central dogma of biology. dna replication dna structure
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Central Dogma of Biology
DNA Replication
DNA Structure
DNA Replication
• is semi-conservative
• is bi-directional
• is semi-discontinuous
• initiation, elongation, termination
• contrasted in prokaryotes and eukaryotes
Is replication semi-conservative?
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
Predictions After One Generation of Replication
Conservative Replication Refuted
Predictions After Two Generations
Dispersive Replication Refuted
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
Is replication unidirectional or bidirectional?
2 unzipping forks1 unzipping fork
Observation
Two PossibleInterpretations
unidirectional replication
bidirectional replication
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
Is replication continuous?
Continuous Replication
The Problem
1. strands are antiparallel
5’
3’
3’
5’
nucleotide
Continuous replication requires that one strand be elongated in a 3’ to 5’ direction
3’
5’
5’3’
5’
3’
5’ 3’
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
Why are lagging strands formed by fragments?
Alternatives: whole molecule unzips first before lagging strand is formed
Alternatives: lagging strand formed of small fragments that get connected
Fragment Formation
3' to 5' strand elongation
5' to 3' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
Practice
Practice
5' to 3' strand elongation
3' to 5' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
L S & L S & L
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
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
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
5' to 3' strand elongation
3' to 5' strand elongation
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
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
5' to 3' strand elongation
3' to 5' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
Why are lagging strands formed by fragments?
Alternatives: whole molecule unzips first before lagging strand is formed
Alternatives: lagging strand formed of small fragments that get connected
Fragment Formation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
5' to 3' strand elongation
3' to 5' strand elongation
Practice
Practice
5' to 3' strand elongation
3' to 5' strand elongation
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
L S & L S & L
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
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
Replication Hypotheses
Ligase+
Ligase-
Continuous Semi-Discontinuous Discontinuous
5' to 3' strand elongation
3' to 5' strand elongation
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
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
Initiation
Inititation at Origin of Replication
Primase and primer formation
Elongation
Elongation - 4 stepsRNA primer
elongation with dNTPs
previous fragment
ligation
elongation with dNTPswith upstream primer removal
Fragment Formation
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
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)
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.
Replication Fork with Primosomesand Separate DNA Polymerases
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
Replication Fork with Replisomes
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
DNA Repair
The double strandedness of DNA enables:
• recognition of mutation sites
• replacement of excised nucleotides with complementary nucleotides
Single Nucleotides or Whole Sequences Can Be Excised and Replaced