Mutations

1
Mutations

• Mutations are inheritable changes in the DNA
• Failure to faithfully store genetic information
• Changes can be to chromosomes or genes
• Current focus changes to DNA sequences.
• This means an alteration in a basepair or
• in the order of the basepairs.

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Types of mutations-1

• Mutations can be classified in many, many ways
• Some ways mutually exclusive, some not.
• Spontaneous vs. Induced
• Spontaneous happens naturally
• Enzymatic errors, especially in copying
• Various chemical reactions
• Induced mutations specifically caused, as by
  researcher
• Treatment with various chemicals, radiation
• Gametic (germ line) vs. somatic
• Gametic mutations can be passed on to next
  generation
• Somatic only affects individual (in metazoans)

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Types of mutations-2

• Morphological
• Change in physical structure, readily observed
• Nutritional/biochemical
• Mutated enzyme results in phenotypic change
• Bacterial auxotrophs sickle cell anemia
• Behavioral mutations
• Regulatory mutations
• Affect control of gene expression rather than
  protein

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Types of mutations-3

• Lethal mutations not easily studied unless
• Conditional mutations expressed depending on
  environmental conditions, especially temperature.
• a way to study lethal mutations permissive and
  restrictive temperatures esp. useful with
  bacteria
• temp sensitive mutations occur
• naturally, continued in offspring
• Siamese cats, Himalayan rabbits

www.tcainc.org/photos/ farpoint/saavik1.jpg
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Types of mutations-4

• Classification of mutations by FUNCTION
• Loss of function knockout or null.
• Hypomorphic lowered expression, leaky
• Hypermorphic greater activity or more visible
  trait
• typically regulatory mutation, results in
  increased expression
• Gain of function e.g. new enzymatic activity
• a factor in evolution
• Dominant negative bad apple spoils the bunch
  e.g. bad protein in multicomponent enzyme

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Detection of mutations

• Bacteria and fungi
• Prototrophs and auxotrophs microbe no longer
  able to synthesize or breakdown particular
  nutrient.
• Change in behavior, e.g. motility
• Various methods in plants and animals
• Humans (not suitable experimental organisms)
• Reliance on pedigrees
• Possible to determine sex linkage, dominance

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Mutations are rare (but not equally so)

• Mutation rate depends on species and on gene
• Hot spot a location in DNA where mutations occur
  significantly more often than the usual 1/ 106.
• Monotonous run of single nucleotide or tandem
  repeats GGGGGGGGG or ATGGATGGATGG
• Methylated cytosines
• methylation is added a CH3 group to something
• Cytosines are methylated to help indicate which
  DNA strand is older (helps with DNA repair).
• Problem occurs when a cytosine is chemically
  damaged by deamination. (more later)

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Mutations

• Our example
• information, 3 letters at a time, read
  consecutively
• Point mutations
• Frameshift mutations Insertion

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more Mutations

• Frameshift deletion
• Transposon mutagensis transposons are segments
  of DNA that can jump into another spot in the
  DNA they have information.

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More types of mutations

• Switch between A G, or C T transition
• Switch between purine and pyrimidine
  transversion
• Silent 3rd position of codon usually means same
  amino acid, so change here has no effect.
• Missense typically a single nucleotide change,
  causes change in amino acid and noticeable
  effect.
• Nonsense change amino acid codon to STOP codon
• Additions, deletions, and stuttering
• Stuttering repeated sequences sometimes copied
  incorrectly enzyme gets confused?

