Mutation

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Mutation
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Dr. Parvin Pasalar Tehran University of Medical
Sciences
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Mutation

• Definition
• An un- repaired damages to DNA
• Causes
• It may be spontaneous or induced because of
  different agents
• Classifications
• are classified on different basis
• Their importance
• Genetic Disease raw material for the
  development

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Different Causes of mutations
Contrary to popular belief Most DNA damage is
caused by endogenous mutagens
Estimated DNA damage/day in human cells SSBs
50,000/day Depurinations
10,000/day Deaminations 600/day
Oxidations 2000/day Alkylations 5000/day
DSBs 50-100/day -
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• What is the scale of our worries?
• We each have 46 chromosome 6 X 109
• bp DNA/cell
• On average, a mistake is made once
• every in 109 bp of DNA copied
• So, we have 6 mistakes/cell/division
• We have 1014 cells in our body that
• divide a minimum of once per year
• So, 6 X 1014 mistakes per year

Or, at least 60 billion mistakes while in
class for Biochemistry today!!!
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Classifications of Mutations
1- Can be spontaneous or induced 2- May be
substitutions or frameshift 3- May occur in
structural or regulatory sequences 4- May be
small (point) or big 5- May have no or severe
effect 6- Somatic or germinal
Types of mutations in ORFs Missense mutation
Base pair substitution results in substitution
of a different amino acid. Nonsense
mutation Base pair substitution results in a
stop codon (and shorter polypeptide). Neutral
mutation Base pair substitution results in
substitution of an amino acid with similar
chemical properties (protein function is not
altered). Silent mutation Base pair
substitution results in the same amino acid (or
nucleotide). Frameshift mutations Deletions or
insertions (not divisible by 3) result in
translation of incorrect amino acids, stops
codons (shorter polypeptides),or read-through of
stop codons (longer polypeptides).
1- Can be spontaneous or induced 2- May be
substitutions or frameshift 3- May occur in
structural or regulatory sequences 4- May be
small (point) or big 5- May have no or severe
effect 6- Somatic or germinal
Substitution The base number remains the same
but the types changes -transitions pu to pu or
py to py -tranversion pu to py or py to
pu Frameshift The base number changes -in
coding regions, insertion or deletion of a nt
that is not a multiple of 3 changes gene coding
sequence Introduces premature stop
codonsprotein truncation
Spontaneous At physiologic rate Induced
Because of the treatment with different agents

• Origins of Spontaneous Mutation
• Errors in DNA replication
• DNA polymerase accuracy
• Errors in DNA recombination
• DNA strands alignment
• Base alterations and base damage
• tautomerization deamination
  depurination
• oxidation alkylation
• Spontaneous frameshift mutations
• mispairing during replication and
  recombination

Qualitative changes Mutation structural
sequences may causes changes in the sequence of
aa of the resulting product (polypeptide)
Quantative changes Mutation in regulatory
sequences does not change the structure of the
product but its amount

• Small Gene mutation are those that change a gene
• Big Chromosomal rearrangements
• - can be inversions, deletions,
• translocations, or amplifications
• -can alter chromosome organization
• and affect gene function
• -can activate gene expression
• -can create novel fusion genes
• -can affect chromosome segregation
• (non dysjunction) during
  meiosis-semi- sterility
• -some types of rearrangements in
• meiosis may be of evolutionary
  benefit

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Different Causes of Mutations

• Biological (normal error rate in DNA metabolic
  processes)
• Physical (Radiation)
• Sunlight
• Chemical (Mutagens, Carcinogens)
• 1- Alkylating agents
• 2- Base analogues
• 3- intercalating agents
• 4- Different chemicals such as
• a- Nitrous acid
• b- Hydroxylamine

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Different Type of DNA damages

• 1- Double-strand breaks (DSBs)
• 2- Single- strand breaks (SSBs)
• 3- Base alteration / damage
• a Oxidation
• b Alkylations
• c Hydrolysis
• depurination
• deaminations

