Chapter 16 *Lecture Outline *See separate FlexArt PowerPoint slides for all
figures and tables pre-inserted into PowerPoint
without notes.
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INTRODUCTION
•  The term mutation refers to a heritable change in the
genetic material
•  Mutations provide allelic variations
–  On the positive side, mutations are the foundation for
evolutionary change needed for a species to adapt to
changes in the environment
–  On the negative side, new mutations are much more likely to
be harmful than beneficial to the individual and often are the
cause of diseases
•  Understanding the molecular nature of mutations is a
deeply compelling area of research.
•  Since mutations can be quite harmful, organisms have
developed ways to repair damaged DNA
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16-2
16.1 CONSEQUENCES OF
MUTATIONS
•  Mutations can be divided into three main types
–  1. Chromosome mutations
•  Changes in chromosome structure
–  2. Genome mutations
•  Changes in chromosome number
–  3. Gene mutations
•  Relatively small change in DNA structure that affects a single
gene
–  Types 1 and 2 were discussed in chapter 8
–  Type 3 will be discussed in this chapter
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16-3
Gene Mutations Change the
Sequence
n
DNA
A point mutation is a change in a single base pair
n
5
3
n
n
n
It can involve a base substitution
AACGCTAGATC 3
TTGCGATCTAG 5
5
3
AACGCGAGATC 3
TTGCGCTCTAG 5
A transition is a change of a pyrimidine (C, T) to
another pyrimidine or a purine (A, G) to another purine
A transversion is a change of a pyrimidine to a purine or
vice versa
Transitions are more common than transversions
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16-4
Gene Mutations Change the
Sequence
n
5
3
DNA
Mutations may also involve the addition or deletion
of short sequences of DNA
AACGCTAGATC 3
TTGCGATCTAG 5
5
3
AACGCTC 3
TTGCGAG 5
Deletion of four base pairs
5
3
AACGCTAGATC 3
TTGCGATCTAG 5
5
3
AACAGTCGCTAGATC 3
TTGTCAGCGATCTAG 5
Addition of four base pairs
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16-5
Gene Mutations Can Alter the
Coding Sequence Within a Gene
n
Mutations in the coding sequence of a structural
gene can have various effects on the polypeptide
n
Silent mutations are those base substitutions that do not
alter the amino acid sequence of the polypeptide
n
n
Due to the degeneracy of the genetic code
Missense mutations are those base substitutions in which
an amino acid change does occur
n
n
Example: Sickle-cell anemia (Refer to Figure 16.1)
If the substituted amino acid has no detectable effect on protein
function, the mutation is said to be neutral. This can occur if the
new amino acid has similar chemistry to the amino acid it replaced
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16-6
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© Phototake/Alamy
Normal red blood cells
© Phototake/Alamy
10 µm"
Sickled red blood cells
10 µm"
(a) Micrographs of red blood cells
NORMAL : NH2 – VALINE – HISTIDINE – LEUCINE – THREONINE – PROLINE – GLUTAMIC ACID – GLUTAMIC ACID...
SICKLE"
CELL " : NH2 – VALINE – HISTIDINE – LEUCINE – THREONINE – PROLINE – VALINE– GLUTAMIC ACID...
(b) A comparison of the amino acid sequence between normal β-globin and sickle-cell β-globin
Figure 16.1
16-7
Gene Mutations Can Alter the
Coding Sequence Within a Gene
n
Mutations in the coding sequence of a structural
gene can have various effects on the polypeptide
n
n
Nonsense mutations are those base substitutions that
change a normal codon to a stop codon
Frameshift mutations involve the addition or deletion of a
number of nucleotides that is not divisible by three
n
n
This shifts the reading frame so that translation of the mRNA
results in a completely different amino acid sequence downstream
of the mutation
Table 16.1 describes all of the above mutations
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16-8
16-9
Gene Mutations outside of coding
sequences can still affect phenotype
n
n
Mutations in the core promoter can change levels of gene
expression
n  Up mutations increase expression. Down mutations
decrease expression
Other important non-coding mutations are in Table 16.2
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16-10
Gene Mutations and Their Effects on
Genotype and Phenotype
n
n
n
In a natural population, the wild-type is the relatively
prevalent genotype. Genes with multiple alleles may
have two or more wild-types.
