PRIMING OF DNA SYNTHESIS BY

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PRIMING OF DNA SYNTHESIS BY SHORT SEQUENCES OF
RNA (RED) DNA POLYMERASE USES THE PRIMERS AS
STARTING POINTS TO SYNTHESIZE PROGENY DNA STRANDS
(GREEN ARROWS)
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Okazakis Experiment Evidence for the RNA Primers
DETECTION OF RNA PRIMERS Okazaki fragments were
iso- lated and labeled with 32P DNA was then
digested with DNase Lanes a-d before
DNase Lanes e-h after DNase Lanes a,e
defective in RNase H Lanes b,f defective in DNA
Pol I Lanes c,g defective in both Lanes d,h
wild-type Primers turned out to be
12 nucleotides long (see ladder)
RNase H digests the RNA strand of an RNA/DNA
hybrid
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Okazaki fragment (11-13 NT)
RNA
RNA
DNA
DNA
RnasH
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DNA Replication

• In 1960s, John Cairns
• Theta replication in E. coli

Labeling with tritium-nucleotide
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E. Gyurasits and R. B. Wake discovered
bi-directional Replication
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Rolling Circle Replication Mode (or sigma mode)
Example single-stranded circular DNA phages
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concatemers
ROLLING-CIRCLE MODEL OF BACTERIOPHAGE l DNA
REPLICATION THIS SCHEME RELATES TO THE SYNTHESIS
OF DOUBLE-STRANDED DNA
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Enzymology of DNA Replication

• Strand separation Helicase
• Helicase Use ATP to separate the two parental
  DNA strands
• Single-strand DNA binding proteins
• Topoisomerases
• Three DNA polymerases

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Francois Jacob in 1968 discovered the
temperature-sensitive mutants in E. coli DNA
replication
Helicase mutant
EFFECTS ON DNA REPLICATION OF TWO
TEMPERATURE-SENSITIVE MUTANTS OF E. coli DNA
HELICASE (dnaB) REPLICATION AT PERMISSIVE
TEMPERATURE BLUE REPLICATION AT NON-PERMISSIVE
TEMPERATURE RED
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LeBowitz and McMacken in 1986 conceived an
experiment to assay the helicase activity
DNA HELICASE ASSAY
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DNA HELICASE ASSAY ALTHOUGH PRIMASE (DnaG) AND
SINGLE- STRAND BINDING PROTEIN (SSB) BOTH
STIMULATE DNA HELICASE (DnaB), NEITHER HAVE
HELICASE ACTIVITY OF THEIR OWN
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Single-Strand DNA Binding Protein

• Proteins do not have helicase activity
• Bind to single-stranded DNA
• Examples
• SSB in E. coli (act as a chain of tetramers)
• Gp32 (gene product 32) in T4 phage (act as a
  chain of monomers)
• Gp5 in M13 phage (act as a chain of dimers)
• Act cooperatively
• Binding of first one facilitates binding of the
  second and so on.
• Stimulate the DNA polymerase activity
• Protect the single-stranded DNA from degradation

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(No Transcript)
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• Stimulate the DNA polymerase activity

STIMULATION OF DNA SYNTHESIS BY THE SSB OF PHAGE
T4 (gp32) TEMPLATE WAS PARTIALLY
SINGLE-STRANDED DNA NUMBERS ON GRAPH REPRESENT
gp32 MOLECULES PER 10 NUCLEOTIDES NOTE THAT TOO
MUCH SSB INHIBITS DNA SYNTHESIS
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• Protect the single-stranded DNA from degradation

Wild type SSB
Temperature sensitive SSB
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Topoisomerases
Unwinding of DNA leads to tightening of the
helix. In the case of circular DNA, a super-coil
may form.
DNA replicates at 500 nt per second, generate
about 50 revolution per second
Cairns recognized the problem in 1963