Transcript Slide 1

‫دانشگاه علوم پزشكي وخدمات بهداشتي‬
‫درماني تهران‬
Dr. Parvin Pasalar
Tehran University of Medical Sciences
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
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
~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
class for Biochemistry today!!!
Classifications of Mutations
of mutations in ORFs:
1- Types
Can
be spontaneous or induced
1- Can be spontaneous or induced
Missense mutation
2- May be substitutions or frameshift
2- Base
substitutions
or frameshift
pairbe
substitution
in substitution
a different amino acid.
May
Small:
Gene
mutationresults
are those
that change
aofgene
3- May occurof
in structural
or regulatory
sequences
• Origins
Spontaneous
Mutation
Spontaneous:
Substitution:
mutation
4-Big:
May
be
small
(point)
or
big
Errors
in
DNA
replication
The
base
number
remains
the
same
the types sequences
changes:
May
Chromosomal
rearrangements
Qualitative
changes:
Mutation
structural
3-•Nonsense
occur
in
structural
orbut
regulatory
At
physiologic
rate
-transitions:
to
pu
or
py
to
pysequence
be
inversions,
deletions,
DNA
polymerase
accuracy
Base
pair
substitution
results
in a
stop
codon
(and shorter
polypeptide).
5-- can
May
have
no or pu
severe
effect
may
causes
changes
in
the
of
aa
of the
sequences
-tranversion:
pu
to py or py to pu
translocations,
or amplifications
•Errors
in DNA recombination
resulting
product (polypeptide)
6- Somatic
or germinal
Neutral
mutation
-can alter
chromosome organization
Induced:
DNA
strands
alignment
Frameshift:
and
affect
gene
function
4-•Base
May
be
small
(point)
or
big
pair substitution
results
in
substitution
of an amino
Because
of
the
treatment
with acid with
Base
alterations
and
base
damage
Quantative
changes:
Mutation
in
regulatory
The
base
number
changes
-can
activate
gene
expression
similar chemical properties (protein function is not altered).
different
agents
tautomerization;
deamination;
depurination;
-increate
coding
regions,
insertion
or deletion
of a nt thatof
is the
-can
novel
fusion
genes
sequences
does
not
change
the
structure
5- Silent
May
have
no
or
severe
effect
mutation
not
a
multiple
of
3
changes
gene
coding sequence
-can
affect
chromosome
segregation
oxidation;
alkylation
product but its amount
Introduces
premature
codons=protein
(non dysjunction)
duringstop
meiosis-semisterility truncation
•Base
Spontaneous
frameshift
pair substitution
results in mutations
the same amino acid (or nucleotide).
6- Somatic
germinal
-some types ofor
rearrangements
in
mispairing
during replication and recombination
Frameshift
mutations:
meiosis
may be of evolutionary benefit
Deletions or insertions (not divisible by 3) result in translation of
incorrect amino acids, stops codons (shorter polypeptides),or readthrough of stop codons (longer polypeptides).
Different Causes of Mutations
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
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
Different Type of DNA damages

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1- Double-strand breaks (DSBs)
2- Single- strand breaks (SSBs)
3- Base alteration / damage
a: Oxidation
b: Alkylations
c: Hydrolysis
depurination
deaminations
DNA Damage, Repair, and Consequences
Damaging agent
Consequences
In hibition of:
•Replication
•Transcription
•Chromosome
segregation
•Mutation
•Chromosom
e aberration
Repair Process
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)
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)
of base alterations
BaseTypes
alteration/damage
Deamination
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
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
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
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:
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)
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
dose- dependent.
Classification:
X rays
Gamma rays
CHEMICAL MUTAGENS

1- Base analogs: resemble purines and pyrimidines

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
• Insertinon of an extra base opposite
intercalated molecule by DNA
polymerase = FRAMESHIFT MUTATION
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
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.
Can we detect Mutagen: Ames Assay
Bruce Nathan Ames
Brith:1928
Ames test: 1970
‫دانشگاه علوم پزشكي وخدمات بهداشتي‬
‫درماني تهران‬
Dr. Parvin Pasalar
Tehran University of Medical Sciences
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?
How to Repair & the un-paired consequences
Damaging agent
Consequences
In hibition of:
•Replication
•Transcription
•Chromosome
segregation
•Mutation
•Chromosom
e aberration
Repair Process
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
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
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
2- Excision Repair Pathways
BER
NER
DNA Ligase
DNAP+ Ligase
DNAP+ Ligase
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
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
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
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
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

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
Syndrome
in Human: Bloom’s
Summary