A forward mutation changes the wild-type genotype
into some new variation
A reverse mutation changes a mutant allele back to
the wild-type
n
It is also termed a reversion
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16-11
n
n
Mutations can also be described based on their
effects on the wild-type phenotype
They are often characterized by their differential
ability to survive
n
Deleterious mutations decrease the chances of survival
n
n
n
n
The most extreme are lethal mutations
Beneficial mutations enhance the survival or reproductive
success of an organism
The environment can affect whether a given mutation is
deleterious or beneficial
Some mutations are conditional
n
n
They affect the phenotype only under a defined set of
conditions
An example is a temperature-sensitive mutation
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16-12
n
n
n
A second mutation will sometimes counteract the
effects of a first mutation
These second-site mutations are called suppressor
mutations or simply suppressors
Suppressor mutations are classified into two types
n
Intragenic suppressors
n
n
Intergenic suppressors
n
n
The second mutant site is within the same gene as the first
mutation
The second mutant site is in a different gene from the first
mutation
Examples of suppressor mutations-Table 16.3
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16-13
16-14
Changes in Chromosome Structure
Can Affect Gene Expression
n
n
A chromosomal rearrangement may affect a gene
because the chromosomal breakpoint occurs within
the gene
A gene may be left intact, but its expression may be
altered because of its new location
n
n
This is termed a position effect
There are two common reasons for position effects:
n
1. Movement to a position next to regulatory sequences
n
n
Refer to Figure 16.2a
2. Movement to a heterochromatic region
n
Refer to Figure 16.2b AND 16.3
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16-15
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B
A
Coding"
sequence
Core"
promoter
B
Gene B
Regulatory"
sequence
Coding"
sequence
Core"
promoter
Regulatory sequences
are often bidirectional
A
Inversion
Gene A
Core promoter"
for gene A is"
moved next to"
regulatory"
sequence of"
gene B.
(a) Position effect due to regulatory sequences
Active"
gene
Gene"
is now"
inactive.
Translocation
Heterochromatic"
chromosome"
(more compacted)
Euchromatic"
chromosome
Translocated"
heterochromatic"
chromosome
Shortened euchromatic"
chromosome
(b) Position effect due to translocation to a heterochromatic!
chromosome
Figure 16.2
16-16
Mutations Can Occur in
Germ-Line or Somatic Cells
n
Geneticists classify animal cells into two types
n
Germ-line cells
n
n
Somatic cells
n
n
All other cells
Germ-line mutations are those that occur directly in a
sperm or egg cell, or in one of their precursor cells
n
n
Cells that give rise to gametes such as eggs and sperm
Refer to Figure 16.4a
Somatic mutations are those that occur directly in a body
cell, or in one of its precursor cells
n
Refer to Figure 16.4b AND 16.5
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16-17
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Germ-line"
mutation
Gametes
Embryo
Somatic"
mutation
The size of the patch
will depend on the
timing of the mutation
The earlier the mutation,
the larger the patch
Therefore, the
mutation can be
passed on to future
generations
Mutation is"
found"
throughout"
the entire"
body.
Mature"
individual
An individual who has
somatic regions that are
genotypically different
from each other is called
a genetic mosaic
Therefore, the mutation cannot be
passed on to future generations
Half of"
the gametes"
carry the"
mutation.
Figure 16.4
Patch of"
affected"
area
(a) Germ-line mutation
None of"
the gametes"
carry the"
mutation.
(b) Somatic cell mutation
16-18
16.2 OCCURRENCE AND CAUSES
OF MUTATION
•  Mutations can occur spontaneously or be induced
•  Spontaneous mutations
–  Result from abnormalities in cellular/biological processes
•  Errors in DNA replication, for example
–  Underlying cause originates within the cell
•  Induced mutations
–  Caused by environmental agents
–  Agents that are known to alter DNA structure are termed
mutagens
•  These can be chemical or physical agents
•  Refer to Table 16.4
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16-19
16-20
Spontaneous Mutations Are Random
Events
n
Are mutations spontaneous occurrences or causally
related to environmental conditions?
