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Chapter 12
DNA Synthesis, Mutation,
and Repair
DNA structure:
A-T
G–C
Antiparallel
Occurs during S phase in eukaryotes
E. coli
Distinguishing between Models of DNA Replication

Three different models of how DNA might replicate were
proposed based on DNA structure.
• Semi-conservative replication (Fig. 12.1a)
• Conservative replication (Fig. 12.1b)
• Dispersive replication (Fig. 12.1c)
Figure 12.1a-c
Alternative Hypotheses for DNA Synthesis
(a) Hypothesis 1:
(b) Hypothesis 2:
(c) Hypothesis 3:
Semi-conservative
replication
Conservative replication
Dispersive replication
Intermediate molecule
Distinguishing Between Models of DNA Replication

The Meselsohn and Stahl experiment determines which
model is correct.
•
15N
was fed to growing E. coli cells to mark DNA, then
cells were switched to 14N.
• DNA replication is semi-conservative: new DNA has
one 15N strand and one 14N strand. (Fig. 12.2)
Figure 12.2a,b
Applying Ideas, Question 2
Be certain that you understand the behavior of DNA replication that gives this pattern
in eukaryotes too.
Apply the three models to this labeling scheme to see what
chromosomal labeling patterns would occur.
3H-thymidine
non-labeled-thymidine
1. One round of DNA
replication in radioactive
solution
2. Mitosis
3. One round of DNA
replication in nonradioactive solution
Figure 12.3a
Structure of dNTPs:
P
What’s happening at the level of the nucleotide?
P
P
5' CH2
Base
O
3'
OH
Figure 12.3b DNA synthesis reaction: addition of nucleotides at the 3’ end
5' end of strand
P
P
Base
CH2
Base
CH2
O
P
O
P
CH2
O
CH2
Base
H20
+
3'
P
P
Synthesis reaction
P
5' CH2
3'
OH
P
P
OH
P
1,2
Base
O
O
Base
CH2
Base
O
base addition
occurs at the 3’
end
base addition
occurs at the 3’
end, next one here
OH
3' end of strand
Cell free in vitro DNA synthesis reactions were used to
identify the enzymes involved in DNA replication.

Proteins were extracted from E. coli and tested for
activity in the cell free system.
• Kornberg et al. determine that:
• DNA replication requires an enzyme; the first discovered
was DNA polymerase I.
• DNA replication requires a DNA template. (Fig. 12.4, 12.8)
Figure 12.4
TESTING TEMPLATE-DIRECTED SYNTHESIS
1. Isolate single
strand of X174
DNA.
X174
virus
2. Make copies of
X174 DNA in vitro
using DNA
polymerase I.
Normal
X174 DNA
Synthetic
X174 DNA
3. Add synthetic
X174 DNA to
E.coli cells growing
in culture.
E. coli
Synthetic
DNA
4. Observe result:
New generation of
X174 particles
appears. Synthetic
X174 DNA is
Note: It will turn out that
infectious.
DNApol I is often
Conclusion: DNA polymerase I catalyzes
template-directed synthesis
involved in DNA repair
Figure 12.6
Classic work of John Cairns in early 1960s: photomicrographs
of E. coli replicating chromosome
Radioactively labeled strand
Two labeled strands =
newly synthesized
Cell free in vitro DNA synthesis reactions were used to
identify the enzymes involved in DNA replication.

Proteins were extracted from E. coli and tested for
activity in the cell free system.
• Cairns and de Lucia: DNA polymerase I is not the main
replication enzyme.
• Kohiyama and Kornberg discover DNA polymerase III, which is
the main replication enzyme.
Figure 12.7a

