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Human Genetics: concepts and applications
5th edition
Ricki Lewis
Chapter 9
DNA Structure and
Replication
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A history of DNA
Friedrich Miescher, 1871
isolated nuclei from pus and
identified nuclein later called nucleic acid.
Archibald Garrod, 1902
linked inheritance of “inborn errors of
metabolism” with the lack of particular
enzyme proteins
9-2
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Discovery of a “transforming principle”
Frederick Griffith, 1928
Pneumonia (Diplococcus pneumoniae) infects mice.
Mice develop pneumonia and die.
Two types of bacteria:
R bacteria rough coat - no pneumonia
S bacteria smooth coat- pneumonia
Coat type is associated with virulence.
9-3
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Griffith’s experiment identifying the “transforming principle”
Bacterial
colonies
+
Rough
nonvirulent
(strain R)
Smooth
virulent
(strain S)
Heat-killed Rough
Heat-killed
smooth
nonvirulent smooth
virulent
(strain R) virulent
(strain S)
(strain S)
Injection
Results
Mouse healthy
9-4
Mouse dies
Mouse healthy
Mouse dies
Live strain S bacteria
in blood sample
from dead mouse
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What is the “transforming principle”?
Oswald Avery, Colin MacLeod and Maclyn McCarty, 1944
Heat-killed S bacteria “transformed” the R bacteria
to a form that causes pneumonia
Conclusion: DNA is the transforming principle allowing R
bacteria to make a smooth coat and allow infection.
9-5
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DNA is the genetic material
Alfred Hershey and Martha Chase, 1953
Viruses can infect the E. coli bacteria.
A virus has protein “head” and DNA core.
Infection occurs when virus injects one of
them into a bacterial cell.
Which one is it?
9-6
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Hershey-Chase experiment demonstrating
DNA is the genetic material
Viral
protein coat
radioactively
labeled
(sulfur)
Viruses
infect
bacteria
Blended and
centrifuged to
Separate cells
from virus
Viral protein
coats
(radioactive)
Bacteria with
viral DNA
(nonradioactive)
9-7
Viral DNA
radioactively
labeled
(phosphorus)
Viral protein
coats
(nonradioactive)
Bacteria with
viral DNA
(radioactive)
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DNA (deoxyribonucleic acid) is
a chain of nucleotides
Nucleotides are composed of:
Sugar - deoxyribose
Phosphate
Base - one of four types:
9-8
adenine (A), thymine (T)
guanine (G), cytosine (C)
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DNA Properties:
Chargaff’s Findings
Amount of A’s = Amount
of T’s
Amount of G’s = Amount
of C’s.
9-9
Other:
Phosphate and sugar is
hydrophilic
Nitrogenous bases are
hydrophobic
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DNA Properties:
Rosalind Franklin’s X-Ray Crystallography
data = Double Helix
9-10
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DNA Structure - Watson and Crick
Built models
9-11
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DNA bases pair via hydrogen bonds
Validated Erwin Chargaff
observations:
# of adenine = # of thymine
# of guanine = # of cytosine
Complementary bases pair:
A and T pair
C and G pair
9-12
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DNA exists as a double helix
T
A
G
C
T
A
C
-Maurice Wilkins and Rosalind Franklin
C
G
X-ray diffraction indicated DNA
has a repeating structure and a
regular width.
G
T
DNA is double-stranded
molecules wound in a double
helix.
A
G
C
A
T
-James Watson and Francis Crick
9-13
G
C
T
A
C
G
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Orientation of DNA
The carbon atoms on the sugar ring are
numbered for reference. The 5’ and 3’
hydroxyl groups (highlighted on the left)
are used to attach phosphate groups.
9-14
The directionality of a DNA strand is due to the
orientation of the phosphate-sugar backbone.
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DNA is a double helix
P
T
A
P
G
5’
3’
P
A sugar and phosphate
“backbone” connects
nucleotides in a chain.
C
DNA has directionality.
PP
P
C
G
A
P
P
Two nucleotide chains
together wind into a helix.
T
P
P
G
Hydrogen bonds between
paired bases hold the two
DNA strands together.
