Chapter 13

Mutation, DNA Repair,…

© John Wiley & Sons, Inc.

 Amino acids in polypeptides are joined by what type of bond?

a) Hydrogen
b) no-Peptide
c) Covalent
d) Ionic
e) Weak
Which of the following is not a true statement about ribosomes?

a) They are structured into large and small subunits

b) They are comprised of RNA and protein
c) The ribosomes in prokaryotes are typically larger than those found in eukaryotes
d) The ribosome subunits associate during the initiation of translation
e) All of these are false statements

Which of the following is a secondary structure often observed in proteins?

a) α helix
b) β sheet
c) σ subunit
d) α helix and β sheet
e) All of these
 Chapter Outline
 Mutation: Source of the Genetic Variability Required for
Evolution
 The Molecular Basis of Mutation
 Mutation: Basic Features of the Process
 Mutation: Phenotypic Effects
 Assigning Mutations to Genes by the Complementation Test
 Screening Chemicals for Mutagenicity: The Ames Test

 DNA Repair Mechanisms

 Inherited Human Diseases with Defects in DNA Repair
 DNA Recombination Mechanisms

© John Wiley & Sons, Inc.

Mutation: Source of the Genetic
Variability Required for Evolution

Mutation
--A change in the genetic material (molecular level)

Mutant
--an organism that exhibits a novel phenotype

© John Wiley & Sons, Inc.

Types of Mutations
– Changes in chromosome number and
structure
– Point mutation--changes at specific
nucleotide in a gene (A,T,C,G)
– Insertion mutations--insert fragment of
DNA
– Deletion mutations--delete fragment of
DNA

© John Wiley & Sons, Inc.

“Virus”
Disruption of FGF -Receptor Function
Fibroblast Growth Factor Signaling is Essential
for Mesoderm Production in Frog Embryos
Dominant mutations in human FGFR-3
(TM domain)

Achondroplasia
 Mutation and Evolution
Mutation is the source of all genetic
variation (e.g.,chromatin remodeling).

Natural selection preserves the

combinations best adapted (or NOT) to
the existing environment.

© John Wiley & Sons, Inc.

 The Molecular Basis of Mutation

Mutations alter the nucleotide sequences of genes in

several ways,

--the substitution of one base pair for another.

(A for T)

--the deletion (or addition) of one or a few base pairs.

( AT…….GC)
Tautomeric Shifts:
--chemical fluctuations,
--conformation states (stable==========unstable)

Py
A:T
.
C:G

Pu

© John Wiley & Sons, Inc.

Tautomeric Shifts Affect Base-Pairing

C:T
.
T:G
© John Wiley & Sons, Inc.
Mutation Caused by Tautomeric Shifts

© John Wiley & Sons, Inc.

 Base Substitutions
A transition replaces a pyrimidine with another
pyrimidine or a purine for another purine.
A transversion replaces a pyrimidine with a
purine or a purine with a pyrimidine.

© John Wiley & Sons, Inc.

Frameshift Mutations: alteration of
the open reading frame (ORF)

© John Wiley & Sons, Inc.

 Mutation Frequency
Frameshift, transition, transversion mutations
are infrequent
– Bacteria and phage: 10–8 to 10–10 per nucleotide
pair per generation
– Eukaryotes: 10–7 to 10–9 per nucleotide pair per
generation
1/107 to 1/109

Silent mutation: UCU=Ser;

UCA, UCC, UCG = Ser

© John Wiley & Sons, Inc.

 Induced Mutations
Induced mutations occur upon exposure to
physical (energy) or chemical (reaction) mutagens.

Muller demonstrated that exposing Drosophila

sperm to X-rays increased the mutation
frequency.

Hermann J. Muller and Edgar Alternburg

measured the frequency (>150 fold increase) of
X-linked recessive lethal mutations in Drosophila.

© John Wiley & Sons, Inc.

 C: crossover suppressor
l: recessive lethal mutation
B: bar-eye mutation

© John Wiley & Sons, Inc.

 The Electromagnetic Spectrum

 X-rays induce mutations through ionization.

(DNA ionization--radical anions and cations-- G.+)

 Ultraviolet light induces mutations through excitation.

© John Wiley & Sons, Inc.

Ionizing Radiation Causes Changes
in Chromosome Structure

Ionizing radiation breaks chromosomes

and can cause deletions, duplications,
inversions, and translocations

© John Wiley & Sons, Inc.

Stability of carbon-containing molecules
Mutagenesis by Ultraviolet Irradiation
 Hydrolysis of cytosine to
a hydrate may cause
mis-pairing during
replication

 Cross-linking of adjacent
thymine forms
thymidine dimers,
which block DNA
replication and activate
DNA repair mechanisms.
© John Wiley & Sons, Inc.
UV-A 320 to 400 nm
UV-B/C <300 nm
Types of Chemical Mutagens
Chemicals that are mutagenic to both
replicating and non-replicating DNA
(e.g., alkylating agents and nitrous acid)

Chemicals that are mutagenic only to

replicating DNA (e.g., base analogs
and acridine dyes)

© John Wiley & Sons, Inc.

