DNA: The Genetic Material Frederick Griffith – 1928 · DNA: The Genetic Material Chapter 14...
Transcript of DNA: The Genetic Material Frederick Griffith – 1928 · DNA: The Genetic Material Chapter 14...
DNA: The Genetic Material
Chapter 14
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
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3
Griffith’s Experiments
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Griffith’s Experiments
5
• 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 bacterial cells
Griffith’s Results
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Avery, MacLeod, & McCarty – 1944
• Repeated Griffith’s experiment usingpurified cell extracts as transformingmaterial
• Removal of all protein did not destroy abilityto transform R strain cells
• DNA-digesting enzymes destroyed alltransforming ability
• Supported DNA as the genetic material
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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
• Only the DNA entered the bacteria and
was used to produce more bacteriophage
• Conclusion: DNA is the genetic material8
Hershey & Chase
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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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Pu
rin
es
Pyri
mid
ines
Adenine Guanine
NH2CC
NN
N
C
H
N
C
CH
O
H
H
OC
NC
H
N
C
NH2
H
CH O
O
C
NC
H
N
CH3C
CH
H
O
O
C
NC
H
N
CH
CH
NH2
CC
NN
N
C
H
N
C
CHH
Nitrogenous Base
4´
5´
1´
3´ 2´
2
8
7 6
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4
5
1
O
P
O–
–O
Phosphate group
Sugar
Nitrogenous base
O CH2
N N
O
N
NH2
OH in RNA
Cytosine
(both DNA and RNA)Thymine
(DNA only)
Uracil
(RNA only)
OH
H in DNA
• Phosphodiesterbond– Bond between
adjacentnucleotides
– Formed betweenthe phosphategroup of onenucleotide and the3! —OH of thenext nucleotide
• Each DNA strandhas a 5! end and a3! end
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BaseCH2
O
5´
3´
O
P
O
OH
CH2
–O O
C
Base
O
PO4
Phosphodiester
bond
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)
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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
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James Watson and Francis Crick – 1953
• Deduced the double helix structure of DNA
using evidence from Chargaff, Franklin,
and others
Double helix
• 2 strands are polymers
of nucleotides
• Phosphodiester
backbone – repeating
sugar and phosphate
units joined by
phosphodiester bonds
• Antiparallel strands
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5´
3´
P
P
P
P
OH
5-carbon sugar
Nitrogenous base
Phosphate group
Phosphodiester bond
O
O
O
O
4´
5´
1´
3´ 2´
4´
5´
1´
3´ 2´
4´
5´
1´
3´ 2´
4´
5´
1´
3´2´
• Complementarity of
bases
• A forms 2 hydrogen
bonds with T
• G forms 3 hydrogen
bonds with C
• Gives consistent
diameter
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A
H
Sugar
Sugar
Sugar
Sugar
T
G C
N
H
N O
H
CH3
H
HN
N N H N
N
N
H
H
H
N O H
H
H N
N H
N
N HN N
Hydrogen
bond
Hydrogen
bond
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DNA Replication
• 3 possible models
1. Conservative model
2. Semiconservative model
3. Dispersive model
2nd
replication
Parental
DNA
Replicated
DNA
KEY
a. Semiconservative replication
1st
replication
b. Conservative replication c. Dispersive replication
1957 Meselson and Stahl - 3 Replication Models
Meselson-Stahl Experiment
1st replication
15N
medium 14N medium
2nd replication
Meselson-Stahl Experiment
(cont.)
15N–14N hybrid DNA
14N–14N (light) DNA
15N–15N (heavy) DNA
CsCl forms a density gradient
during centrifugation, with the
highest density at the bottom
of the tube.
DNA molecules move to positions
where their density equals that of the
CsCl solution and form bands. Shown
are the positions of differently labeled
DNA molecules. Experimentally the
bonds are detected by absorbance of
UV light.
Meselson-Stahl Experiment
(cont.)
15N–14N
hybrid DNA15N–15N
(heavy) DNA
DNA from15N medium
DNA after one
replication in 14N
15N–14N
hybrid DNA
DNA after two
replications in 14N
14N–14N
(light) DNA
Meselson-Stahl Experiment
Semiconservative
Conservative
Dispersive
The results support the semiconservative model.
Matches
results
Does not
match
results
Does not
match
results
Two replications in 14N15N medium One replication in 14N
Direction of
replication
OldNew
Complementary base
pairing in the DNA double
helix: A pairs with T, G
pairs with C.
The two chains unwind
and separate.
Each “old” strand is a
template for the addition
of bases according to the
base-pairing rules.
The result is two DNA
helices that are exact
copies of the parental
DNA molecule with one
“old” strand and one
“new” strand
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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
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Enzymes of DNA Replication
• DNA polymerases assemble nucleotides into a
chain
• Require 3! end (e.g., RNA primer)
• Synthesize in 5!-to-3! direction only
• Helicase unwinds the DNA
• Primase synthesizes RNA primer (starting point
for nucleotide assembly by DNA polymerases)
• Nuclease removes primers
• DNA ligase closes remaining single-chain nicks
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
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Assembling Antiparallel Strands Enzyme ActivitiesUnwinding enzyme
(helicase)
Primase
RNA
primers
Replication fork
Overall
direction of replication
1 Helicase unwinds the DNA,
and primases synthesize
short RNA primers.
RNA
Leading strand
Lagging strand
DNA polymerase
DNA polymerase
RNADNA
2 RNA primers are used as
starting points for the
addition of DNA nucleotides
by DNA polymerases.
Primer being extended
by DNA polymerase
DNA polymerase
Newly synthesized primer
DNA unwinds further, and
leading strand synthesis
proceeds continuously, while
a new primer is synthesized
on the lagging strand
template and extended by
DNA polymerase III.
3
DNA
polymerase
Nick Another type of DNA
polymerase (I) removes the
RNA primer, replacing it with
DNA, leaving a nick between
the newly synthesized
segments.
4
DNA ligase Nick is closed
by DNA ligase.5
Lagging strand
Leading strand
Newly
synthesized
primer
DNA polymerase
Primer being extended
by DNA polymerase III
6 DNA continues to unwind,
and the synthesis cycle
repeats as before:
continuous synthesis of
leading strand and synthesis
of a new segment to be
added to the lagging strand.
Prokaryotic Replication
• E. coli model
• Single circular molecule of DNA
• Replication begins at one origin of
replication
• Proceeds in both directions around the
chromosome
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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! exonuclease activity
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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
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
strand38
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• Telomeres composed of short repeatedsequences of DNA
• Telomerase – enzyme makes telomere oflagging 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 oftelomerase
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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
Excision repair
• Targets multiple types of lesions
• 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
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