Download DNA: The Genetic Material

Chapter 14
Lecture Outline
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DNA: The Genetic Material
Chapter 14
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Frederick Griffith – 1928
• Studied Streptococcus pneumoniae, a
pathogenic bacterium causing pneumonia
• 2 strains of Streptococcus
– S strain is virulent
– R strain is nonvirulent
• Griffith infected mice with these strains
hoping to understand the difference
between the strains
3
• Griffith’s results
– Live S strain cells killed the mice
– Live R strain cells did not kill the mice
– Heat-killed S strain cells did not kill the
mice
– Heat-killed S strain + live R strain cells
killed the mice
4
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Live Nonvirulent
Strain of
S. pneumoniae
Live Virulent
Strain of S. pneumoniae
Polysaccharide
coat
Mice die
a.
Mice live
b.
5
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Mixture of Heat-killed Virulent
and Live Nonvirulent
Strains of S. pneumoniae
Heat-killed Virulent
Strain of S. pneumoniae
+
Mice die
Their lungs contain live
pathogenic strain of
S. pneumoniae
Mice live
c.
d.
6
• Transformation
– Information specifying virulence passed
from the dead S strain cells into the live
R strain cells
• Our modern interpretation is that genetic
material was actually transferred between
the cells
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Avery, MacLeod, & McCarty – 1944
• Repeated Griffith’s experiment using
purified cell extracts
• Removal of all protein from the transforming
material did not destroy its ability to
transform R strain cells
• DNA-digesting enzymes destroyed all
transforming ability
• Supported DNA as the genetic material
8
Hershey & Chase –1952
• Investigated bacteriophages
– Viruses that infect bacteria
• Bacteriophage was composed of only
DNA and protein
• Wanted to determine which of these
molecules is the genetic material that is
injected into the bacteria
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• Bacteriophage DNA was labeled with
radioactive phosphorus (32P)
• Bacteriophage protein was labeled with
radioactive sulfur (35S)
• Radioactive molecules were tracked
• Only the bacteriophage DNA (as indicated
by the 32P) entered the bacteria and was
used to produce more bacteriophage
• Conclusion: DNA is the genetic material
10
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35S-Labeled
Bacteriophages
+
Phage grown in radioactive 35S,
which is incorporated into phage coat
Virus infect
bacteria
32P-Labeled
Blender separates
phage coat from bacteria
Centrifuge forms
bacterial pellet
35S
in supernatant
Bacteriophages
+
Phage grown in radioactive 32P.
which is incorporated into phage DNA
Virus infect
bacteria
Blender separates
Centrifuge forms
phage coat from bacteria bacterial pellet
32P
in bacteria pellet
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DNA Structure
• DNA is a nucleic acid
• Composed of nucleotides
– 5-carbon sugar called deoxyribose
– Phosphate group (PO4)
• Attached to 5′ carbon of sugar
– Nitrogenous base
• Adenine, thymine, cytosine, guanine
– Free hydroxyl group (—OH)
• Attached at the 3′ carbon of sugar
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Nitrogenous Base
Nitrogenous base
NH2
1
8
Phosphate group
2
O
N
9
P
O
4
N
N C C
H
N
C
H
N C
C
N C C
N
H
N C
C
NH2
C
H
N
H
3
H
Adenine
CH2
N
Guanine
5′
O–
1′
4′
3′
2′
OH in RNA
OH
Sugar
O
NH2
O
H in DNA
Pyrimidines
–O
5
O
NH2
6
Purines
7N
H
C
H
C
C
N
N
C
O
H
Cytosine
(both DNA and RNA)
H3C
C
H
C
C
N
O
N
H
H
C
C
O
H
C
H
Thymine
(DNA only)
C
N
N
H
C
O
H
Uracil
(RNA only)
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• Phosphodiester
bond
– Bond between
adjacent nucleotides
– Formed between the
phosphate group of
one nucleotide and
the 3′ —OH of the
next nucleotide
• The chain of
nucleotides has a 5′to-3′ orientation
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5′
PO4
Base
CH2
O
C
O
Phosphodiester
bond
–O
O
P
O
Base
CH2
O
OH 3′
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Chargaff’s Rules
• Erwin Chargaff determined that
– Amount of adenine = amount of thymine
– Amount of cytosine = amount of guanine
– Always an equal proportion of purines
(A and G) and pyrimidines (C and T)
15
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Rosalind Franklin
• Performed X-ray diffraction
studies to identify the 3-D
structure
– Discovered that DNA is helical
– Using Maurice Wilkins’ DNA
fibers, discovered that the
molecule has a diameter of
2 nm and makes a complete
turn of the helix every 3.4 nm
a.
b.