Chemical Mutagens

© John Wiley & Sons, Inc.

 Alkylating Agents
 chemicals that donate alkyl groups to other molecules.

 induce transitions, transversions, frameshifts, and chromosome

aberrations (anomaly).

 Alkylating agents of bases can change base-pairing properties.

(GC to AT)

 can also activate errors during repair processes.

© John Wiley & Sons, Inc.

A Base Analog: 5-Bromouracil
--similar structures
--incorporated into DNA
--increase frequency of mis-pairing

© John Wiley & Sons, Inc.

Mutagenic Effects
of 5-Bromouracil

© John Wiley & Sons, Inc.

Nitrous Acid Causes Oxidative Deamination
of Bases

© John Wiley & Sons, Inc.

 Intercalation of an Acridine Dye
Causes Frameshift Mutations

--(+) charges molecules

--Incorporated into DNA
--DNA is more rigid
--Change conformation
(non-bending)

© John Wiley & Sons, Inc.

 Hydroxylamine
(NH2OH)
Hydroxylamine is a hydroxylating (OH) agent.

Hydroxylamine hydroxylates the amino group

.
of cytosine and leads to G:C A:T transitions.

© John Wiley & Sons, Inc.

 Mutations Induced by Transposons
(Repeats)
Fragmets/segments of DNA that are capable to shift / translocate/move from one location to another

© John Wiley & Sons, Inc.

 Expansion of Trinucleotide Repeats
Simple tandem repeats are repeated
sequence of one to six nucleotide pairs (CGG,
CAG and CTG).

Trinucleotide repeats can increase in copy

number and cause inherited diseases (Fragile
X Syndrome, Huntington disease,
Spinocerebellar ataxia)

© John Wiley & Sons, Inc.

• Mutations are induced by
• chemicals,
• ionizing irradiation,
• ultraviolet light, and
• endo(exo)genous transposable genetic elements.

• Point mutations are of three types:

(1) Transitions—purine for purine and pyrimidine for pyrimidine substitutions,
(2) Transversions—purine for pyrimidine and pyrimidine for purine substitutions,
and
(3) Frameshift mutations—additions or deletions of one or two nucleotide pairs,
which alter the reading frame of the gene distal to the site of the mutation.

Chromatin remodeling==Epigenetics

© John Wiley & Sons, Inc.

Mutation: Basic Features of the Process

Mutations occur in all organisms from viruses to

humans.

They can occur spontaneously or be induced by

mutagenic agents.

Mutation is usually a random, non-adaptive process.

© John Wiley & Sons, Inc.

Mutation: Somatic or Germinal
Germinal mutations occur in germ-line
cells and will be transmitted through the
gametes to the progeny.
puberty (10-14 years)
Somatic mutations occur in somatic
cells; the mutant phenotype will occur
only in the descendants of that cell and
will not be transmitted to the progeny.

© John Wiley & Sons, Inc.

 Mutation: Spontaneous or Induced

Spontaneous mutations occur without a

known cause due to unknown agents in the
environment.

Induced mutations result from exposure or

organisms to mutagens, physical and
chemical agents that cause changes in
DNA, such as ionizing irradiation, ultraviolet
light, or certain chemicals.
© John Wiley & Sons, Inc.
 Factors Influencing the Rate
of Spontaneous Mutations
Accuracy of the DNA replication
machinery
Efficiency of the mechanisms for the
repair of damaged DNA
Degree of exposure to mutagenic
agents in the environment

© John Wiley & Sons, Inc.

 Mutation: Usually a Random, Non-
adaptive Process
Is mutation random (intrinsic) or directed by the
environment?

Replica plating was used to identify the presence

of antibiotic (chemical) resistant bacteria prior to
treatment with an antibiotic (chemicals).

Environmental stress does not cause mutations

but selects for mutants that are best adapted to the
environmental stress.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.
Mutation: A Reversible Process

Forward mutation—mutation of a wild-

type allele to a mutant allele.
Reverse mutation (reversion)—a
second mutation that restores the
original phenotype.
– Back mutation—a second mutation at the
same site.
– Suppressor mutation—a second mutation
at a different location in the genome.
© John Wiley & Sons, Inc.
Phenotype is restored

Phenotype is not suppressed

© John Wiley & Sons, Inc.

Mutation: Phenotypic
Effects
The effects of mutations on
phenotype range from no
observable change to lethality.

DNA/RNA/ polypeptide

© John Wiley & Sons, Inc.

