Transcript Cell and Molecular Biology

Cell and Molecular Biology
DNA mutation and
repair
Behrouz Mahmoudi
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DNA Damage
Mechanisms for maintaining genetic stability associated
with DNA replication in E. Coli
Machanism
Cumulative error frequency
Base pairing
~10-1 - 10-2
DNA polymerase actions (including base
~10-5 - 10-6
selection, 3'->5' proofreading)
Accessory proteins (e.g. SSBP)
~10-7
Post-replication mismatch correction
~10-10
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Spontaneous alterations:
(a) Mismatches: Occurs
during DNA synthesis (i.e.
replication, repair, or
recombination)
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(b) Tautomeric shifts
Nucleotides spontaneously under go a transient
rearrangement of bonding, e.g. a shift from
NH2 (amino form) to NH (imino form) or C=O
(keto) to C-OH (enol). Therefore, if any base in
a template strand exists in its rare tautomeric
form during DNA replication, misincorporation
in the daughter strand can result.
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Base Pairing of Imino A-C
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(c) Deamination
Three of the four bases normally present in DNA
(cytosine, adenine, and guanine) contain amino
group (NH2). The loss of the amino group
(deamination) can occur spontaneously and
result in the conversion of the affected bases to
uracil, hypoxanthine, and xanthine,
respectively.
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(d) Loss of bases
Depurination and depyrimidination:
The loss of purines or pyrimidines
from DNA usually occurs at
acidic pH; however, it can also
happen in physiological pH
(~10,000 purine per day in
mammalian cell; ~500
pyrimidine/day). This will results
in breaking the 3' phosphodiester
bond called b-elimination.
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Induced Mutations
(a) Physical agents that damage DNA:
--- Ionizing radiation: OH, O2-, H2O2,
damage base and sugar residues.
--- UV radiation: Cyclobutane pyrimidine
dimers, Thymidine dimers (T-T) dimer
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Chemical Agents
(b) Chemical agents that damage DNA:
--- Alkylating agents: Alkylating agents are
electrophilic compounds with affinity for
nucleophilic centers in organic macromolecules.
These include a wide variety of chemicals, many of
which are proven or suspected carcinogens (such as
nitrous acid, hydroxylamine, and ethylmethane
sulfonate, EMS), Adding alkyl group to hydrogenbonding oxygen of G or T, resulting in G-T mispairing
G-C ---> G*T --->A-T
T-A
--->T*-G
--->
CG
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Base-analogue Agents
A base analogue is a substance other
than a standard nucleic acid base
that can be incorporated into a
DNA molecule by the normal
process of polymerization. Such a
substance must be able to pair with
the base on the complementary
strand being copies, or the 3'->5'
editing function will remove it.
For example, 5-bromouracil is an
analogue of thymine and might
cause an A-T to G-C transition
mutation.
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Base Analogue
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Intercalating Agents:
Intercalating agents: Substances whose
dimensions are roughly the same as those of a
purine-pyrimidine pair. In aqueous solutions,
these substances form stacked arrays, and are
also able to stack with a base-pair by insertion
between two base-pairs. This may result in
frameshift mutation.
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Model of intercalating agent
induced mutagenesis
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Metabolite Mutagens
Chemicals that are metabolized to
electrophilic reagents: Aflatoxins,
benzo[a]pyrene
A mutagen is a physical or chemical agent
that causes mutations to occurs.
Mutagenesis is the process of producing a
mutation.
Mutant refers to an organism or a gene that
is different from the normal or wild type.
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Reversion and the
Ames test:
Mutants may have second
mutation and become wild
type again.
Reversion was used as a
means of detecting
mutagens and carcinogensthe Ames test
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DNA Repair Mechanisms
(1) Repair by direct reversal: The simplest
mechanism. e.g. UV induced T-T dimer is
recognized by photolyase and is cleaved into
intact thymine (light dependent). This is
called photoactivation
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Excision Repair
(2) Excision Repair:
The most
ubiquitous repair
mechanism, which
can deal with a
large variety of
structural defects in
DNA.
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Recombinational Repair
(3) Recombinational repair (Postreplicational
repair): Occurs before excision repair has
happened or when excision repair can not
fix the problem
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The SOS response
(4) The SOS response: The SOS response
system is only active in response to some
signal such as a blocked of replication fork. In
E. Coli, recA and lexA govern the expression
of a number of other genes involved in DNA
repair. This is an error-prone DNA repair
mechanism and result in higher than normal
mutagenesis.
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SOS DNA Repair
1. DNA damage
2. RecA converted to RecA*
3. RecA* facilitated LexA self-cleavage
4. Increased synthesis of SOS proteins
5. Error prone repair induced
6. DNA damage repaired
7. RecA* returned to RecA
8. LexA no longer self-cleaved
9. LexA repressed SOS genes
10. LexA repress lexA gene expression
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Type of Mutations(I)
I. Point mutation:
A. Base substitution
Change in DNA
Transition: One purine replaced by a different
purine;or one pyrimidine replaced by a
diferent pyrimidine
A
G
T
C
Transversion: A purine replaced by a pyrimidine
or vice versa
A
T
C
G
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Type of Mutations (II)
Change in protein
1. Silent mutation: altered codon codes for the
same a.a.
GAG (Glu) --->GAA (Glu)
2. Neutral mutation: altered codon codes for
functional similar a.a. GAG--->GAC (Asp)
3. Missense mutation: altered codon codes for
different dissimilar a.a. GAG ---> AAG (Lys)
4. Nonsense mutation: altered codon becsomes
a stop codon
GAG ---> UAG (stop)
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Type of Mutations (III)
B. Frameshift mutation: addition or deletion of one
base-pair result in a shift of reading frame and
alter amino acid sequence
1. Wild type:
ATG ACC AGG TC
Met
2. Base addition:
Thr
Gln
Val
ATG ACA GGT C
Met
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Arg
ATG ACA CAG GTC
Met
3. Base deletion:
Thr
Thr
Gly
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Type of Mutations (IV)
II. Insertion
III. Deletion
IV. Translocation
V. Inversion
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