Chapter 7: DNA Mutation, DNA Repair, and Transposable Elements

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Chapter 7 DNA Mutation, DNA Repair, and
Transposable Elements

• Linnea Fletcher Ph.D.
• BIOL 2316

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Principal Points

• Review and answer
• What is a mutation? Name at least 5 different
  types of mutations.
• What agents can cause mutations, name two.
• Explain the difference between a simple
  reversion and a suppressor mutation
• What is a bacterial auxotroph? How can they be
  generated? How can they be selected using replica
  plating?
• How are mutations repaired in the cell
• What is the Ames test and how is it used to
  determine if a chemical is mutagenic?
• In Eukaryotes, what can happen when a mutation
  is not repaired?
• What is a transposable element? How are
  bacterial and eukaryotic transposable elements
  the same how are they different?

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What can happen when a mutation occurs in the
DNA (e.g. sickle cell anemia)
Figure 7.1 Concept of a mutation in the
protein-coding region of a gene. (Note that not
all mutations lead to altered proteins and that
not all mutations are in protein-coding regions.)

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Mutations Defined

• A mutation in a cell that is not passed to the
  next generation is a somatic mutation
• A mutation in a germ-line cell is passed to the
  next generation
• Mutation rate mutation per nucleotide pair or
  gene per generation
• Mutation frequency of a particular mutation
  per 100,000 organisms
• Do problem 7.10

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• Describe the differences between the following
  point mutations
• transition mutation (purine/pyrimidine to
  purine/pyrimidine)
• transversion mutation (purine/pyrimidine to
  pyrimidine/purine)
• missense mutation (different amino acid)
• nonsense mutation (stop)
• neutral mutation (does not affect phenotype)
• silent mutation (same amino acid)
• frameshift mutation (changes everything after it)
• Which of the above point mutations would never
  affect the protein that the gene encodes?
• Which of the above point mutations COULD result
  in no change in the protein being coded for?
• Why do frameshift mutations usually result in a
  nonfunctional protein?

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A nonsense mutation and its effect on translation
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Explain how a tRNA suppressor mutation works and
differentiate between an intrageneic and an
intergenic suppressor mutation.
(Intrageneic-within the same gene) intergenic
(different gene-usually a tRNA gene)
What is a reverse mutation?
A tRNA Tyr gene has mutated so that the tRNAs
anticodon is changed from 3-AUG-5 to 3-AUC-5,
which can read a UAG nonsense codon, inserting
tyrosine in the polypeptide chain at that codon.
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Spontaneous Mutations

• What are some causes of spontaneous mutations?
  (DNA replication as well as during the G phases
  of the cell cycle)
• What is the spontaneous mutation rate per gene
  per generation for eukaryotes? (10-4 to 4 x10-6
  per gene per generation)
• How does the cell keep this rate so low? (DNA
  repair and self-correcting polymerases)
• What types of mutations can result from
  replication errors by DNA polymerase and during
  what phase of the cell cycle can this happen?
• What are the most common sources of spontaneous
  mutations, and what are the direct consequences
  of such changes?
• Looping out of the DNA/skipping of the DNA pol
• Chemical depurination, deamination,
• Describe a point mutation that is most frequently
  found in mutational hot spots of a genome.
• Methylated cytosine is deaminated to produce a
  thymine (this change is NOT detected by repair
  systems)

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Bases are Normally In the keto Formif in The
enol form Base pair incorrectly
Figure 7.6 Normal WatsonCrick and
nonWatsonCrick base pairing in DNA. (a) Normal
WatsonCrick base pairing between the normal
forms of T and A and of C and G. (b)
NonWatsonCrick base pairing between normal
forms of pyrimidines and rare tautomeric forms of
purines. (c) NonWatsonCrick base pairing
between rare tautomeric forms of pyrimidines and
normal forms of purines.
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Figure 7.7 Production of a mutation as a result
of a mismatch caused by wobble base pairing. The
details are explained in the text.
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Figure 7.7 Production of a mutation as a result
of a mismatch caused by wobble base pairing. The
details are explained in the text.
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Spontaneous Chemical Changes
Figure 7.9 (a) Deamination of cytosine to uracil.
(b) Deamination of 5-methylcytosine (5mC) to
thymine.
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Induced Mutations

• What are some examples of some physical mutagens?
  (radiation or chemicals)
• Why are the types of mutations different when
  caused from UV light, compared to ionizing
  radiation? (ionizing produces ions breaking
  covalent bonds (e.g. sugar-phosphate backbone-the
  effect is accumulative) (the rings absorb UV
  light and are changed chemically-form T-T dimers)
• What are the direct consequences of formation of
  a thymine dimer? (bulge in the DNA)
• What are 3 general classes of chemical mutagens?
• (base analogs, base-modifying, and intercalating)
• In what parts of the cell cycle do these
  different classes of mutagens manifest
  themselves? (base analogs and intercalating work
  during replication base-modifying-anytime)
• What type of mutation do base analogs induce?
  (take the place of the base cause transitions)
• What other types of disruptions can a base analog
  effect? What is an example of this and how is it
  used medicinally? AZT is an analog of T (stops
  replication)
• What are 3 types of base-modifying agents that
  cause mutations, and what are their direct
  effects? (deamination, hydroxylation, alkylation)

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Can you pick the right answer?