Courtesy of Cold Spring Harbor Laboratory Archives
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James Watson and Francis
Crick – 1953
• Deduced the structure of DNA using
evidence from Chargaff, Franklin, and
others
• Did not perform a single experiment
themselves related to DNA
• Proposed a double helix structure
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Double helix
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5´
Phosphate group
P
• 2 strands are polymers
of nucleotides
• Phosphodiester
backbone – repeating
sugar and phosphate
units joined by
phosphodiester bonds
• Wrap around 1 axis
• Antiparallel
5
O
1
4
3
Phosphodiester bond
2
P
5
O
4
1
3
P
2
5
O
1
4
5-carbon sugar
3
2
Nitrogenous base
P
5
O
1
4
3
2
OH
3
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2nm
5′
A
T
G
Minor
groove
3′
3.4nm
C
T
G
A
T
0.34nm
C
G
Major
groove
G
A
G
T
C
Major
groove
Minor
groove
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3′
5′
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Hydrogen
bond
• Complementarity of
bases
• A forms 2 hydrogen
bonds with T
• G forms 3 hydrogen
bonds with C
• Gives consistent
diameter
O
N
H
N
G
N
H
H
H
N
N
H
N
Sugar
H
C
N
N
H
Sugar
H
Hydrogen
bond
H
N
H
N
N
Sugar
A
N
CH3
O
H
H
N
H
T
N
N
H
Sugar
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DNA Replication
3 possible models
1. Conservative model
2. Semiconservative model
3. Dispersive model
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Conservative
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Conservative
Semiconservative
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Conservative
Semiconservative
Dispersive
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Meselson and Stahl – 1958
• Bacterial cells were grown in a heavy
isotope of nitrogen, 15N
• All the DNA incorporated 15N
• Cells were switched to media containing
lighter 14N
• DNA was extracted from the cells at
various time intervals
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Meselson and Stahl’s Results
• Conservative model = rejected
– 2 densities were not observed after round 1
• Semiconservative model = supported
– Consistent with all observations
– 1 band after round 1
– 2 bands after round 2
• Dispersive model = rejected
– 1st round results consistent
– 2nd round – did not observe 1 band
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DNA
E. coli
15N
medium
14N
medium
0 min
0 rounds
E. coli cells grown
in 15N medium
Cells shifted to
14N medium and
allowed to grow
20 min
1 round
40 min
2 rounds
Samples taken at
three time points
and suspended in
cesium chloride
solution
Samples are centrifuged
0 rounds
1 round
2 rounds
0
1
2
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Top
Bottom
Rounds of
replication
From M. Meselson and F.W. Stahl/PNAS 44(1958):671
DNA Replication
• Requires 3 things
– Something to copy
• Parental DNA molecule
– Something to do the copying
• Enzymes
– Building blocks to make copy
• Nucleotide triphosphates
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• DNA replication includes
– Initiation – replication begins
– Elongation – new strands of DNA are
synthesized by DNA polymerase
– Termination – replication is terminated
29
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Template Strand
HO
New Strand
3′
Template Strand
HO
5′
G
5′
P
C
G
O
O
Sugar–
phosphate
backbone
3′
P
C
New Strand
O
O
P
P
P
T
A
P
T
A
O
O
O
O
P
P
P
A
DNA polymerase III
O
T
O
P
A
P
P
P
C
O
G
P
C
O
G
O
O
P
P
3′
P
OH
A
A
O
P
T
P
P
O
P
O
T
A
OH
O
OH
P
P
Pyrophosphate
A
O
5′
P
P
O
3′
P
O
T
O
5′
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5
3
5
RNA polymerase makes primer
5
3
3
5
DNA polymerase extends primer
• DNA polymerase
– Matches existing DNA bases with
complementary nucleotides and links them
– All have several common features
• Add new bases to 3′ end of existing strands
• Synthesize in 5′-to-3′ direction
• Require a primer of RNA
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Prokaryotic Replication
• E. coli model
• Single circular molecule of DNA
• Replication begins at one origin of
replication
