central dogma of biology. dna replication dna structure
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Central Dogma of Biology
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DNA Replication
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DNA Structure
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DNA Replication
• is semi-conservative
• is bi-directional
• is semi-discontinuous
• initiation, elongation, termination
• contrasted in prokaryotes and eukaryotes
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Is replication semi-conservative?
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DNA Replication
• the two DNA parent strands can unzip (breaking weak H bonds)• the resulting single strands are “sticky” and can act as templates • template strands order the elongation of new strands• new strands are elongated by the addition of complementary dNTPs
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Three Hypotheses
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Predictions After One Generation of Replication
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Conservative Replication Refuted
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Predictions After Two Generations
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Dispersive Replication Refuted
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Another test of semi-conservative replication
chromatid 1on left
chromatid 2on left
dNTP*
dNTP
dNTP
A
A B C D
chromatid 1on left
chromatid 2on right
dNTP*
dNTP
dNTP
A
chromatid 1on left
chromatid 2on left
dNTP*
dNTP
dNTP
A
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Is replication unidirectional or bidirectional?
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2 unzipping forks1 unzipping fork
Observation
Two PossibleInterpretations
unidirectional replication
bidirectional replication
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Test: pulse-chase experiment
Allow replication over a short time course, with replacement of precursors:
cold,then hot (very radioactive), then warm (less radioactive)
unidirectional replication
bidirectional replication
predictions
observations
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Is replication continuous?
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Continuous Replication
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The Problem
1. strands are antiparallel
5’
3’
3’
5’
nucleotide
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Continuous replication requires that one strand be elongated in a 3’ to 5’ direction
3’
5’
5’3’
5’
3’
5’ 3’
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2. but new strands are only synthesized in a 5’ to 3’ orientation
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3' to 5' strand elongation
5' to 3' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
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Why are lagging strands formed by fragments?
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Alternatives: whole molecule unzips first before lagging strand is formed
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Alternatives: lagging strand formed of small fragments that get connected
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Fragment Formation
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3' to 5' strand elongation
5' to 3' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
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Practice
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Practice
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5' to 3' strand elongation
3' to 5' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
L S & L S & L
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With Functional Ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
ContinuousSemi-Discontinuous
Discontinuous
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With functional ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
observed
ContinuousSemi-Discontinuous
Discontinuous
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Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
5' to 3' strand elongation
3' to 5' strand elongation
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With non-functional ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
ContinuousSemi-Discontinuous Discontinuous
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With non-functional ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
observed
ContinuousSemi-Discontinuous Discontinuous
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5' to 3' strand elongation
3' to 5' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
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Why are lagging strands formed by fragments?
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Alternatives: whole molecule unzips first before lagging strand is formed
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Alternatives: lagging strand formed of small fragments that get connected
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Fragment Formation
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Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
5' to 3' strand elongation
3' to 5' strand elongation
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Practice
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Practice
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5' to 3' strand elongation
3' to 5' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
L S & L S & L
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With Functional Ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
ContinuousSemi-Discontinuous
Discontinuous
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With functional ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
observed
ContinuousSemi-Discontinuous
Discontinuous
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Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
5' to 3' strand elongation
3' to 5' strand elongation
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With non-functional ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
ContinuousSemi-Discontinuous Discontinuous
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With non-functional ligase
amountof new
sequences (radioactivity)
W X
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Y
Sequence Size
Centrifuge Tube
top bottom
amountof new
sequences (radioactivity)
Sequence Size
Centrifuge Tube
top bottom
Z
observed
ContinuousSemi-Discontinuous Discontinuous
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Initiation
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Inititation at Origin of Replication
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Primase and primer formation
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Elongation
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Elongation - 4 stepsRNA primer
elongation with dNTPs
previous fragment
ligation
elongation with dNTPswith upstream primer removal
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Fragment Formation
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DNA and RNA Polymerasesproofreading requires a previous nucleotide
A
G
C
T
A
A
A
T
T
A
G
C
T
A
A
A
U
U
DNAPolymerase
RNApolymerase elongation
proof-reading
elongation
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DNA Proof-reading
A
G
C
T
A
A
G
T
T
A
G
C
T
A
A
T
TG
A
A
G
C
T
A
A
A
T
T
G
A
G
C
T
A
A
G
A
T
T
Proof-reading requires the presence of a previous nucleotide before a new one can be added …RNA polymerase has no such requirement (primase either)
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Why are there RNA primers?
• New strands must be started in isolation (no 3’ OH on a previous nucleotide to act as an anchor)
• Only RNA polymerases (like primase) can begin in the open like that
• Why have no similar DNA polymerases evolved? Without proof-reading, a DNA primer would be a mutational hot-spot …better to have an RNA primer because the hot spot is more readily recognized (ribose nucleotides) and removed.
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Replication Fork with Primosomesand Separate DNA Polymerases
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Problems
• Kinetics of replication don’t reflect the kind of delay expected for disassociation and reassociation of the lagging polymerases
• SEM images show variable sized small loops associated with replication forks
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Replication Fork with Replisomes
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Replisome
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Why did DNA replace RNA as the hereditary molecule?
• Mutations usually include only one strand at first
• A double stranded nucleic acid stores information in the unaffected strand that can be used to correct mutations in the affected strand
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DNA Repair
The double strandedness of DNA enables:
• recognition of mutation sites
• replacement of excised nucleotides with complementary nucleotides
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Single Nucleotides or Whole Sequences Can Be Excised and Replaced