Mutation and Repair

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

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