 Types of Mutations
 Isoalleles have no effect on phenotype or small
effects that can be recognized only by special
techniques.

 Null alleles result in no gene product or totally non-

functional gene products.

 Recessive (Dominant) lethal mutations affect genes

required for growth of the organisms and are lethal in
the homozygous state.

© John Wiley & Sons, Inc.

 X-linked Recessive Lethal
Mutations Alter the Sex Ratio

Monoploid: (Recessive or Dominant) positive mutational effect (phenotype)

Diploid: (Recessive)--positive mutational effect (phenotype)--homozygous
X-linked .....hemizygous
© John Wiley & Sons, Inc.
 Recessive Mutations Often
Block Metabolic Pathways

(Recessive) lethal
mutations

Neutral mutations ( no effect on phenotype)

Expression of Wild-type and
Mutant Alleles

© John Wiley & Sons, Inc.

 Mutations in Human Globin Genes
Adult hemoglobin (Hemoglobin A) contains two 
chains and two  chains.

Hemoglobin in patients with sickle-cell anemia

(Hemoglobin S) differs from Hemoglobin A at only
one position.

The sixth amino acid in the  chain is glutamic acid

in Hemoglobin A (HBBA) and is valine in
Hemoglobin S (HBBS). This substitution is caused
by mutation of a single base pair (T:A substitution).

© John Wiley & Sons, Inc.

 Tay-Sachs Disease
Tay-Sachs disease
is an autosomal
recessive disease.
The mutation
causing Tay-Sachs
disease is in the
gene encoding
hexosaminidase A.

not linked to sex chromosome © John Wiley & Sons, Inc.

 Conditional Lethal Mutations
(Experimental)

Conditional lethal mutations are

– Lethal in the restrictive condition but
– Viable in the permissive condition.

Mutants with conditional lethal alleles can be

propagated under the permissive condition,
and the phenotype can be studied under
restrictive condition.

© John Wiley & Sons, Inc.

 Conditional Lethal Mutants
Auxotrophs are unable to synthesize an
essential metabolite that is synthesized by
prototrophs. Auxotrophs can grow only when
the essential metabolite is supplied in the
medium.
Temperature-sensitive mutants will grow at
one temperature but not at another.
Suppressor-sensitive mutants are viable
only when a second genetic factor, a
suppressor, is present.
© John Wiley & Sons, Inc.
 Morphogenesis in
Bacteriophage T4

# =gene

 This pathway was identified using mutants, electron

microscopy, and biochemistry.
 Temperature-sensitive and suppressor-sensitive
© John Wiley & Sons, Inc.
http://www.endocytosis.org/Dynamin/Shibire.html
 Assigning Mutations to genes
by the Complementation test

The complementation or trans

test can be used to determine
whether two mutations are located
in the same chromosome or in two
different chromosomes.

Genetic tool to demonstrate: One gene....One polypeptide

© John Wiley & Sons, Inc.
Assigning Mutations
coupling

Double heterozygote:
Two mutations (m1 and m2)
Wild-type (m1+ and m2+)
Co-exists in one or two
chromosomes

repulsion
Arrangement (organized)

© John Wiley & Sons, Inc.

 Apr and W are X-linked mutations
recessive mutations

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.
(head) (tail)

© John Wiley & Sons, Inc.

 ?

© John Wiley & Sons, Inc.

 Screening Chemicals for mutagenicity:
The Ames test
The Ames test provides a simple and inexpensive method for
detecting the mutagenicity of chemicals (Carcinogens)
-intracellular
-extracellular
-enviromental

Auxotrophic prototrophic
© John Wiley & Sons, Inc.
© John Wiley & Sons, Inc.
DNA Repair Mechanisms

Living organisms contain many

enzymes that scan their DNA for
damage and initiate repair
processes when damage is
detected.

© John Wiley & Sons, Inc.

 DNA Repair Mechanisms in E. coli

Light-dependent repair (photo-reactivation).

Excision repair.
Mismatch repair.
Post-replication repair.
Error-prone repair system (SOS response).

© John Wiley & Sons, Inc.

 UV Light-
Dependent Repair:
Photolyase
Cleaves Thymine
Dimers.

--No endonuclease
--No Poly
--No ligase

© John Wiley & Sons, Inc.

 Excision Repair (steps)
A DNA repair endonuclease or endonuclease-
containing complex recognizes, binds to, and
excised the damaged base or bases.

A DNA Polymerase fills in the gap, using the

undamaged complementary strand of DNA as a
template.

DNA ligase seals the break left by DNA

polymerase.

© John Wiley & Sons, Inc.

 Types of Excision Repair
Base excision repair pathways
remove abnormal or chemically
modified bases.

Nucleotide excision repair pathways

remove larger defects, such as thymine
diners.

