Transcript Causes

DNA Repair and
Dr. Asad Vaisi-Rayggani
Department of Clinical Biochemistry
Kermanshah University of Medical
Sciences
Mutation
• Definition:
• Mutations are inheritable changes in the DNA
sequence. They can result from replication errors,
from damage to the DNA, or from errors introduced
during repair of damage. Mutations that are changes
of a single base pair are called point mutations.
• Causes:
It may be spontaneous or induced because of
different agents
• Classifications:
are classified on different basis
Enzymes Repair Damaged DNA
• A human has 1014 nucleated cells each with 3x
109 base pairs of DNA. If about 1016 cell
divisions occur in a lifetime and
• 10−10 mutations per base pair per cell
generation escape repair,
• there may eventually be as many as one
mutation per 106 bp in the genome.
• Fortunately,most of these will probably occur in
DNA that does not encode proteins or will not
affect the function of encoded proteins and so
are of no consequence.
• In addition,spontaneous and chemically induced
damage to DNA must be repaired.
• Damage to DNA by
environmental, physical, and
chemical agents may be
classified into four types
• (Table 36–8).
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
-
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
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 pairs with A
Mutagenesis as a tool !
.
1- Sterilization:
Induction of mutation
to sterile germs.
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)
• Transitions are point mutations in which
one purine is substituted for another (i.e.,
A for G or G for A) or
• One pyrimidine is substituted for another(
i.e.,T for C or C for T). Deamination of C to
form U,
Point mutations can also be characterized by
their effect on a coding sequence
• Missense mutations are point mutations
that change a single base pair in a codon
such that the codon now encodes a
different amino acid
• Nonsense mutations are point mutations
that change a single base pair in a codon
to a stop codon that terminates translation
• (Figure4 .24b)
• Nonsense mutations usually have more
severe effects than missense mutations,
because they lead to synthesis of truncated
(and generally unstable) polypeptides
• Silent or synonymous mutations do not alter
the amino acid encoded; these include
• many changes in the third nucleotide of a
codon. Some silent mutations may, however,
• Have serious consequenceis they alter the
splicing pattern of the gene.
• Insertions or deletions of one or more
base pairs( if the number of base pairs is
not a multiple of 3) lead to frame shift that
disrupt the coding of a protein
• acridines and proflavin, intercalate into the
• DNA; that is, they insert between adjacent
base pairs. This usually leads to insertions
or deletions of a single base pair, and thus
a frame shift
Can we detect Mutagen: Ames Assay
Bruce Nathan Ames
Brith:1928
Ames test: 1970
For carcinogens, based on their mutagencity
Salmonula having a mutant that in active an
enzyme of the His biosynthetic
90% found in eukaryotic
DNA Damage, Repair, and 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 inter strand
cross-links
5.
Tolerance mechanisms
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
Mismatch Repair
• Faulty(Damaged) mismatch repair
has been linked to hereditary
nonpolyposis colon cancer
(HNPCC), one of the most common
inherited cancers.
• Genetic studies linked HNPCC in
some families to a region of
chromosome 2.
• The gene located, designated
hMSH2, was subsequently shown to
encode the human analog of the E
• Mutations of hMSH2 account for
50–60% of HNPCC cases.
Base Excision-Repair
• The depurination of DNA, which happens
spontaneously owing to the thermal lability
of the purine Nglycosidic bond, occurs at a
rate of 5000–10,000/cell/d at 37 °C.
• Specific enzymes recognize a depurinated
site and replace the appropriate purine
directly, without interruption of the
phosphodiester backbone.
Uracil
apurinic or apyrimidinic
endonuclease
to excise the abasic sugar
Nucleotide Excision-Repair
• This mechanism is used to replace
regions of damaged DNA up to 30
bases in length.
• Common examples of DNA damage
include ultraviolet (UV) light, which
induces the formation of cyclobutane
pyrimidine-pyrimidine dimers, and
• smoking, which causes formation of
• benzo[a]pyrene-guanine adducts.
• Ionizing radiation,cancer
chemotherapeutic agents, and a
variety of chemicals found in the
environment cause base
modification,
• strand breaks, cross-linkage between
bases on opposite strands or
between DNA and protein, and
numerousother defects.
This gap is then filled
in by a polymerase
(δ/ε in humans) and
religated.
Genetics of NER in Humans
1- Xeroderma Pigmentosum
Occurrence: 1-4/106 population
Sensitivity: sunlight (ultraviolet)
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 skin cancer is
1000- to 2000-fold increased
Biochemical defect : NER
Genetic: five genes (A, B and XPB, D & G)
autosomal recessive
factor TFIIH Transcription
Summary
The tumor suppressor p53
• , a protein of MW 53 kDa, plays a key role
in both G1 and G2 checkpoint
control.
• Normally a very unstable protein, p53 is
a DNA binding transcription factor,
one of a family of related proteins, that is
somehow stabilized in response to DNA
damage,
• perhaps by direct p53-DNA interactions
• Increased levels of p53 activate
transcription of an ensemble of genes that
collectively serve to delay transit through
the cycle.
• One of these induced proteins, p21CIP,
is a potent CDK-cyclin inhibitor (CKI)
that is capable of efficiently
• inhibiting the action of all CDKs.
• Clearly, inhibition of CDKs will halt
progression through the cell cycle
• If DNA damage is too extensive to repair,
the affected cells undergo apoptosis
(programmed cell death) in a
p53-dependent fashion.