n
n
This is a question that biologists have asked themselves
for a long time
Jean Baptiste Lamarck
n
n
Proposed that physiological events (e.g. use and disuse) determine
whether traits are passed along to offspring
Charles Darwin
n
Proposed that genetic variation occurs by chance
n
Natural selection results in better-adapted organisms
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16-21
Mutation Rates and Frequencies
n
The term mutation rate is the likelihood that a gene
will be altered by a new mutation
n
n
n
The mutation rate for a given gene is not constant
n
n
It is commonly expressed as the number of new mutations
in a given gene per cell generation
It is in the range of 10-5 to 10-9 per generation
It can be increased by the presence of mutagens
Mutation rates vary substantially between species
and even within different strains of the same species
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16-26
Mutation Rates and Frequencies
n
Within the same individual, some genes mutate at a
much higher rate than other genes
n
Some genes are larger than others
n
n
Some genes have locations within the chromosome that
make them more susceptible to mutation
n
n
This provides a greater chance for mutation
These are termed hot spots
Note: Hot spots can be also found within a single gene
n
Specific bases or regions that are more likely to be the site of a
mutation within a gene
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16-27
Mutation Rates and Frequencies
n
The mutation frequency for a gene is the number of
mutant genes divided by the total number of genes
in a population
n
n
If 1 million bacteria were plated and 10 were mutant
-5
n  The mutation frequency would be 1 in 100,000 or 10
The mutation frequency depends not only on the
mutation rate, but also on the
n
n
Timing of the mutation
Likelihood that the mutation will be passed on to future
generations
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16-28
Causes of
Spontaneous Mutations
n
Spontaneous mutations can arise by three types of
chemical changes
n
1. Depurination
n
2. Deamination
n
3. Tautomeric shift
The most common
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16-29
Causes of
Spontaneous Mutations
n
Depurination involves the removal of a purine
(guanine or adenine) from the DNA
n
The covalent bond between deoxyribose and a purine
base is somewhat unstable
n
It occasionally undergoes a spontaneous reaction with water that
releases the base from the sugar
n
This is termed an apurinic site
n
Fortunately, apurinic sites can be repaired
n
However, if the repair system fails, a mutation may result during
subsequent rounds of DNA replication
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16-30
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5′
3′
5′
C G
A T
T A
C G
G C
C G
A T
T A
C
G C
Depurination
5′
3′
3′
Apurinic site
3′
5′
5′
3′
(a) Depurination
3′
5′
C G
A T
T A
C
G C
3′
DNA replication
3′
5′
5
3′
C G
A T
T A
X
G C
5′
(b) Replication over an apurinic site
Figure 16.8
Three out of four (A, T and G)
are the incorrect nucleotide
C G
A T
T A
C G
G C
3′
Spontaneous depurination
There s a 75% chance
of a mutation
X could be"
A, T, G, or C
5′
16-31
n
Deamination involves the removal of an amino group
from the cytosine base
n
The other bases are not readily deaminated
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NH2
H
H
N
O
O
+
N
H 2O
H
H
N
O
+
N
Sugar
Sugar
Cytosine
Uracil
NH3
H
Figure 16.9 (a) Deamination of cytosine
n
DNA repair enzymes can recognize uracil as an
inappropriate base in DNA and remove it
n
However, if the repair system fails, a C-G to A-T mutation will result
during subsequent rounds of DNA replication
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16-32
n
Deamination of 5-methyl cytosine can also occur
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NH2
H
CH3
N
O
O
+
N
H 2O
H
Sugar
5-methylcytosine
CH3
N
+
O
N
NH3
H
Sugar
Thymine
Figure 16.9 (b) Deamination of 5-methylcytosine
n
n
Thymine is a normal constituent of DNA
This poses a problem for repair enzymes
n
n
They cannot determine which of the two bases on the two DNA
strands is the incorrect base
For this reason, methylated cytosine bases tend to create
hot spots for mutation
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16-33
n
A tautomeric shift involves a temporary change in
base structure (Figure 16.10a)
n
The common, stable form of thymine and guanine is the
keto form
n
n
The common, stable form of adenine and cytosine is the
amino form
n
n
At a low rate, A and C can interconvert to an imino form
These rare forms promote AC and GT base pairs
n
n
At a low rate, T and G can interconvert to an enol form
Refer to Figure 16.10b
For a tautomeric shift to cause a mutation it must
occur immediately prior to DNA replication
n
Refer to Figure 16.10c
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16-34
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Common
O
H
Rare
OH
N
N
Tautomeric shift
N
N
H
H 2N
N
N
H
Guanine
H 2N
N
N
Sugar
Sugar
Keto form
Enol form
N
NH
N
N
Tautomeric shift
H
H
H
N
N
Adenine
Sugar
Amino form
N
N
H
H
N
Sugar
Imino form
Common
Rare
O
H
N
Common
O
OH
CH3
Tautomeric shift
H
Thymine
N
O
N
Sugar
Sugar
Keto form
Enol form
N
H
Rare
NH
H
N
Figure 16.10
CH3
N
NH2
O
N
H
Tautomeric shift
Cytosine
H
H
N
O
N
Sugar
Sugar
Amino form
Imino form
H
(a) Tautomeric shifts that occur in the 4 bases found in DNA
16-35
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H
H 3C
H
H
O
N
N
Sugar
H
N
O
N
N
O
H
Thymine (enol)
H
N
Sugar
N
H
Guanine (keto)
H
N
H
N H
N
Sugar
N
N
N
N
N
O
Cytosine (imino)
H
Sugar
H
Adenine (amino)
(b) Mis–base pairing due to tautomeric shifts
Figure 16.10
16-36
Temporary
tautomeric shift
Shifted back to
its normal fom
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5′
Base"
mismatch
3′
5′
A thymine base"
undergoes a"
tautomeric shift prior"
to DNA replication.
