Download 2-Mutation

‫دانشگاه علوم پزشكي وخدمات بهداشتي‬
‫درماني تهران‬
Dr. Parvin Pasalar
Tehran University of Medical Sciences
1
Objectives:

To know and explain about:
Mutation and its causes
 Different basis of Mutation classification
 Mutagens
 Positive aspects of mutations
 Ames test
 Repair systems
Diseases related to repair system defect

2
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
Depurinations
Deaminations
Oxidations
Alkylations
DSBs
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~50,000/day
~10,000/day
~600/day
~2000/day
~5000/day
~50-100/day
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
this class today!!!
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Classifications
of substitution Mutations:
Classifications
of Mutations
Missense
mutation
1- Can be
spontaneous or induced
May be
substitutions results
or frameshift
Base2-pair
substitution
in substitution of a different
amino
acid.
3- MaySubstitution:
occur
in structural
or regulatory
sequences
Origins
of
Spontaneous
Mutation
•
Spontaneous:
base
number
remains the same but the types changes:
4- MayThe
be
(point)
or big
• Errors
insmall
DNA
replication
At
physiologic
rate
Nonsense
mutation pu to pu or py
-transitions:
to py

Small:
Gene
mutation
areeffect
those that change a gene
DNA
polymerase
accuracy
5May
have
no
or severe
-tranversion: pu to py or py to pu
•Errors
in DNA
recombination
Base
substitution
resultsMutation
in a stop structural
codon (and
shorter
6-pair
Somatic
or germinal
Qualitative
changes:
sequences
Induced:
 Big: Chromosomal
rearrangements
polypeptide).
DNA
strands
alignment
Frameshift:
causes deletions,
changes in the sequence of aa of the
- canmay
be inversions,
Because
of
thedamage
treatment with
The
base
number
changes
• Base
alterations
and
base
resulting
product
(polypeptide)
translocations,
or
amplifications
Neutral mutation
-in coding
regions,
insertion or deletion
of a nt that is
different
agents
tautomerization;
deamination;
depurination;
-can
alter
chromosome
organization
not affect
a multiple
of 3 changes gene coding sequence
and
gene
function
oxidation;
alkylation
Quantative
changes:
Mutation
in regulatory
Base pair
substitution
results
in codons=protein
substitution
of
an amino acid
Introduces
premature
stop
truncation
-can activate
expression
• with
Spontaneous
frameshift
mutations
similargene
chemical
properties
(protein
function
is not
sequences
does
not
change
the
structure
of
the
-can
create novel fusion
genes
altered).
mispairing
during
replication and recombination
but its amount
-canproduct
affect chromosome
segregation
(non dysjunction) during meiosis-semi- sterility
Silent mutation
of rearrangements
in in the same amino acid (or
Base-some
pair types
substitution
results
meiosis may be of evolutionary benefit
nucleotide).
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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
•Chromosom
e 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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Types of base alterations and supressor tRNA
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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
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Deamination
Depurination
Deamination
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
of electric
• -consists
radiation
was discovered in the 1890s
and magnetic
waves
-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 (TV’s, smoke detectors,
Scanners)
Types of radiation:
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Long wave length
Visible
UV
Ionizing
1. Ultraviolet (UV) radiation
• Definition:
•Wavelength < 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 < 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 dosedependent.
Classification:
X rays
Gamma rays
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CHEMICAL MUTAGENS

1- Base analogs: resemble purines and pyrimidines

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bromouracil (BU) & aminopurine
CHEMICAL MUTAGENS
2- intercalating agents
They are:
• Flat, multiple ring molecules, that can
interact with and insert between DNA
bases.
acridine orange
ethidium bromide
proflavin
• It Causes:
• DNA to be stretched
• Insertion of an extra base opposite
intercalated molecule by DNA
polymerase = FRAMESHIFT MUTATION
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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
Brith:1928
Ames test: 1970
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‫دانشگاه علوم پزشكي وخدمات بهداشتي‬
‫درماني تهران‬
Dr. Parvin Pasalar
24
Tehran University of Medical Sciences
25
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 & what is the un-repaired consequences
Damaging agent
Consequences
In hibition of:
•Replication
•Transcription
•Chromosome
segregation
•Mutation
•Chromosom
e 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
4. Recombinational repair
- multiple pathways
- double strand breaks and interstrand
cross-links
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1- Direct reversal: photoreactivation
T T
Damage Recognized:
Thymine dimers
6-4 photoproduct
Gene Products Required:
Photolyase
Related disease:
Photolyase not yet found in
placental mammals
Visible light
T T
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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 and 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
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DNAP+ Ligase
3- Mismatch Repair( MMR) 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- 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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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.
Is defected in: Bloom’s
Syndrome
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Summary
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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- Cockayne’s Syndrome
Occurrence: 1 per/ 106 population
Sensitivity: sunlight
Disorder:
arrested development,
mental retardation,
dwarfism, deafness, optic atrophy,
intracranial calcifications
Biochemical defect : NER
Genetic: five genes (A, B and XPB, D & G)
autosomal recessive
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MMR Mutations in
Hereditary 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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