C
P
P
3’
9-15
C
G
P
DNA strands are antiparallel.
5’
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Roles of the genetic material
“A genetic material must carry out two jobs:
duplicate itself and control the development of
the rest of the cell in a specific way.”
-Francis Crick
9-16
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Replication
the process of making new copies of the DNA molecules
Potential mechanisms:
Conservative
Semiconservative
Dispersive
organization of DNA strands
old/old + new/new
old/new + new/old
mixed old and new on each strand
Test: Meselson and Stahl grew E. coli in media with
heavy nitrogen and then in media with lighter nitrogen.
Nitrogen becomes part of the DNA molecule as
replication occurs.
9-17
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Meselson and Stahl’s replication experiment
Conclusion: Replication is semiconservative.
9-18
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Replication as a process
Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two molecules are made.
Each has one new and one old
DNA strand.
9-19
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Chromatin = DNA and associated proteins
DNA winds around
histone proteins
(nucleosomes).
Other proteins wind
DNA into more tightly
packed form, the
chromosome.
Unwinding portions of
the chromosome is
important for mitosis,
replication and making
RNA.
9-20
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Replication
3’
3’
5’
5’
3’
5’
3’
5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
9-21
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Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
9-22
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Replication
Overall direction
of replication
3’
5’
3’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
9-23
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Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
9-24
3’
5’
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Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
9-25
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Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
9-26
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Replication
3’
5’
3’
5’
3’
5’
3’ 5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
9-27
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Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
9-28
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Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
9-29
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Replication
3’
3’
5’
3’
5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate
backbone.
9-30
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Replication
3’
5’
3’
3’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’ 5’
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
3’
5’
3’
5’
5’
3’
3’
5’
3’
3’
5’
3’
5’
5’
3’5’
3’
5’
9-31
3’
5’
3’
3’
5’
3’
5’
3’
5’
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Enzymes in DNA replication
Helicase unwinds
parental double helix
DNA polymerase
binds nucleotides
to form new strands
9-32
Binding proteins
stabilize separate
strands
Exonuclease removes
RNA primer and inserts
the correct bases
Primase adds
short primer
to template strand
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone
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Polymerase Chain Reaction (PCR)
Is a method in which multiple repetitions of DNA
replication are performed in a test tube.
Mix in test tube:
9-33
DNA template
DNA to be amplified
Primers
one complementary to each strand
Nucleotides
A, G, C, and T
DNA polymerase
heat stable form
from thermophilic bacteria
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Polymerase Chain Reaction (PCR)
DNA replication in a test tube
DNA template is denatured with heat to separate strands.
5’
3’
G
C
5’
9-34
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’
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Polymerase Chain Reaction (PCR)
DNA template is denatured with heat to separate strands.
5’
3’
5’
9-35
G
A
A
C
T
A
G
C
G
C
T
T
G
A
T
C
G
C
3’
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Polymerase Chain Reaction (PCR)
Each DNA primer anneals, binding to its
complementary sequence on the template DNA
5’
3’
G
C
A
T
A
T
5’
C
T
A
G
C
G
3’
5’
3’
C
5’
9-36
T
T
G
A
T
G
C
C
G
G
C
3’
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Polymerase Chain Reaction (PCR)
DNA polymerase creates a new strand of DNA
complementary to the template DNA starting from the
primer.
5’
3’
G
C
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’
5’
5’
3’
G
C
5’
9-37
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’
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Polymerase Chain Reaction (PCR)
9-38
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A gene: molecular definition
A gene is a segment of DNA
which directs the formation of RNA
which in turn directs formation of a protein.
The protein (or functional RNA) creates the
phenotype.
Information is conveyed by the sequence of the
nucleotides.
9-39
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Polymerase Chain Reaction (PCR)
9-40
Denaturation
DNA template is denatured with
high heat to separate strands.
Annealing
Each DNA primer anneals, binding
to its complementary sequence
on the template DNA
Extension
DNA polymerase creates a
new strand of DNA complementary
to the template DNA
starting from the primer.
Multiple rounds of denaturation-annealing-extension are
performed to create many copies of the template DNA
between the two primer sequences.