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DNA Damage, Repair, and Consequences
Damaging agent
Consequences

• In hibition of
• Replication
• Transcription
• Chromosome segregation

• Mutation
• Chromosome aberration

Repair Process
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Base alteration/damage
a Oxidation It is caused by 1- Normal
metabolism 2- ROS (reactive oxygen species)
such as O2-, H2O2, OH. 3- Ionizing
radiation 4- Chemicals It causes
Base-mispairing (i.e., oxoG can pair with C
or A)
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Base alteration/damage
b Alkylation It is caused by Transfer of
methyl or ethyl group to DNA bases It
causes Base-mispairing (ie., O6-methylG
mispairs with T)
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Base alteration/damage
Deamination
Types of base alterations

• C Hydrolytic damage
• Deamination
• It is caused by
• Conversion of amino groups of A, G, and C to
  keto groups.
• It causes
• changes in base pairing properties
• Depurination
• It is caused by
• Base loss (hydrolysis)
• It causes
• -breaking of base sugar bond
• -creates abasic site

Depurination
Deamination
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Induced Mutagenesis

• Physical (Radiation)
• UV
• Ionizing
• Chemical (Mutagens, Carcinogens)
• 1- Alkylating agents
• 2- Base analogues
• 3- intercalating agents
• 4- Different chemicals such as
• a- Nitrous acid
• b- Hydroxylamine

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PHYSICAL MUTAGENS / RADIATION
EM spectrum -consists of electric and
magnetic waves

• radiation was discovered in the 1890s
• -Roentgen discovered X-rays in 1895
• -Becquerel discovered radiation in 1896
• -Marie and Pere Curie discovered
• radioactive elements in 1898
• first discovered mutagenic agent known
• -effects on genes first reported in 1920s
• in Drosophila (Muller)

BIOLOGICALLY SIGNIFICANT
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PHYSICAL MUTAGENS / RADIATION

• Sources of radiation
• 1- Natural sources of radiation
• -cosmic, terrestrial, atmosphere
• 2- Anthropogenic
• -medical testing devices
• -nuclear testing and power plants
• -other products (TVs, smoke detectors,
• Scanners)
• Types of radiation
• Long wave length
• Visible
• UV
• Ionizing

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1. Ultraviolet (UV) radiation

• Definition
• Wavelength lt 320 nm
• Less energetic than IR (non-ionizing)
• It is preferentially absorbed
• by aromatic compound
• It causes
• covalent attachment of adjacent pyrimidines in
  one strand
• bulky lesions can block replication, and
  transcription
• can stimulate mutation

• Classification
• UV-C 180-290 nm, (germicidal)
• UV-B 290-320 nm, (major lethal/mutagenic
  fraction in sunlight)
• UV-A 320 nm-visible light (near UV produces
  few pyrimidine dimers, but can produce reactive
  oxygen radicals)

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2. Ionizing radiation (IR)
Definition Wavelength lt 180 nm
More energetic than UV It produces ROS
that 1- react with DNA and other
biological molecules. 2- Make breaks in one
or both strands mutations and gross chromosomal
rearrangements. 3- Increases
recombination rate death if unrepaired.
4- Crosslinking of DNA to itself or proteins.
5- ROS affects rapidly dividing cells
effects are dose- dependent. Classification
X rays Gamma rays
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CHEMICAL MUTAGENS

• 1- Base analogs resemble purines and pyrimidines
  
• bromouracil (BU) aminopurine

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CHEMICAL MUTAGENS
2- intercalating agents

• They are
• Flat, multiple ring molecules, that can
  interact with and insert between DNA bases.
• It Causes
• DNA to be stretched
• Insertinon of an extra base opposite
  intercalated molecule by DNA polymerase
  FRAMESHIFT MUTATION

acridine orange ethidium bromide proflavin
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CHEMICAL MUTAGENS