• Proceeds in both directions around the
chromosome
• Replicon – DNA controlled by an origin
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Replisome
Origin
Termination
Termination
Replisome
Origin
Termination
Origin
Termination
Termination
Origin
Origin
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• E. coli has 3 DNA polymerases
– DNA polymerase I (pol I)
• Acts on lagging strand to remove primers
and replace them with DNA
– DNA polymerase II (pol II)
• Involved in DNA repair processes
– DNA polymerase III (pol III)
• Main replication enzyme
– All 3 have 3′-to-5′ exonuclease activity –
proofreading
– DNA pol I has 5′-to-3′ exonuclase activity
34
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Supercoiling
Replisomes
No Supercoiling
Replisomes
DNA gyrase
• Unwinding DNA causes torsional strain
– Helicases – use energy from ATP to unwind
DNA
– Single-strand-binding proteins (SSBs) coat
strands to keep them apart
– Topoisomerase prevent supercoiling
• DNA gyrase is used in replication
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Semidiscontinous
• DNA polymerase can synthesize only in 1
direction
• Leading strand synthesized continuously
from an initial primer
• Lagging strand synthesized
discontinuously with multiple priming
events
– Okazaki fragments
36
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5′
3′
Lagging strand
(discontinuous)
First RNA primer
5′
3′
Open helix and
replicate further
Open helix
and replicate
Second RNA primer
5′
3′
3′
3′
5′
5′
RNA primer
5′
3′
Leading strand
(continuous)
RNA primer
5′
3′
37
• Partial opening of helix forms replication
fork
• DNA primase – RNA polymerase that
makes RNA primer
– RNA will be removed and replaced with
DNA
38
Leading-strand synthesis
– Single priming event
– Strand extended by DNA pol III
• Processivity –  subunit forms “sliding
clamp” to keep it attached
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a.
b.
a-b: From Biochemistry by Stryer. © 1975, 1981, 1988, 1995 by Lupert Stryer. Used with permission of W.H. Freeman and Company
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Lagging-strand synthesis
– Discontinuous synthesis
• DNA pol III
– RNA primer made by primase for each
Okazaki fragment
– All RNA primers removed and replaced by
DNA
• DNA pol I
– Backbone sealed
• DNA ligase
• Termination occurs at specific site
– DNA gyrase unlinks 2 copies
40
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5′
DNA ligase
Lagging strand
(discontinuous)
RNA primer
DNA polymerase I
Okazaki fragment
made by DNA
polymerase III
Primase
Leading strand
(continuous)
3′
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Replisome
• Enzymes involved in DNA replication form
a macromolecular assembly
• 2 main components
– Primosome
• Primase, helicase, accessory proteins
– Complex of 2 DNA pol III
• One for each strand
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Replication fork
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New bases
β clamp (sliding clamp)
Leading strand
3
5
Single-strand binding
proteins (SSB)
Clamp loader
DNA gyrase
Open β clamp
5
3
Parent
DNA
Helicase
Primase
DNA
polymerase III
Lagging strand
Okazaki fragment
New bases
3
5
DNA
polymerase I
DNA ligase
RNA primer
43
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DNA polymerase III
Leading strand
Helicase
DNA gyrase
5´
3´
5´
3´
Clamp loader
5´
3´
5´
3´
First Okazaki
fragment
RNA primer
RNA primer
Primase
Single-strand
binding proteins
(SSB)
β clamp
5´
3´
Lagging strand
Loop
grows
Second Okazaki
fragment nears
completion
5´
3´
RNA primer
1. A DNA polymerase III enzyme is active on each strand. Primase
synthesizes new primers for the lagging strand.
2. The “loop” in the lagging-strand template allows replication to
occur 5´-to- 3´ on both strands, with the complex moving to the left.
5´
3´
5´
3´
DNA polymerase III
DNA polymerase I
Lagging
strand
releases
5´
3´
β clamp
releases
3. When the polymerase III on the lagging strand hits the previously
synthesized fragment, it releases the β clamp and the template
strand. DNA polymerase I attaches to remove the primer.