5′
3′
5′
T G
3′
5′
5′
3′
5′
5′
3′
T A
3′
5′
3′
3′
5′
T A
3′
Mutation
C G
A second round"
3′
of DNA replication" 5′
occurs.
T A
3′
T A
3′
5′
3′
5′
T A
5′
3′
DNA molecules found"
in 4 daughter cells
(c) Tautomeric shifts and DNA replication can cause mutation
Figure 16.10
16-37
Mutations Due to Trinucleotide
Repeats
n
Several human genetic diseases are caused by an
unusual form of mutation called trinucleotide repeat
expansion (TNRE)
n
n
These diseases include
n
n
n
The term refers to the phenomenon that a sequence of 3
nucleotides can increase from one generation to the next
Huntington disease (HD)
Fragile X syndrome (FRAXA)
Refer to Table 16.5 for these and other examples
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16-38
16-39
n
Certain regions of the chromosome contain
trinucleotide sequences repeated in tandem
n
n
In normal individuals, these sequences are transmitted from
parent to offspring without mutation
However, in persons with TNRE disorders, the length of a
trinucleotide repeat has increased above a certain critical size
n
n
n
Disease symptoms occur
In some diseases, it also becomes prone to expansion
This phenomenon is shown here with the trinucleotide repeat CAG
CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG
n = 11
CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG
n = 18
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16-40
n
In some cases, the expansion is within the coding
sequence of the gene
n
n
Typically the trinucleotide expansion is CAG (glutamine)
Therefore, the encoded protein will contain long tracks of
glutamine
n
n
n
This causes the proteins to aggregate with each other
This aggregation is correlated with the progression of the disease
In other cases, the expansions are located in
noncoding regions of genes
n
n
Some of these expansions are hypothesized to cause
abnormal changes in RNA structure
Some produce methylated CpG islands which may silence
the gene
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16-41
n
There are two particularly unusual features that
some TNRE disorders have in common
n
1. The severity of the disease tends to worsen in future
generations
n
n
This phenomenon is called anticipation
2. Anticipation usually depends on whether the disease is
inherited from the father or mother
n
n
n
In Huntington disease, the TNRE is more likely to occur if inherited
from the father
In myotonic muscular dystrophy, the TNRE is more likely to occur if
inherited from the mother
This suggests that TNRE can occur more frequently during
oogenesis or spermatogenesis, depending on the gene involved.
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16-42
n
The DNA cause of TNRE is not fully understood
n
TNREs contain at least one C and one G
n
n
This allows formation of a hairpin
During DNA replication, a hairpin can lead to an increase
or decrease in the length of the DNA
n
n
n
Polymerase can slip off DNA
Hairpin forms and pulls strand back
DNA polymerase hops back on
n
n
n
See Figure 16.12 for details
These changes can occur during gamete formation
n
n
Begins synthesis from new location
offspring will have very different numbers of repeats
Can also increase repeats in somatic cells
n
This can increase severity of the disease with age
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16-43
TNRE sequences can form hairpins
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One DNA strand with a trinucleotide repeat sequence
T G C C A A G C A T T C T G C T G C T G C T G C T G C T G T C A A A G C A T T
Trinucleotide (CTG) repeat
Hairpin formation
T C A A A G C A T T
Hairpin with CG base pairing
T
T
C T G C T G
C T G C T G
T G C C A A G C A T T
(a) Formation of a hairpin with a trinucleotide (CTG) repeat sequence
Figure 16.12a
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16-44
Mechanisms of trinucleotide repeat expansion or deletion
One DNA template strand prior to DNA replication
One DNA template strand prior to DNA replication
TNRE
TNRE
DNA replication begins"
and goes just past the TNRE.