© John Wiley & Sons, Inc.

 Base Excision Repair

AP:apyrimidinic site © John Wiley & Sons, Inc.

 ( )

© John Wiley & Sons, Inc.

Nucleotide Excision Repair

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.
 Mismatch Repair in E. coli
 Mismatching or mispairing of G and T
(DNA polymerase/exonuclease proofreading activity)

 The A in GATC sequences is methylated subsequent to DNA

replication.

 In newly replicated DNA, the parental strand is methylated, but

the new strand is not. This difference allows the mismatch repair
system to distinguish the new strand from the old strand.

 The mismatched nucleotide is excised from the new strand and

replaced with the correct nucleotide, using the methylated
parental strand as a template.

© John Wiley & Sons, Inc.

 Exonuclease

QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Endonuclease
 Mismatch Repair in E. coli
MutS recognizes mismatches and binds to
them to initiate the repair process.
MutH and MutL join the complex.
MutH cleaves the unmethylated strand at
hemimethylated GATC sequences on either
side of the mismatch.
Excision requires MutS, MutL, MutU (DNA
helicase II), and an exonuclease.
DNA polymerase III fills in the gap, and DNA
ligase seals the nick.
Me Me

Me Me
© John Wiley & Sons, Inc.
 Post-replication Repair in E. coli
 A thymine dimer in the template strand blocks replication
(DNA Polymerase III does not recognize thymidine dimer)

 DNA Polymerase III restarts DNA synthesis past the dimer,

leaving a gap in the nascent strand.

 RecA binds to the single strand of DNA at the gap and

mediates base pairing with the homologous segment of the
sister double helix to fill the gap.

 DNA polymerase fills the gap in the sister double helix, and
DNA ligase seals the nick.

© John Wiley & Sons, Inc.

The SOS Response in E. coli
 If DNA is heavily damaged by mutagenic agents, the
SOS response, which involves many DNA
recombination, DNA repair, and DNA replication
proteins, is activated.

 DNA dependent DNA Polymerase V replicates DNA

in damaged regions, but sequences in damaged
regions cannot be replicated accurately.

 This error-prone system eliminates gaps but increases

the frequency of replication errors (Pol II, IV and V are
low-fidelity polymerases)

© John Wiley & Sons, Inc.

 Induction of the SOS Response
 In the absence of DNA damage, LexA binds to DNA
regions that regulate transcription of SOS response
genes and keeps their expression levels low.

 When extensive DNA damage occurs, RecA binds to

single-stranded regions of DNA in damaged regions.

 This activates RecA, which stimulates LexA to

inactivate itself. When LexA is inactivated, the SOS
response genes are expressed.

© John Wiley & Sons, Inc.

Inherited Human Diseases
with Defects in DNA Repair

Several inherited human disorders

result from defects in DNA repair
pathways.

© John Wiley & Sons, Inc.

Xeroderma Pigmentosum (XP)
 Individuals with XP are
sensitive to sunlight (UV light).

 The cells of individuals with XP

are deficient in the repair of
UV-induced damage to DNA.

 Individuals with XP may

develop skin cancer or
neurological abnormalities.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.
DNA Recombination Mechanisms

Recombination between homologous

DNA molecules involves the activity of
numerous enzymes that
1-cleave,
2-unwind,
3-stimulate single-strand invasions of
double helices (RecA proteins),
4-repair, and
5-join strands of DNA.
© John Wiley & Sons, Inc.
 Recombination
 In eukaryotes, crossing over is associated with the
formation of the synaptonemal complex during
prophase of meiosis I (Chiasmata).

 Crossing over involves the breakage of parental

chromosomes and rejoining of the parts in new
combinations.

 The Holliday model and the double-strand break

model are two explanations of the molecular basis of
recombination.

© John Wiley & Sons, Inc.

The Holliday Model: single strand break model
The Holliday Model: single strand break model

--DNA-dependent ATPase.

--can hold a single strand and

double strand together.

--DNA synapsis reaction

between a DNA double helix and a
homologous region of single
stranded DNA.

--catalyzes branch migration begins.

 The Recombination
Intermediate is a Chi Structure

© John Wiley & Sons, Inc.

http://web.mit.edu/engelward-lab/animations/DSBR.html
5) Which of the following is considered a point mutation?
1. Substitution of the base A for the base C
2. Deletion of the base T
3. Insertion of the base G

a) 1
b) 2
c) 3
d) 1 and 2
e) All of these

Which base pair combinations can form when nitrogenous bases are present in their rare imino
or enol states?

a) A:T
b) C:G
c) A:C
d) A:T and C:G
e) C:G and A:C
Which of the following enzymes performs light dependent repair?

a) DNA gyrase
b) DNA polymerase
c) DNA photolyase
d) RNA polymerase
e) DNA helicase