Chapter 14
DNA
1
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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
2
http://o.quizlet.com/i/GEJK81oHlTEYutTzmSQK6Q.jpg
Griffith’s Experiment
• Griffith infected mice with these strains
hoping to understand the difference
between the strains
3
Griffith’s Experiment
Live Nonvirulent
Strain of
S. pneumoniae
Live Virulent
Strain of S. pneumoniae
Polysaccharide
coat
Mice die
Mice live
4
a.
b.
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Griffith’s Experiment
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.
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5
• 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
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
7
Avery, MacLeod, & McCarty – 1944
• Repeated Griffith’s experiment using
purified cell extracts
8
http://biology.kenyon.edu/courses/biol114/KH_lecture_images/How_DNA_works/FG11_02.JPG
Avery, MacLeod, & McCarty – 1944
• Results:
– 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
9
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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Hershey & Chase –1952
• Bacteriophage DNA was labeled with
radioactive phosphorus (32P)
• Bacteriophage protein was labeled with
radioactive sulfur (35S)
• Radioactive molecules were tracked
11
Hershey & Chase –1952
• Results:
– 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
13
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
14
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)
17
Representation of Chargaff’s Data
Table (1952)
Organis
m
%A
%G
%C
%T
A/T
G/C
%GC
%AT
φX174
24.0
23.3
21.5
31.2
0.77
1.08
44.8
55.2
Maize
26.8
22.8
23.2
27.2
0.99
0.98
46.1
54.0
Octopus
33.2
17.6
17.6
31.6
1.05
1.00
35.2
64.8
Chicken
28.0
22.0
21.6
28.4
0.99
1.02
43.7
56.4
Rat
28.6
21.4
20.5
28.4
1.01
1.00
42.9
57.0
Human
29.3
20.7
20.0
30.0
0.98
1.04
40.7
59.3
Grassho
pper
29.3
20.5
20.7
29.3
1.00
0.99
41.2
58.6
Sea
Urchin
32.8
17.7
17.3
32.1
1.02
1.02
35.0
64.9
Wheat
27.3
22.7
22.8
27.1
1.01
1.00
45.5
54.4
Yeast
31.3
18.7
17.1
32.9
0.95
1.09
35.8
64.4
E. coli
24.7
26.0
25.7
23.6
1.05
1.01
51.7
48.3
http://en.wikipedia.org/wiki/Chargaff%27s_rules
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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
19
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
20
Antiparallel Nature of DNA
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/dsDNA.jpg
22
DNA Replication
3 possible models
1. Conservative model
2. Semiconservative model
3. Dispersive model
25
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Conservative
26
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Conservative
Semiconservative
27
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Conservative
Semiconservative
Dispersive
28
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
29
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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
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 Replication
• Requires 3 things
• Something to copy
• Parental DNA molecule
• Something to do the copying
• Enzymes
• Building blocks to make copy
• Nucleotide triphosphates
32
• DNA replication includes
• Initiation – replication begins
• Elongation – new strands of DNA are
synthesized by DNA polymerase
• Termination – replication is terminated
33
DNA Replication Enzymes
• Bacteria
• Primase – makes RNA primer
• DNA polymerase III – synthesizes DNA
• DNA polymerase I – erases primer and fills gap
• Helicase – unwinds double helix
• Gyrase – relieves torque
• DNA ligase – links okazaki fragments, repair
• Eukaryotic
• Similar to prokaryotic but more complex
• Primase – complex of RNA and DNA polymerase
• DNA polymerase delta and DNA polymerase epsilon
• Telomerase – fixes telomeres on ends of chromosomes
34
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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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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
37
• Bacteria
• 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
39
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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
40
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
41
• Partial opening of helix forms replication
fork
• DNA primase – RNA polymerase that
makes RNA primer
– RNA will be removed and replaced with
DNA
43
Leading-strand synthesis
– Single priming event
– Strand extended by DNA pol III
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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
44
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
45
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
47
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51
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
52
• 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)
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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
54
http://www.scientificamerican.com/media/inline/telomeres-telomerase-and_1.jpg
• 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
56
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
58
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
59
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
60
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
62