Hairpin forms in template strand"
prior to DNA replication.
DNA"
polymerase
DNA polymerase slips off"
the template strand and a"
hairpin forms.
DNA replication occurs and"
DNA polymerase slips over"
the hairpin.
DNA polymerase resumes"
DNA replication.
DNA repair occurs.
DNA repair occurs.
TNRE is longer.
TNRE is shorter.
OR
TNRE is the same length.
(b) Mechanism of trinucleotide repeat expansion
Figure 16.12b and c
OR
TNRE is the same length.
(c) Mechanism of trinucleotide repeat deletion
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16-45
Experiment 16A: X-Rays,
the First Mutagens
•  In 1927, Hermann Müller devised an approach to
show that X-rays can induce mutations in
Drosophila melanogaster
–  Muller reasoned that a mutagenic agent might cause
some genes to be defective
–  His experimental approach focused on the ability of a
mutagen to cause defects in X-linked genes that result in
a recessive lethal phenotype
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16-46
•  Müller used a strain of fruit flies that enabled
him to detect X-linked recessive lethal
mutations
–  He set up his crosses in such a way that a
female that inherited a new X-linked recessive
lethal allele would not be able to produce any
male offspring
–  Müller reasoned that if X-rays were indeed
mutagens
•  Then exposure to X-rays would increase the
number of females unable to produce sons
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16-47
Types of Mutagens
n
n
An enormous array of agents can act as mutagens
that permanently alter the structure of DNA
The public is concerned about mutagens for two
main reasons:
n
n
n
1. Mutagens are often involved in the development of
human cancers
2. Mutagens can cause gene mutations that may have
harmful effects in future generations
Mutagenic agents are usually classified as chemical
or physical mutagens
n
Refer to Table 16.6
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16-55
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16-56
Mutagens Alter DNA Structure in
Different Ways
n
Chemical mutagens come into three main types
n
1. Base modifiers
n
2. Intercalating agents
n
3. Base analogues
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16-57
n
Base modifiers covalently modify the structure of a
nucleotide
n
n
For example, nitrous acid, replaces amino groups with
keto groups (–NH2 to =O)
This can change cytosine to uracil and adenine to
hypoxanthine
n
n
n
These modified bases do not pair with the appropriate nucleotides
in the daughter strand during DNA replication
Refer to Figure 16.15
Some chemical mutagens disrupt the appropriate pairing
between nucleotides by alkylating bases within the DNA
n
Examples: Nitrogen mustards and ethyl methanesulfonate (EMS)
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16-58
Template strand
H
After replication
H
NH2
N
H
O
HNO2
N
N
N
Sugar
O
Sugar
Cytosine
H
N
N
H
N
N
H
Sugar
N
O
H
Uracil
These mispairings
create mutations in the
newly replicated strand
Adenine
H
N
H
N
H
NH2
O
H
H
N
HNO2
N
N
Sugar
N
Sugar
N
N H
H
N
N
H
Adenine
N
H
Hypoxanthine
O
Sugar
Cytosine
Figure 16.15 Mispairing of modified bases
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16-59
n
Intercalating agents contain flat planar structures
that intercalate themselves into the double helix
n
n
n
This distorts the helical structure
When DNA containing these mutagens is replicated, the
daughter strands may contain single-nucleotide additions
and/or deletions resulting in frameshifts
Examples:
n
n
Acridine dyes
Proflavin
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16-60
n
Base analogues become incorporated into daughter
strands during DNA replication
n
For example, 5-bromouracil is a thymine analogue
n
It can be incorporated into DNA instead of thymine
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
H
Br
O
N
N
Sugar
H
H
N
N
N
5-bromouracil"
(keto form)
Figure 16.16
N
Sugar
Sugar
H
This tautomeric shift
occurs at a relatively
high rate
H
H
N
O
O
H
H
N
N
N
5-bromouracil"
(enol form)
Adenine
Normal pairing
O
N
N
N
O
Br
H
Sugar
N
H
Guanine
Mispairing
(a) Base pairing of 5BU with adenine or guanine
