Replication

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Replication RNA Synthesis Decoding the Genetic
Code
Noel Murphy
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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
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DNA is the Genetic Material

• Therefore it must
• Replicate faithfully.
• 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

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Replication
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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."

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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
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MODELS OF DNA REPLICATION
(a) Hypothesis 1
(b) Hypothesis 2
(c) Hypothesis 3
Semi-conservative replication
Conservative replication
Dispersive replication
Intermediate molecule
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Testing Models for DNA replication
Matthew Meselson and Franklin Stahl (1958)
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Meselson and Stahl
Semi-conservative replication of DNA
Isotopes of nitrogen (non-radioactive) were used
in this experiment
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Generations 0 0.3 0.7 1.0 1.1 1.5 1.9 2.5
3.0 4.1 0 and 1.0 mixed 0 and 4.1 mixed
HH HL LL HL
Equilibrium Density Gradient Centrifugation Detect
ion 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
HH
HL
LH
HL
LL
LL
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DNA replication
Nucleotides are successively added using
deoxynucleoside triphosphosphates (dNTPs)
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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.
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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.

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Replication can be Uni- or Bidirectional
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John Cairns
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.
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Evidence points to bidirectional replication
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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

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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 3OH end
(primer) In vitro assay for DNA synthesis
Used the assay to purify a DNA polymerizing
enzyme DNA polymerase I
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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
-therefore DNA synthesis occurs only in the 5 to
3 direction
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THIS LEADS TO A CONCEPTUAL PROBLEM
Consider one replication fork
Continuous replication
Discontinuous replication
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Evidence for the Semi-Discontinuous replication
model was provided by the Okazakis (1968)
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Evidence for Semi-Discontinuous
Replication (pulse-chase experiment)
Harvest the bacteria at different times after the
chase
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
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Results of pulse-chase experiment
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DNA replication is semi-discontinuous
Continuous synthesis
Discontinuous synthesis
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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

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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

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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
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• 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.

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Proof reading activity of the 3 to 5
exonuclease. DNAPI stalls if the incorrect ntd
is added - it cant 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.
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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
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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

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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

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So if its 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 (Cairns- deLucia
  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

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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)

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DNA Polymerase III

• The "real" replicative polymerase in E. coli
• Its fast up to 1,000 dNTPs added/sec/enzyme
• Its highly processive gt500,000 dNTPs added
  before dissociating
• Its accurate makes 1 error in 107 dNTPs added,
  with proofreading, this gives a final error rate
  of 1 in 1010 overall.