Download Chapter 13 Mutation, DNA Repair, and Recombination

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)  helix
b) sheet
c)  subunit
d)  helix and 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
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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
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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
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“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.
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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
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Tautomeric Shifts Affect Base-Pairing
C:T
.
T:G
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Mutation Caused by Tautomeric Shifts
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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.
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Frameshift Mutations: alteration of
the open reading frame (ORF)
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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
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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.
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C: crossover suppressor
l: recessive lethal mutation
B: bar-eye mutation
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The Electromagnetic Spectrum
 X-rays induce mutations through ionization.
(DNA ionization--radical anions and cations-- G.+)
 Ultraviolet light induces mutations through excitation.
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Ionizing Radiation Causes Changes
in Chromosome Structure
Ionizing radiation breaks chromosomes
and can cause deletions, duplications,
inversions, and translocations
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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.
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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)
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Chemical Mutagens
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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.
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A Base Analog: 5-Bromouracil
--similar structures
--incorporated into DNA
--increase frequency of mis-pairing
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Mutagenic Effects
of 5-Bromouracil
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Nitrous Acid Causes Oxidative Deamination of
Bases
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Intercalation of an Acridine Dye
Causes Frameshift Mutations
--(+) charges molecules
--Incorporated into DNA
--DNA is more rigid
--Change conformation
(non-bending)
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Hydroxylamine
(NH2OH)
Hydroxylamine is a hydroxylating (OH) agent.
Hydroxylamine hydroxylates
the
amino
group
.
of cytosine and leads to G:C A:T transitions.
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Mutations Induced by Transposons
(Repeats)
Fragmets/segments of DNA that are capable to shift / translocate/move from one location to another
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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)
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•
•
•
•
•
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
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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.
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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.
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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.
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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
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Mutation: Usually a Random, Nonadaptive 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.
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Mutation: A Reversible Process
Forward mutation—mutation of a wildtype 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.
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Phenotype is restored
Phenotype is not suppressed
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Mutation: Phenotypic
Effects
The effects of mutations on
phenotype range from no
observable change to lethality.
DNA/RNA/
polypeptide
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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 nonfunctional gene products.
 Recessive (Dominant) lethal mutations affect genes
required for growth of the organisms and are lethal in
the homozygous state.
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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
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Recessive Mutations Often
Block Metabolic Pathways
(Recessive) lethal
mutations
Neutral mutations ( no effect on phenotype)
Expression of Wild-type and
Mutant Alleles
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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).
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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
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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.
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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.
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Morphogenesis in
Bacteriophage T4
# =gene
 This pathway was identified using mutants, electron
microscopy, and biochemistry.
 Temperature-sensitive and suppressor-sensitive
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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
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Assigning Mutations
coupling
Double heterozygote:
Two mutations (m1 and m2)
Wild-type (m1+ and m2+)
Co-exists in one or two
chromosomes
Arrangement (organized)
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repulsion
Apr and W are
recessive mutations
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X-linked mutations
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(head)
(tail)
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?
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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
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DNA Repair Mechanisms
Living organisms contain many
enzymes that scan their DNA for
damage and initiate repair
processes when damage is
detected.
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DNA Repair Mechanisms in E. coli
Light-dependent repair (photo-reactivation).
Excision repair.
Mismatch repair.
Post-replication repair.
Error-prone repair system (SOS response).
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UV LightDependent Repair:
Photolyase
Cleaves Thymine
Dimers.
--No endonuclease
--No Poly
--No ligase
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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.
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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.
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Base Excision Repair
AP:apyrimidinic site
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(
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)
Nucleotide Excision Repair
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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.
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Exonuclease
Qu ic kTime ™ a nd a
TIFF (Unc omp res se d) d ec omp res so r
a re n ee de d to se e t his p ic tu re.
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
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Me
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.
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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)
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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.
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Inherited Human Diseases
with Defects in DNA Repair
Several inherited human disorders
result from defects in DNA repair
pathways.
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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.
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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.
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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.
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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
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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