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16-61
In this way, 5-bromouracil can promote a change
of an AT base pair into a GC base pair
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5′
5′
3′"
A 5BU
3′
3′
A T
DNA"
replication
3′
5′
5′
5′
5′
3′
G 5BU
3′
5′
3′
G C
DNA"
replication
3′
5′
5′
3′
G or A 5BU
3′
5′
(b) How 5BU causes a mutation in a base pair during DNA replication
Figure 16.16
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16-62
n
Physical mutagens come into two main types
n
n
n
1. Ionizing radiation
2. Nonionizing radiation
Ionizing radiation
n
n
n
n
n
Includes X-rays and gamma rays
Has short wavelength and high energy
Can penetrate deeply into biological molecules
Creates chemically reactive molecules termed free radicals
Can cause
n
n
n
n
n
Base deletions
Oxidized bases
Single nicks in DNA strands
Cross-linking
Chromosomal breaks
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16-63
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
H
O
n
Nonionizing radiation
n
n
n
n
n
Includes UV light
Has less energy
Cannot penetrate deeply
into biological molecules
Causes the formation of
cross-linked thymine
dimers
Thymine dimers may
cause mutations when that
DNA strand is replicated
O
P
O
CH2
O–
H
H
H
N
CH3
H
H
Thymine
CH3
O
O
P
O
CH2
O–
H
H
O
O
H
H
N
N
H
H
O
Thymine
H
Ultraviolet"
light
O
O
P
O
O
H
O
CH2
O–
H
H
N
O
O
O
H
H
N
H
CH3
H
H
CH3
O
O
P
O
CH2
O–
Figure 16.17
N
O
H
H
H
O
O
H
H
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
N
H
N
H
O
Thymine dimer
16-64
16.3 DNA REPAIR
•  Since most mutations are deleterious, DNA repair
systems are vital to the survival of all organisms
–  Living cells contain several DNA repair systems that can
fix different type of DNA alterations
•  In most cases, DNA repair is a multi-step process
–  1. An irregularity in DNA structure is detected
–  2. The abnormal DNA is removed
–  3. Normal DNA is synthesized
•  Refer to Table 16.7
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16-67
16-68
Damaged Bases Can Be
Directly Repaired
n
In a few cases, the covalent modifications of
nucleotides can be reversed by specific enzymes
n
Photolyase can repair thymine dimers
n
n
n
n
It splits the dimers restoring the DNA to its original condition
Uses energy of visible light
Refer to Figure 16.19a
O6-alkylguanine alkyltransferase repairs alkylated bases
n
It transfers the methyl or ethyl group from the base to a cysteine
side chain within the alkyltransferase protein
n
n
Surprisingly, this permanently inactivates alkyltransferase!
Refer to Figure 16.19b
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16-69
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
O
H
Thymine dimer
CH3
N
O
H 3C
O6-methylguanine
N
H
O
CH3
SH
CH2
N
N
H
O
N
H
DNA"
backbone
H
H 3C
N
NH2
N
Alkyltransferase
Alkyltransferase catalyzes"
the removal of the methyl"
group onto itself.
CH3
O
CH3
N
N
DNA"
backbone
DNA photolyase"
cleaves the 2"
bonds between"
the thymine dimer.
O
H
O
N
Guanine
H
O
H
N
N
CH2
H
O
N
H
H
N
O
The normal structure of the 2 thymines is restored.
(a) Direct repair of a thymine dimer
N
N
S
NH2
The normal structure of guanine is restored.
(b) Direct repair of a methylated base
Figure 16.19 Direct repair of damaged bases in DNA
16-70
Base Excision Repair Removes a
Damaged DNA
n
Base excision repair (BER) involves a category of
enzymes known as DNA N-glycosylases
n
n
Depending on the species, this repair system can
eliminate abnormal bases such as
n
n
n
These enzymes can recognize an abnormal base and
cleave the bond between it and the sugar in the DNA
Uracil; Thymine dimers
3-methyladenine; 7-methylguanine
Refer to Figure 16.20
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16-71
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5′
3′
C T C
C
C
C C T
G
A A
G A
G A
C G A G T U G C T G C T G G A
3′
5′
U
N-glycosylase recognizes an abnormal"
base and cleaves the bond between the"
base and the sugar.
5′
3′
C T C
C
C
C C T
G
A A
G A
G A
G A G
G
G
G G A
C
T
C T
C T
3′
5′
Apyrimidinic"
nucleotide
AP endonuclease recognizes a missing"
base and cleaves the DNA backbone on"
the 5′ side of the missing base.