• Base analogs may cause mutations because
• they modify the chemical structure and properties
  of the normal base.
• they insert themselves between adjacent bases on
  the DNA strand and cause an extra base to be
  inserted during replication.
• they remove amino groups from bases, causing them
  to pair with the wrong base during replication.
• they may exist in alternate chemical states that
  pair with different DNA bases than the normal
  state during replication.
• Both A and C

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What type of radiation does a tanning booth
produce? Is it safe?
Figure 7.10 Production of thymine dimers by
ultraviolet light irradiation. The two components
of the dimer are covalently linked in such a way
that the DNA double helix is distorted at that
position.
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• Intercalating agents- proflavin, acridine, and
  ethidium bromide insert themselves between
  adjacent bases in one or both strands of the DNA
  double helix causing it to relax
• During replication, an extra base must be
  inserted opposite the agent (addition)
• If it inserts itself into the new DNA in place of
  a base, then when the DNA replicates the agent is
  lost resulting in a deletion
• Site-Specific Mutagenesis in vitro generation of
  specific mutations

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Intercalating Mutations (e.g. EtBr)
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Ames Test A Screen for Potential Carcinogens

• Describe the mutants used in the Ames test, and
  how a negative and a positive results appears in
  this test.
• Why is a liver extract called S9 used in the Ames
  test? (provides the enzymatic detoxification
  pathway found in mammalian liver)
• Describe some controls that must be used in an
  Ames test to avoid a false-positive result (e.g.
  Just the S9 extract bacteria without the
  chemical, to make sure the extract is not
  mutagenic by itself)
• Describe some controls that must be used in an
  Ames test to avoid a false-negative result. (e.g.
  Bacteria alone is both a negative and positive
  control to make sure that the (his-)bacteria
  (without his) does not revert on its own to wt,
  and with his, to make sure that it is alive)
• Why must an Ames test be followed up by animal
  studies in order to verify a result?
• Why are Ames tests used first, before animal
  studies?

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Ames Test (know this test and its controls)
The chemical Causes a reversion Mutation in the
bacteria So it now can grow Without histidine
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• How are mutants detected by scientists?
• Visible
• Nutritional (auxotrophic)
• What is replica plating, and how is it used to
  detect auxitrophic mutants?
• What is the most common type of conditional
  mutant, and what would explain how this works?
  (temperature sensitive changes the shape of the
  protein)
• Resistance Mutations (plate on media containing
  the chemical)

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Replica-plating technique to screen for mutant
strains of a colony-forming microorganism.
How would you Select for Trp- Auxotrophic
mutant? (doesnt make tryptophan Requires it)
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DNA Repair

• DNA repair mechanisms are placed into different
  categories on the basis of the way they operate
• Direct correction or direct reversal- reversing
  the damage
• Excise the damaged areas and then repair the gap
  by new DNA synthesis

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Direct Reversal of DNA Damage

• Mismatch Repair by DNA Polymerase Proofreading
  (exonuclease activity-how was it shown that it is
  important?)
• Repair of UV-Induced Pyrimidine Dimers (reverted
  by exposure to near-UV light-activates photolyase
  not found in humans)
• Repair of Alkylation Damage (by O6-methylguanine
  methyltransferase encoded by ada gene)

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Base Excision Repair and Repair Involving
Excision of Nucleotides

• Repair glycosylase enzyme recognizes the damaged
  base and removes it, the baseless-sugar is then
  excised, the gap is filled by a repair DNA pol
  and ligase
• Excision Repair (dark repair or nucleotide
  exision repair NER)-not photoactivated, repairs
  T dimers and other distortions in the DNA

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Figure 7.16 Nucleotide excision repair (NER) of
pyrimidine dimer and other damage induced
distortions of DNA.
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• Repair by Methyl-Directed Mismatch Repair
  (recognizes mismatched nucleotides, excises, and
  carries out repair synthesis)
• In humans, mutations in any one of the four human
  mismatch repair genes confers a phenotype of
  hereditary predisposition to a form of colon
  cancer called hereditary nonpolyposis colon
  cancer (OMIM 120435)

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Figure 7.17 Mechanism of mismatch repair. The
mismatch correction enzyme recognizes which
strand the base mismatch is on by reading the
methylation state of a nearby GATC sequence. If
the sequence is unmethylated, a segment of that
DNA strand containing the mismatch is excised and
new DNA is inserted.
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OMIM 278700) XP xeroderma pigmentosum
Caused by homozygosity For a recessive mutation
in A repair gene.
One example of a DNA-repair genetic disease
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Transposons

• Fall into two general classes with respect to how
  they move.
• One class encodes proteins that move the DNA
  element directly to a new position or replicate
  the DNA.
• Found in both prokaryotes and eukaryotes
• The other class are related to retroviruses in
  that they encode a reverse transcriptase for
  making DNA copies of their RNA transcripts, which
  then integrate at new sites in the genome.
• Found only in eukaryotes.

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• Transposable elements are important because they
  can insert into sites where there is no sequence
  homology (nonhomologous recombination)

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Prokaryotes

• What are two types of transposons in prokaryotes
  and how do they differ? (IS and Tn)
• What enzyme is required for the transposition of
  an IS element?
• How is a composite transposon different from a
  noncomposite transposon?
• How does the replicative transposition mechanism
  differ from the conservative mechanism of
  transposition?

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• EUKARYOTIC TRANSPOSITION
• What is cytogenetics, and how was it used to find
  jumping genes in eukaryotes?
• In what ways are eukaryotic transposable elements
  similar to those found in prokaryotes?
• What can determine the stability of a
  newly-inserted transposable element in plants?

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• What genes do Ty elements in yeast carry, and
  what are their purposes?
• In what ways is the yeast Ty element similar to a
  retrovirus?
• Why are Ty elements classified as retroposons?

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• What type of transposons are SINEs and LINEs?
• What percentage of the human genome is composed
  of LINEs, and what is the most common one?
• Are human LINEs still able to transpose? How do
  we know this?
• What is the most common SINE in the human genome
  and about how frequently does it occur there?
• Are human SINEs still able to transpose? How do
  we know this?