Characteristics of replication in E. coli:
•
At the replication fork, DNA polymerase III builds the
new strands in the 5’-3’ direction.
•
New nucleotides are only added to 3’ hydroxyl groups
of other nucleotides. (creates a problem)
Formation of the leading strand
3'
5'
DNA polymerase III
5'
Newly
synthesized
leading strand
3'
5'
Replication fork
Figure 12.7b
Formation of lagging strand
3'
5'
Lagging
strands
5'
3'
3'
DNA polymerase III
5'
3'
5'
Okazaki
fragments
5'
3'
3'
5'
Gap
DNA polymerase III
beginning synthesis
of new fragment
Figure 12.8
Completely single stranded
3'
5'
G
A
A
T
C
T
G
Completely double stranded
3'
G A A T C T
G
C
Polymerase
inactive
5'
C
Polymerase
inactive
C
T
T
A
G
5'
A
C
G
3'
Single strand as template plus 3' end
to start synthesis
3'
5'
G A A T C T
G C
Polymerase
active
C
5'
T
T
OH
3'
The new strands are initiated by adding nucleotides
to a short RNA primer because there is no DNA on
which to build. (Fig. 12.7)
Figure 12.9
(error prone start, remove RNA, remove errors)
2
Topoisomerase
nicks DNA to
relieve tension
from unwinding
3 Pol III synthesizes leading strand
1 Helicase opens helix
4 Primase synthesizes RNA primer
(what’s the story here?)
5
6
Pol I excises RNA primer; fills gap
7
Pol III elongates primer;
produces Okazaki fragment
1
DNA ligase links Okazaki
fragments to form
continuous strand
Laboratory Analysis and Manipulation
of DNA Sequences
• Polymerase Chain Reaction (PCR): amplifies
DNA from primers, produces numerous copies.
(Fig. 12.11a,b)
Mullis
• Dideoxysequencing: a method for determining
the exact nucleotide sequence of any DNA. (Fig.
12.12)
Figure 12.11a
Polymerase Chain Reaction: developed by Kary Mullis
Nobel Prize 1993
Primers are required to run PCR: bit of a problem!
CCCCATGCTTACAAGCAAGT
Primer
5'
3'
3'
5'
Primer
Region of DNA to be
amplified by PCR
ATCCTATGGTTGTTTGGATGGGTG
Figure 12.11 steps 1-3
POLYMERASE CHAIN REACTION
3'5'
1. Start with a solution
containing template DNA,
synthesized primers, and
an abundant supply of
the four dNTPs.
5'3'
3'
5'
2. Denaturation
Heating leads to
denaturation of the
double-stranded DNA.
5'
3'5'
5'
3'
5'
5'3'
3. Primer binding
At cooler temperatures,
the primers anneal to
the template DNA by
complementary base
pairing.
Figure 12.11 steps 4-6
5'
5'3'
3'5'
3'5'
5'3'
5'3'
4. Extension
During incubation,
DNA polymerase
synthesizes
complementary DNA
strand starting at
the primer.
5. Repeat cycle of three
steps (2-4) again,
doubling the copies
of DNA.
6. Repeat cycle again,
up to 20-30 times, to
produce millions of
copies of template DNA.
Figure 12.11 steps 1-3
POLYMERASE CHAIN REACTION
3'5'
1. Start with a solution
containing template DNA,
synthesized primers, and
an abundant supply of
the four dNTPs.
5'3'
3'
5'
2. Denaturation
Heating leads to
denaturation of the
double-stranded DNA.
5'
3'5'
5'
3'
5'
5'3'
3. Primer binding At
cooler temperatures,
the primers anneal to
the template DNA by
complementary base
pairing.
Figure 12.11 steps 4-6
often used to amplify
small DNA samples
left at a crime scene
5'
5'3'
3'5'
3'5'
5'3'
5'3'
4. Extension
During incubation,
DNA polymerase
synthesizes
complementary DNA
strand starting at the
primer.
5. Repeat cycle of three
steps (2-4) again,
doubling the copies of
DNA.
1-6
6. Repeat cycle again,
up to 20-30 times, to
produce millions of
copies of template DNA.
Laboratory Analysis of DNA Sequences