• 3- Nitrous acid
• cause deaminations
• C ? U, meC ? T
• A ? hypoxanthine
• 4-Nitrosoguanidine
• cause base alkylation
• methyl and ethyl
• methanesulfonate
• 5-Hydroxylamine
• Hydroxylates amino-gp of C
• C pairs with A

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Mutagenesis as a tool !
1- Sterilization Induction of mutation to
sterile germs. 2- Making small changes in
protein sequence. Site-specific in vitro
mutagenesis is a method by which mutant alleles
can be synthesized in the lab and transformed
into cell culture and animals.
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Can we detect Mutagen Ames Assay
Bruce Nathan Ames Brith1928 Ames test 1970
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Repair
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Dr. Parvin Pasalar Tehran University of Medical
Sciences
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(No Transcript)
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Some questions

• 1- How much of DNA synthesis in a prokaryote is
  because of Replication?
• 2- Why DNA is double stranded?
• 3- Why we are diploid?

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How to Repair the un-paired consequences
Damaging agent
Consequences

• In hibition of
• Replication
• Transcription
• Chromosome segregation

• Mutation
• Chromosome aberration

Repair Process
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DNA Repair Pathways
1. Direct reversals
2. Excision repair a. Base excision repair
(BER) b. Nucleotide excision repair (NER)
3. Mismatch repair - replication errors
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1- Direct reversal photoreactivation
Damage Recognized Thymine dimers 6-4 photoproduct
Gene Products Required Photolyase
Related disease Photolyase not yet found in
placental mammals
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2- Excision Repair Pathways

• a. Base Excision Repair
• damaged bases are removed as free bases
• primarily responsible for removal of oxidative
• and alkylation damages
• most genes in pathway are essential
• thought to have an important role in aging

• b. Nucleotide Excision Repair
• damaged bases are removed as oligonucleotides
• primarily responsible for removal of UV-induced
• damage and bulky adducts
• also removes 20 of oxidative damage
• deficient in human disorders

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2- Excision Repair Pathways
BER
NER
DNA Ligase
DNAP Ligase
DNAP Ligase
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Genetics of NER in Humans
1- Xeroderma Pigmentosum Occurrence 1-4/106
population Sensitivity sunlight Disorder
multiple skin disorders malignancies of the
skin neurological and ocular abnormalities
Biochemical defect early step of NER Genetic
seven genes (A-G), autosomal recessive
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Genetics of NER in Humans
2- Cockaynes Syndrome Occurrence 1 per/ 106
population Sensitivity sunlight Disorder
arrested development, mental retardation, dwarfi
sm, deafness, optic atrophy, intracranial
calcifications Biochemical defect NER Genetic
five genes (A, B and XPB, D G) autosomal
recessive
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3- Mismatch Repair in E. coli Decision between
right wrong (methyl-directed)
Before replication both strands of GATC are
methylated
Shortly afte replication it is hemimethylated
After a while it becomes fully methylated again

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3- Mismatch Repair (MMR) in E. coli
Damage Recognized Base-base mismatch (except
C-C) Small insertion/deletion loops (IDLs)
Gene Products Required (11) MutS (damage
recognition) MutL MutH (endonuclease) MutU (DNA
helicase) Exonucleases (ExoI, ExoVII, ExoX,
RecJ) DNA polymerase III Single strand binding
protein (SSB) DNA Ligase
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MMR Mutations inHereditary Nonpolyposis Colon
Cancer (HNPCC)

• MMR mutations in 70 of families
• Population prevalence 1 2851 (15-74 years)
• 18 of colorectal cancers under 45 years
• 28 of colorectal cancers under 30 years

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4- Recombinational repair

• Definition Using of another DNA molecule(
  homologous) as template
• Function The system is important in normal C.O
• When it is used Double Strand Breaks
  interstrand cross-links
• The consequence Gene Conversion.
• Defect in Human Blooms Syndrome

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Summary