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Mutagens that which causes mutations

• Base analogs e.g. 5-bromouracil. In equilibrium
  between keto and enol forms
• In keto form, looks like T
• In enol form looks like C
• Used one way, but when copied, mispairing can
  occur.
• Modifying agents chemically change bases
• HNO2 nitrous acid deaminates (amino to keto)
• See upcoming slide deamination
• Alkylating agents (ethylmethane sulfonate) add
  methyl or ethyl group to bases (-CH3 or CH2CH3),
  cause mispairing during synthesis

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Loss of a purine, a natural process
Can lead to an incorrect base being added a
mutation.
saturn.roswellpark.org/.../ AP_site_generation.gif
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Pyrimidines and deamination
Deamination Loss of an amine group, replacement
w/ a keto group.
Deamination of cytosine makes uracil recognized
as wrong and repaired. Deamination of 5-methyl
cytosine produces thymine, which is normal
results in a transition mutation.
14
Frameshift mutations

• Cause misalignment during DNA replication caused
  by intercalating agents such as ethidium bromide
  or acridine orange

http//www.photobiology.com/photoiupac2000/pierard
/intintercal.jpg
15
Radiation

• UV light at 265 nm
• causes thymine dimers covalent connections
  between adjacent thymines. Hurried repair makes
  mistakes.
• Ionizing radiation
• short wavelength, high energy radiation, e.g.
  x-rays, gamma radiation.
• Causes ss, ds breaks in DNA.

http//academic.brooklyn.cuny.edu/ biology/bio4fv/
page/molecular 20biology/mutation-prym-dimers.jpe
g
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Ionizing Radiation

• Major damage is from free radicals
• Most abundant substance in cell is water
  radiation produces radicals that attack DNA,
  causing breaks.
• The effects of radiation are a matter of
  considerable scientific and political
  controversy.
• Effects of high levels of radiation are well
  understood, but effects of low levels are very
  difficult to study.
• Brief soapbox after Chernobyl tragedy, people
  vacated many square miles around damaged reactor.
  Now, endangered animals making a comeback despite
  radiation.

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Repair of DNA damage

• Despite the constant bombardment of DNA with
  radiation and chemicals, cells possess repair
  mechanisms.
• Repair systems exist for
• UV light damage
• Chemical changes to bases
• Loss of bases
• Incorrect copying
• Ss and ds breaks in DNA

http//earthobservatory.nasa.gov/Library/UVB/Image
s/dna_mutation.gif
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Repair of Thymine dimers
Photoreactivation Enzyme uses a photon of blue
light to separate thymines from each other.
(When using UV as a mutagen, put cells in dark
afterwards!) (in E. coli) Excision Repair DNA
repair enzymes recognize a distorted DNA helix
(such as caused by thymine dimers). The entire
local section of DNA is removed and replaced.
In all prokaryotes eukaryotes.
http//www-personal.ksu.edu/bethmont/excisio3.gif

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Repair of chemical changes

• Deamination of cytosine
• as shown previously, converts cytosine to uracil
• the enzyme uracil glycosylase cuts off uracil,
  leaving deoxyribose as part of backbone, creating
  an AP site
• AP apurinic or apyrmidinic, meaning purine etc.
  NOT there.
• AP repair, mechanism that specifically fixes such
  places.

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Creation of an AP site
saturn.roswellpark.org/.../ AP_site_generation.gif
21
AP repair- continued
Activity of uracil glycosylase or spontaneous
loss of base from DNA can create an AP site. An
endonuclease cuts out the remaining
sugar-phosphate and replaces it with a
complete nucleotide.
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Bulky excision repair

• Like in repair of UV-induced damage, cells sense
  bulges, kinks, or similar damage to DNA
• Chunk of DNA containing the damaged area is
  excised, replaced by DNA polymerase I enzyme (or
  equivalent)
• 13 bases removed in bacteria
• Eukaryotes (always more elaborate) take out 28
  nucleotides
• In humans, failure in this repair system causes
  disease xeroderma pigmentosum with increased risk
  of skin cancer.

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Mismatch repair
If Proofreading misses Other enzymes recognize
that the wrong base pair is in place, cuts out
incorrect one and replaces it. Which one is
incorrect? Presumably the newest one the one
on the DNA chain with the least amount of methyl
cytosines.
cmgm.stanford.edu/.../DNA20Repair20-20Doug/
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SOS Repair

• Especially in bacteria, when damage to DNA is
  severe, an emergency system goes into effect
  where damage is repaired rapidly, but sloppily.
  Introduces many mutations, some possibly fatal,
  but DNA damage would surely be fatal otherwise.