DNA Replication I: Basic Mechanism and Enzymology

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DNA Replication I Basic Mechanism and Enzymology
Liuqing Yang
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Contents

• General Features of DNA Replication
• Enzymology of DNA Replication
• DNA Damage and Repair

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Semicomservative Replication
There are three hypothesis about DNA replication
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Mix DNA containing nomal isotope of nitrogen(14N)
and DNA labeled with a heavy isotope of nitrogen
(15N), then, subject it to cesium chloride
density gradient centrifugation
Two DNAs localization
The amounts of DNA
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Three hypothesis of DNA replication
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The result of cesium chloride density gradient
centrifugation Left light DNA Right
heavy DNA
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Behaviors of heat-denatured DNAs in
ultracentrifugation
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Semidiscontinuous Replication
Three hypothesis of DNA replication
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• DNA polymerase of all natural replicating
  machines can make DNA in only one direction 5
  3.
• So Reiji Okazaki concluded that both strands
  could not replicate continuously

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• He proposed a Semidiscontinuous Replication
  model
• One stand (the leading stand) continuously in
  direction, the other srand (the lagging strand)
  would have to be made discontinuously as show in
  previous figure

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• Okazaki made two predictions derived from his
  model
• Lable newly synthesized DNA and check it in very
  short periods, we should get DNA in short pieces
• Eliminate DNA ligase, this short pieces of DNA
  ought to be detectable even with ralatively long
  period
• Then his research team tested these predictions
  experimentally

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Priming of DNA Synthesis

• The replication of M13 phage DNA by an E.coli
  extract is inhibited by a inhibitor of E.coli RNA
  polymerase
• Tuneko Okazaki, wife and scientific colleague of
  Reiji Okazaki, found that DNase can not
  completely destroy Okazaki fragments, it leaved
  little pieces of RNA 10-12 bases long

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The explanation of previously two phenomena is
that the DNA replication needs a RNA
primer, which is 10-12 bases long. M13 phage DNA
uses the E.coli RNA polymerase to make
RNA primers for its replication.
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Tuneko Okazaki used the capping enzyme, guanylyl
transferase, and alpha-32PGTP to lable the
5-ends of only intact primer, after DNase
Incubation, then subject it to
gel electrophoresis a-d before DNase
incubation e-h after DNase incubation a and e
cells were defective in RNase H b and f cells
were defective in the nuclease activity of
DNA pol I c and g cells were defective in
both d and h wild type
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Bidirectional Replication
The theta mode of DNA replication in E.coli
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Experimental demonstration of bidirectional DNA
replication first, use low radioactivity DNA
precursor, after the bubble had grown somewhat,
a more radioactivity DNA precursor added Right
diograph interpretation
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Bidirectional DNA replication in eukaryotes Low
radioactivity DNA precursor added after high
radioactivity
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Autoradiograph of replicating embryonic Triturus
vulgaris DNA Note the constant size and shape of
the pairs of streaks, suggesting that all the
coresponding replicons bigan replicating at the
same time
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Rolling Circle Replication

• The E.coli phages with single-stranded circular
  DNA genomes, such as phineX174, use a relatively
  simple form of rolling circle replication in
  which a double-stranded replicative form (RFI)
  give rise to many copies of a single-stranded
  progeny DNA

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Schematic representation of rolling circle
replication
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Strand Separation

• Helicase can harnesses the chemical energy of ATP
  to separate the two parental DNA strands at the
  replicating fork
• Use a E.coli strain carrying a temperature-sensiti
  ve mutations in the dna B gene, when the
  temperature rise to the nonpermissive level, the
  DNA synthesis halt immediately
• We would expect if the dna B encodes helicase

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DNA helicase assay
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Dna B alone had halicase activity This was
stimulated by Dna G and SSB
substrates
products
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Single-Strand DNA-Binding Proteins

• SSBs do not catalyze strands separation, they
  bind selectively to single-stranded DNA as soon
  as it forms and prevent its annealling
• The T4 phage SSB is gp32, the M13 phage SSB is
  gp5, all of these proteins act cooperatively (the
  binding of one protein facilitates the binding of
  the next)

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SSBs also have other functions, Such
as Stimulation of DNA synthesis
Right figure stimulation of DNA synthesis By the
T4 phage SSB, gp32
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Temperature-sensitivity of DNA Synthesis in
cells infected by T4 Phage with a
temperature- Sensitive mutation in the SSB
Gene Blue gene 23 mutant Red gene
23 and 32 mutant Yellow gene 32 and 49 mutant
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Prevent DNA degradation Degradation of T4 DNA in
cells infected by T4 phage with
a temperature-sensitive mutation in gene 32
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Topoisomerases

• Topoisomerases can release the positive
  supercoils, which were caused by the proceeding
  of the replication fork. It can also introduce
  negative supercoils into DNA

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Assay for a DNA Topoisomerases
Incubate relexed circular ColE1 DNA with
varying amounts of E.coli gyrase
(topoisomerases) Lane 1 positive
control Lane 2 no gyrase Lane 3-10 gyrase
increasing Lane 11 no ATP Lane 12 no
spermidine (???) Lane 13 no
MgCl2 Lane 14 supercoiled DNA incubated with
gyrase without ATP as a ctr
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DNA polymerase I

• Pol I (a single 102-KD polypeptide chain)
• DNA polymerase activity
• 3 5 exonuclease activity (proofreading)
• 5 3 exonuclease activity

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• Pol I cleaved by mild proteolytic treatment into
  two polypeptide
• Klenow fragment has DNA polymerase activity and
  proofreading activity, be often used to sequence
  a DNA
• A small fragment has 5 3 exonuclease activity
• The whole Pol I is used to lable a probe in vitro

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• A pol I laked mutant still has DNA replicating
  activity strongly suggests that Pol I is not the
  DNA replicating enzyme
• Following experiments indicated that Pol III is
  the enzyme replicates E.coli DNA

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The Pol III Holoenzyme
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Identify which subunit responsible for the DNA
polymerase activity
Separation of pol III and the alpha unit from a
cell that over-expresses the alpha unit
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Purification and identify the function of the E
subunit
purification
Proofreading function
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Damage Caused by Alkylation of Bases
Alkyl groups uaually are electrophiles(????),
they can seek centers of negative charge
in other molecules This figure shows
electron-rich centers in DNA
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Mispair caused by Alkylation
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Damage caused by UV
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DNA repair machanisms

• Directly undoing DNA damage
• Excision repair
• Double-srand break repair
• Mismatch repair

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Two models of Directly undoing DNA damage
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Two types of excision repair
Base excision repair
Nucleotide excision repair
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Double-strand break repair

• Homologous recombination
• Nonhomologous end-jioning

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Mismatch repair in E.coli
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Coping with DNA without repair it

• Recombination repair (post replication repair)
• Error-prone bypass
• Error-free bypass (human cells)

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An overview of error-prone Bypass process
In human error-free Bypass these two
bases Displaced by AA
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Thank you !