44
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5´
3´
Clamp loader
5´
3´
5´
3´
DNA ligase
patches “nick”
Leading strand
replicates
continuously
5´
3´
DNA polymerase I
detaches after
removing RNA primer
4. The clamp loader attaches the β clamp and transfers this
to polymerase III, creating a new loop in the lagging-strand
template. DNA ligase joins the fragments after DNA
polymerase I removes the primers.
5´
3´
Loop
grows
New bases
5´
3´
5. After the β clamp is loaded, the DNA polymerase III on
the lagging strand adds bases to the next Okazaki fragment.
45
Eukaryotic Replication
• Complicated by
– Larger amount of DNA in multiple
chromosomes
– Linear structure
• Basic enzymology is similar
– Requires new enzymatic activity for
dealing with ends only
46
• Multiple replicons – multiple origins of
replications for each chromosome
– Not sequence specific; can be adjusted
• Initiation phase of replication requires more
factors to assemble both helicase and primase
complexes onto the template, then load the
polymerase with its sliding clamp unit
– Primase includes both DNA and RNA polymerase
– Main replication polymerase is a complex of DNA
polymerase epsilon (pol ε) and DNA polymerase delta
(pol δ)
47
Telomeres
• Specialized structures found on the ends
of eukaryotic chromosomes
• Protect ends of chromosomes from
nucleases and maintain the integrity of
linear chromosomes
• Gradual shortening of chromosomes with
each round of cell division
– Unable to replicate last section of lagging
strand
48
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Replication first round
5´
3´
3´
5´
Leading strand (no problem)
Lagging strand (problem at the end)
5´
3´
3´
5´
Last primer
Origin
Leading
strand
Primer removal
3´
5´
5´
Lagging
strand
3´
Replication second round
Removed primer
cannot be replaced
5´
3´
3´
5´
5´
3´
3´
5´
Shortened template
49
• Telomeres composed of short repeated
sequences of DNA
• Telomerase – enzyme makes telomere section
of lagging strand using an internal RNA template
(not the DNA itself)
– Leading strand can be replicated to the end
• Telomerase developmentally regulated
– Relationship between senescence and telomere
length
• Cancer cells generally show activation of
telomerase
50
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5́
T
T
G
3́
Synthesis by telomerase
Telomerase
5́
3́
T
A
T
A
G
C
G
G
C
Telomere
extended
by telomerase
G
C
C
A
A
C
Template RNA is
part of enzyme
Telomerase moves and
continues to extend telomere
5́
3́
T
T
G
G
G
Now ready
to synthesize
next repeat
G
T
T
G
A
A
C
C
C
C
A
A
C
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DNA Repair
• Errors due to replication
– DNA polymerases have proofreading ability
• Mutagens – any agent that increases the
number of mutations above background
level
– Radiation and chemicals
• Importance of DNA repair is indicated by
the multiplicity of repair systems that have
been discovered
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DNA Repair
Falls into 2 general categories
1. Specific repair
– Targets a single kind of lesion in DNA and
repairs only that damage
2. Nonspecific
– Use a single mechanism to repair multiple
kinds of lesions in DNA
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Photorepair
• Specific repair mechanism
• For one particular form of damage caused
by UV light
• Thymine dimers
– Covalent link of adjacent thymine bases in
DNA
• Photolyase
– Absorbs light in visible range
– Uses this energy to cleave thymine dimer
54
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DNA with adjacent thymines
T
A
T
A
UV light
Helix distorted by
thymine dimer
Thymine dimer
A
A
Photolyase binds
to damaged DNA
Photolyase
A
A
Visible light
Thymine dimer
cleaved
T
A
T
A
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Excision repair
• Nonspecific repair
• Damaged region is removed and replaced
by DNA synthesis
• 3 steps
1. Recognition of damage
2. Removal of the damaged region
3. Resynthesis using the information on the
undamaged strand as a template
56
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Damaged or incorrect base
Excision repair enzymes recognize damaged DNA
Uvr A,B,C complex
binds damaged DNA
Excision of damaged strand
Resynthesis by DNA polymerase
DNA polymerase
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