allow replication
after the chase
(i.e. seconds)
To continue
Bacteria are
replicating
smallest
Isolate their DNA
Separate the strands
(using alkali conditions)
Run on a sizing gradient
Radioactivity will only
be in the DNA that was
made during the pulse
largest
Results of pulse-chase experiment
Pulse
Chase
3’
3’
5’
Primer
smallest
5’
Direction of
unwinding
3’
5’
3’
largest
5’
3’
***
5’
DNA replication is semi-discontinuous
Continuous synthesis
Discontinuous synthesis
Features of DNA Replication
• DNA replication is semiconservative
– Each strand of template DNA is being copied.
• DNA replication is bidirectional
– Bidirectional replication involves two replication forks,
which move in opposite directions
• DNA replication is semidiscontinuous
– The leading strand copies continuously
– The lagging strand copies in segments (Okazaki fragments)
which must be joined
The Enzymology
of DNA Replication
• In 1957, Arthur Kornberg demonstrated the
existence of a DNA polymerase - DNA
polymerase I
• DNA Polymerase I has THREE different
enzymatic activities in a single polypeptide:
• a 5’ to 3’ DNA polymerizing activity
• a 3’ to 5’ exonuclease activity
• a 5’ to 3’ exonuclease activity
The 5’ to 3’ DNA polymerizing activity
Subsequent
hydrolysis of
PPi drives the
reaction forward
Nucleotides are added at the 3'-end of the strand
Why the exonuclease activities?
• The 3'-5' exonuclease activity serves a
proofreading function
• It removes incorrectly matched bases, so that
the polymerase can try again.
Proof reading activity
of the 3’ to 5’ exonuclease.
DNAPI stalls if the incorrect
ntd is added - it can’t add the
next ntd in the chain
Proof reading activity is slow
compared to polymerizing
activity, but the stalling of
DNAP I after insertion of an
incorrect base allows the
proofreading activity to
catch up with the polymerizing
activity and remove the
incorrect base.
DNA Replication is Accurate
(In E. coli: 1 error/109 -1010 dNTPs added)
How?
1) Base-pairing specificity at the active site
- correct geometry in the active site occurs only with
correctly paired bases BUT the wrong base still gets
inserted 1/ 104 -105 dNTPs added
2) Proofreading activity by 3’-5’ exonuclease
- removes mispaired dNTPs from 3’ end of DNA
- increases the accuracy of replication 102 -103 fold
3) Mismatch repair system
- corrects mismatches AFTER DNA replication
Is DNA Polymerase I the principal
replication enzyme??
In 1969 John Cairns and Paula deLucia isolated a
mutant bacterial strain with only 1% DNAP I
activity (polA)
- mutant was super sensitive to UV radiation
- but otherwise the mutant was fine i.e. it could
divide, so obviously it can replicate its DNA
Conclusion:
• DNAP I is NOT the principal replication
enzyme in E. coli
Other clues….
- DNAP I is too slow (600 dNTPs added/minute –
would take 100 hrs to replicate genome instead of
40 minutes)
- DNAP I is only moderately processive
(processivity refers to the number of dNTPs added
to a growing DNA chain before the enzyme
dissociates from the template)
Conclusion:
• There must be additional DNA polymerases.
• Biochemists purified them from the polA mutant
So if it’s not the chief replication enzyme
then what does DNAP I do?
- functions in multiple processes that require
only short lengths of DNA synthesis
- has a major role in DNA repair (CairnsdeLucia mutant was UV-sensitive)
- its role in DNA replication is to remove
primers and fill in the gaps left behind
- for this it needs the nick-translation activity
The DNA Polymerase Family
A total of 5 different DNAPs have been reported
in E. coli
• DNAP I: functions in repair and replication
• DNAP II: functions in DNA repair (proven in
1999)
• DNAP III: principal DNA replication enzyme
• DNAP IV: functions in DNA repair (discovered in
1999)
• DNAP V: functions in DNA repair (discovered in
1999)
DNA Polymerase III
The "real" replicative polymerase in E. coli
• It’s fast: up to 1,000 dNTPs added/sec/enzyme
• It’s highly processive: >500,000 dNTPs added
before dissociating
• It’s accurate: makes 1 error in 107 dNTPs added,
with proofreading, this gives a final error rate of 1
in 1010 overall.