Mutation and Repair
Objective
At the end of this
lecture, student will be able to
• Explain mutation and repair mechanisms
DNA
Mutation and Repair
DNA Damage
Mechanisms for maintaining genetic stability associated with
DNA replication in E. Coli
Mechanism
Cumulative error frequency
Base pairing ~10-1 – 10-2
DNA polymerase actions (including base selection, 3′->5′
proofreading) ~10-5 –
10-6
Accessory proteins (e.g. SSBP) ~10-7
Post-replication mismatch correction ~10-10
Spontaneous
alterations:
(a) Mismatches:
Occurs during DNA synthesis (i.e. replication, repair, or recombination)
(b) Tautomeric shifts
• Nucleotides spontaneously undergo 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.
Base Pairing of Imino
A-C
(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.
(d) Loss of bases
• Depurination & depyrimidination: The loss of purines
or pyrimidines from DNA usually occurs at acidic pH;
• This will results in breaking the 3’ phosphodiester bond
called b- elimination.
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
(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 hydrogen-bonding oxygen of
G or T, resulting in G-T mispairing
G-C —> G*T —>A-T
T-A —>T*-G —> CG
(c) 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.
Base Analogue
(d) Intercalating
agents:
• Substances whose dimensions are roughly the same as those
of a purine-pyrimidine pair
• In aqueous solutions, these substances form stacked arrays
• Able to stack with a base-pair by insertion between two
base-pairs. This may result in frameshift mutation.
Model of
intercalating agent induced mutagenesis
Type of Mutations
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 à G T à C
Transversion: A
purine replaced by a pyrimidine or vice versa
A à T C à G
Change in protein
1. Silent mutation:
altered codon codes for the same a.a.
2. Neutral mutation:
altered codon codes for functional similar a.a.
GAG (Glu) —>GAA (Glu)
3. Missense mutation:
altered codon codes for different dissimilar a.a.
GAG—>GAC (Asp)
4. Nonsense mutation:
altered codon becsomes a stop codon
GAG —> AAG (Lys)
GAG —> UAG (stop)
B. Frameshift
mutation: addition or deletion of one base-pair result in a shift of
reading frame and alter amino acid sequence
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.
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
carcinogens- the Ames test
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
(2) Excision Repair:
The most ubiquitous repair mechanism, which can deal with a large variety of
structural defects in DNA.
(3) Recombinational
repair (Postreplicational repair): Occurs before excision repair has
happened or when excision repair cannot fix the problem
(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.
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
Summary
• Mutation can be of endogenous or exogenous origin
• Mismatches: Occurs during DNA synthesis
• Intercalating agents are those substances whose dimensions
are roughly the same as those of a purine-pyrimidine pair
• Addition or deletion of one base-pair result in a shift of
reading frame and alter amino acid sequence known as frameshift mutation