5′
3′
C T C
C
C
C C T
G
A A
G A
G A
G A G
G
G
G G A
C
T
C T
C T
Depending on whether a purine
or pyrimidine is removed, this
creates an apurinic and an
apyrimidinic site, respectively
3′
5′
Nick
In E. coli, DNA polymerase I uses its 5′
3′"
exonuclease activity to remove the damaged"
region and then fills in the region with normal"
DNA. DNA ligase seals the region.
5′
3′
G C T C A A C G A C G A C C T
G A G
G
G
G G A
C
T T
C T
C T
3′
5′
Nick-translated region
In eukaryotes such as humans, DNA"
polymerase β can remove the apyrimidinic"
nucleotide and replace it with the correct"
nucleotide. DNA ligase seals the region.
5′
3′
C T C
C
C
C C T
G
A A
G A
G A
G A G
G
G
G G A
C
T T
C T
C T
5′
3′
In eukaryotes such as humans,"
DNA polymerase δ or ε can"
synthesize a short segment of"
DNA, which generates a flap.
5′
3′
C T C
C
C
C C T
G
A A
G A
G A
G A G
G
G
G G A
C
T T
C T
C T
3′
Flap
5′
Flap is removed by flap"
endonuclease. DNA ligase"
seals the region.
5′
3′
G C T C A A C G A C G A C C T
G A G
G
G
G G A
C
T T
C T
C T
Figure 16.20
Base Excision Repair
3′
5′
16-72
Nucleotide Excision Repair Removes
Damaged DNA Segments
n
n
An important general process for DNA repair is
nucleotide excision repair (NER)
This type of system can repair many types of DNA
damage, including
n
n
n
Thymine dimers and chemically modified bases
missing bases, some types of crosslinks
NER is found in all eukaryotes and prokaryotes
n
However, its molecular mechanism is better understood in
prokaryotes
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16-73
Nucleotide Excision Repair Removes
Damaged DNA Segments
n
In E. coli, the NER system requires four key proteins
n
These are designated UvrA, UvrB, UvrC and UvrD
n
Named as such because they are involved in Ultraviolet light repair
of pyrimidine dimers
n
n
They are also important in repairing chemically damaged DNA
UvrA, B, C, and D recognize and remove a short segment
of damaged DNA
n
DNA polymerase and ligase finish the repair job
n
Refer to Figure 16.21
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16-74
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Thymine dimer
5′
3′
A
3′
T T
A
5′
B
The UvrA/UvrB complex tracks along the"
DNA in search of damaged DNA.
5′
3′
A
T T
3′
A
B
5′
After damage is detected, UvrA"
is released, and UvrC binds.
5′
3′
T T
3′
B
5′
UvrC
UvrC makes cuts on both"
sides of the thymine dimer.
Figure 16.21
16-75
Typically, the cuts are 4-5 nucleotides from the 3 end of the
damage, and 8 nucleotides from the 5 end
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Cut
Cut
5′
3′
T T
3′
B
5′
UvrC
UvrD, which is a helicase, removes"
the damaged region. UvrB and"
UvrC are also released.
5′
3′
3′
5′
DNA polymerase fills in the gap,"
and DNA ligase seals the gap.
No thymine dimer
Figure 16.21
5′
3′
3′
5′
16-76
Nucleotide Excision Repair Removes
Damaged DNA Segments
n
Several human diseases have been shown to involve
inherited defects in genes involved in NER
n
These include xeroderma pigmentosum (XP), Cockayne
syndrome (CS) and PIBIDS
n
n
n
A common characteristic of all three syndromes is an increased
sensitivity to sunlight
Figure 16.22 shows an individual affected with XP
Xeroderma pigmentosum can be caused by defects in
seven different NER genes
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16-77
Mismatch Repair Systems Detect and
Correct A Base Pair Mismatch
n
n
A base mismatch is another type of abnormality in
DNA
The structure of the DNA double helix obeys the AT/
GC rule of base pairing
n
n
However, during DNA replication an incorrect base may be
added to the growing strand by mistake
DNA polymerases have a 3 to 5 proofreading
ability that can detect base mismatches and fix them
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16-78
Mismatch Repair Systems Detect and
Correct A Base Pair Mismatch
n
n
n
If proofreading fails, the mismatch repair system
comes to the rescue
Mismatch repair systems are found in all species
An important aspect of these systems is that they are
specific to the newly made strand
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16-79
Mismatch Repair Systems Detect and
Correct A Base Pair Mismatch
n
The molecular mechanism of mismatch repair has
been studied extensively in E. coli
n
Three proteins, MutL, MutH and MutS detect the mismatch
and direct its removal from the newly made strand
n
n
The proteins are named Mut because their absence leads to a
much higher mutation rate than normal
A key characteristic of MutH is that it can distinguish
between the parental strand and the daughter strand
n
n
Prior to replication, both strands are methylated
Immediately after replication, the parental strand is methylated
whereas the daughter is not!