Second basic method used in
sequence analysis of DNA
• Dideoxysequencing: a method for
determining the exact
nucleotide sequence of any DNA.
(Fig. 12.12)
Basis for the human genome initiative
(Sequencing the Human Genome)
Figure 12.12a,b
DNA sequencing: one method is dependent on a dideoxynucleotide pool
1
Figure 12.12c
Different-length strands can be lined up by size to determine
DNA sequence.
1
Mutation and DNA Repair Mechanisms

Mutations are created by chemicals, radiation, errors
in meiosis and mistakes in DNA replication.
• Mutations can be deleterious, beneficial, or silent.
(Fig. 12.15, 12.17)
• Mutations in an individual are usually deleterious, may
cause disease and death. (Fig. 12.16a,b)
• Mutations in a population are a source of genetic diversity
that allows evolution to occur.
Figure 12.15
Mismatch (about 1/1000 base additions)
A A C T G G C
Wild type
T T G A C C G
A A C T G G C
A A C T A G C
MUTANT
3'
5'
A A C T G G C
T T G A T C G
DNA replication
T T G A C C G
5'
3'
A A C T G G C
T T G A T C G
DNA replication
A A C T G G C
Wild type
Parental DNA
T T G A C C G
T T G A C C G
First generation
progeny
A A C T G G C
Wild type
T T G A C C G
Second generation
progeny
Figure 12.17 upper
General Categories of Mutations
Mutation
type
Insertion
Deletion
Definition
Example
Consequence
G
Addition of any
number or
nucleotides due to
an error in DNA
synthesis
Removal of any
number of
nucleotides due to
an error in DNA
synthesis
Original
sequence:
Mutant
sequence:
Original
sequence:
Mutant
sequence:
ACC
CAT
GAT
GTA
ACC
CGA
GAT
TGT A
ATA
ATA
ACC
ATA
ATA
A
CAT
GAT
GTA
ACC
ATG
GTC
TA
Addition of 1 or 2 bases
disrupts reading frame.
Usually results in a
dysfunctional gene product.
Deletion of 1 or 2 bases
disrupts reading frame.
Usually results in a
dysfunctional gene product.
Figure 12.17 lower
Mutation
type
Definition
Gene
duplication
Addition of a small
chromosome segment
due to an error during
crossing over at
meiosis I.
Chromosome
inversion
Change in a
chromosome segment
when DNA breaks in
two places, flips, and
rejoins.
Example
A
B
C
D
A
B
C
D
Consequence
Genes
A
B
C
D
A A A
B B B
C C D
D C
D
Mutant
A
B
D
C
A
B
C
D
A
B
C
D
Produces an extra copy of
one or more genes. If point
mutations occur in extra
DNA, it can produce a new
product.
Changes gene order along
chromosome. Other types of
chromosome breaks can
lead to deletion or addition
of chromosome segments.
Figure 12.16
Consequences of mutations: Well-studied
example: DNA point mutation can lead to
a different amino acid sequence.
Phenotype
Start of coding sequence
CAC
DNA
sequence GTG
GTG
CAC
GAC
CTG
TGA
ACT
GGA
CCT
CTC
GAG
CTC
GAG
Normal
Amino
acid
sequence
Valine
CAC
DNA
sequence GTG
Histidine
GTG
CAC
Leucine
GAC
CTG
Threonine
Glutamic
Proline Glutamic
acid
acid
TGA
ACT
GGA
CCT
CAC
GTG
Normal red blood cells
CTC
GAG
Mutant
Amino
acid
sequence
Histidine
Valine
Threonine
Leucine
Proline
Valine
Glutamic
acid
Sickled red blood cells
Mutation and DNA Repair Mechanisms