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16-80
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The MutS protein finds a mismatch. The MutS/MutL complex binds"
to MutH, which is already bound to a hemimethylated sequence.
m
GAT C
C TAG
Parental"
strand
MutH
Newly"
made"
strand
MutL
Acts as a linker between
MutS and MutH
MutS
T
G
Incorrect"
base
MutH makes a cut in the"
nonmethylated strand. MutU"
separates the DNA strands at the"
cleavage site and an exonuclease"
digests the nonmethylated strand"
just beyond the base mismatch.
m
T C
GA
MutH cleavage site
G
Figure 16.23 Methyl-directed mismatch repair in E. coli
16-81
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m
T C
GA
MutH cleavage site
G
DNA polymerase fills in"
the vacant region. DNA"
ligase seals the ends.
m
GATC
C TAG
The mismatch has been"
repaired correctly.
C
G
Figure 16.23 Methyl-directed mismatch repair in E. coli
16-82
Double-Strand Breaks in DNA Can Be
Repaired by Recombination
n
DNA Double-Strand Breaks are very dangerous
n
n
Breakage of chromosomes into pieces
Caused by ionizing radiation and chemical mutagens
n
n
n
n
Also caused by reactive oxygen species which are the byproducts of
cellular metabolism
10-100 breaks occur each day in a typical human cell
Breaks can cause chromosomal rearrangements and
deficiencies
They may be repaired by two systems known as
homologous recombination repair (HRR) and
nonhomologous end joining (NHEJ)
n
Refer to Figures 16.24 and 16.25
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16-83
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Double-strand break
5′
3′
3′
An identical"
3′
region"
between"
sister"
5′
chromatids
5′
5′
3′
End processing
5′
3′
3′
5′
3′
5′
5′
3′
Strand exchange
Figure 16.24
5′
3′
3′
5′
3′
5′
5′
3′
16-81
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5′
3′
3′
5′
3′
5′
5′
3′
DNA synthesis
5′
3′
3′
5′
3′
5′
5′
3′
Resolution and ligation
Figure 16.24
5′
3′
3′
5′
3′
5′
5′
3′
16-85
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Double-strand break
End binding
End-binding proteins
End bridging
Protein cross-bridge
Recruitment of additional"
proteins and end processing
Proteins for DNA processing
Gap filling and ligation
Figure 16.25
16-86
Repair of Actively Transcribed DNA
n
Not all DNA is repaired at the same rate
n
n
Actively transcribed genes in eukaryotes and prokaryotes
are more efficiently repaired than is nontranscribed DNA
The targeting of DNA repair enzymes to actively
transcribing genes has several biological advantages
n
Active genes are more loosely packed
n
n
n
May be more vulnerable to DNA damage
Transcription may make DNA more susceptible to damage
DNA regions that contain active genes are more likely to
be important for survival than nontranscribed regions
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16-87
Repair of Actively Transcribed DNA
n
In E. coli, a protein known as transcription-repair
coupling factor (TRCF) mediates transcription
coupled DNA repair
n
n
It targets the NER system to actively transcribed genes
with damaged DNA
Refer to Figure 16.26
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16-88
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA polymerase
Thymine dimer
T T
A thymine dimer has"
caused RNA polymerase"
to stall during transcription.
TRCF functions as a helicase"
and removes RNA polymerase"
from the damaged region.
TRCF"
(contains a"
binding site"
for UvrA)
RNA"
polymerase
The UvrA/UvrB complex is"
recruited to the damaged"
region. TRCF is released.
A
A
B
TRCF
Figure 16.26
The region is repaired as described in Figure 16.21.
16-89
n
n
In E. coli, translesion synthesis occurs under
extreme conditions that promote damage to DNA
This is termed the SOS response
n
n
It results in the up-regulation of several genes that repair
DNA, restore replication and prevent premature division
The damaged DNA that has not been repaired is
replicated by DNA polymerases II, IV and V
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16-93