DNA Repair Mechanisms
• DNA polymerase I proofreads and corrects point mutations
during replication.
• Other excision repair systems scan newly formed DNA and
correct remaining mutations. (Fig. 12.13a,b)
• Repair enzymes identify the correct template strand by its
methyl groups. (Fig. 12.14a,b)
• Defects in repair system enzymes are implicated in a variety
of cancers. (Fig. 12.18a-c)
Figure 12.13
Mismatched
bases.
3'
T
G
T
C
C
A
C
A
G
G
A
5'
T
C
G
C
G
5'
OH 3'
But, how does the “system” know which is the correct
sequence and which is the mutant sequence?
Polymerase III can 3'
repair mismatches.
5'
1
5'
T
G
T
C
C
A
C
A
G
G
A
T
C
G
C
Figure 12.14b
METHYLATION-DIRECTED MISMATCHED BASE REPAIR
Mismatch
Answer
1. Where a mismatch occurs, the
correct base is located on the
methylated strand: the incorrect base
occurs on the unmethylated strand.
2. Enzymes detect mismatch and nick
unmethylated strand.
3. DNA polymerase I excises
nucleotides on unmethylated strand.
4. DNA polymerase I fills in
gap in 5' 3' direction.
5. DNA ligase links new and
old nucleotides.
Repaired Mismatch
Figure 12.18a
Interesting and common: UV-induced thymine dimers caused DNA to kink
P
CH2
DNA strand
with adjacent
P
thymine
bases
CH
P
N
O
N Thymine
H
H
O
N Thymine
CH3
H
O
O
N
N
O
UV light
Kink
P
N
2
CH2
O
CH3
O
H
P
H
O
O
CH2 O
H
O
N
H
P
Thymine
dimer
CH3
H
N
O
CH3
Case Study: Xeroderma Pigmentosum
quoted from http://www.mssc.edu/biology/B305/GTS/ws99/xero/xero.html
by Sherrie Smith (underlines by HN)

“Clinical Signs of the Disorder
Xeroderma Pigmentosum is a very rare genetic defect. It is caused by a defect in ultraviolet radiation induced
DNA repair mechanisms, and is characterized by severe sensitivity to all sources of ultraviolet radiation,
especially sunlight. There are less than one thousand known cases of XP worldwide. XP sufferers are
grouped according to the capacity of their body to repair DNA. Groups A, C, D, and Variant make up over
90% of all cases. Group A has the lowest level of DNA repair and the most severe symptoms. There is a wide
range of symptoms: blindness and deafness, blistering or freckling on minimal sun exposure, developmental
disabilities, dwarfism and hypergonadism, increased skin and eye cancers, and mental retardation. There is
no cure. DNA damage is cumulative and irreversible. Treatment is limited to avoidance of exposure to UV
radiation by staying indoors with sunlight blocked out, and the use of protective clothing, sunscreens, and
eyeglasses. It’s also very important to avoid other known carcinogens.1

Inheritance Pattern
Xeroderma Pigmentosum is a rare human autosomal recessive disease.2 The first symptoms usually occur
between one and two years of age. Children will have a history of severe burns on small amounts of sunlight
exposure. Others have numerous freckle-like spots on sun-exposed body parts. Later symptoms include
premature aging, skin cancers, eye problems, and neurologic abnormalities.

Mechanism of the Disease Process
Tests have shown that normal skin fibroblasts can repair ultraviolet radiation damage to DNA by inserting new
bases into DNA, XP sufferers lack this capacity , or have a much-reduced capacity for repair. In a study of
Japanese patients, they were found to have a splice site mutation where reduced amounts of mRNA of
reduced size was found on Northern Blot Anaylsis.”
Figure 12.18b
Nature of XP cellular responses
Vulnerability of cells to UV light damage
Percentage of cells surviving
100
Normal individuals
10
Individuals with XP
1
Dose of UV light
Figure 12.18c
60
50
(counts per minute)
Amount of radioactive thymidine incorporated
Ability of cells to repair damage
DNA damage repaired by normal individuals
40
30
20
10
No DNA repair in XP individuals
0
Dose of UV light
Laboratory Applications of DNA Sequences

DNA sequence analysis is used to
compare genes within and
between species to determine
function and evolutionary
relatedness. Use for endangered
species restoration.

Used in numerous forensic
applications:
http://www.ornl.gov/hgmis/elsi/forensics.html#5
1