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1 0 TH W E E K

DNA damage, repair & Mutagenesis

Gihan E-H Gawish, MSc, PhD


Ass. Professor
Molecular Genetics and Clinical
Biochemistry
KSU
DNA damage, repair & mutagenesis

Mutagenesis
Mutation: replication fidelity, mutagens,
mutagenesis

DNA damage
DNA lesions: oxidative damage, alkylation, bulky
adducts

DNA repair
Photoreaction, alkyltransferase, excision repair,
mismatch repair, hereditary repair defects
DNA damage, repair & Mutagenesis

1 Mutagenesis

Mutation
Replication fidelity
Mutagens: chemical & physical
Mutagenesis: direct & indirect
Mutation

Replication Mutagenesis
Fidelity
Mutagens
1 Mutaagenesis

1-1 Mutation
Permanent, heritable alterations
in the base sequence of DNA

Reasons
1. Spontaneous errors in DNA replication or meiotic
recombination
2. A consequence of the damaging effects of physical
or chemical mutagens on DNA
1 Mutaagenesis

Point mutation
(a single base change)
Transition : Purine or pyrimidine is replaced
by the other AG T C

Transversion: a purine is replaced by a


pyrimidine or vice verse
A T or C T A or G
G T or C C A or G
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Effects of a point mutation

Phenotypic
effects
Noncoding DNA
Nonregulatory DNA Silent mutation No
3rd position of a codon

Coding DNA altered Missense mutation Yes or No

Coding DNA stop codon


truncated protein Nonsense mutation Yes
1 Mutaagenesis

Insertions or deletions
The addition or loss of one or more bases in a DNA region

Frameshift mutations
The translation of a protein encoded gene is
frameshifted , then changed the C-terminal side of the
mutation is completely changed.
Examples of deletion mutations
1 Mutaagenesis

1-2 Replication fidelity


Important for preserve the genetic information
from one generation to the next

Mutation relevant
1. Spontaneous errors in DNA replication is very rare, one
error per 1010 base in E. coli.
1 Mutaagenesis

Molecular mechanisms for the


replication fidelity

1. DNA polymerase: Watson-Crick base pairing


2. 3 5 proofreading exonuclease.
3. RNA priming: proofreading the 5 end of the
lagging strand
4. Mismatch repair
1 Mutaagenesis

Proofreading
by
E. coli polymerase
1 Mutaagenesis

Mutagens
Mutation relevant
Cause DNA damage that can be converted to mutations.
1 Mutaagenesis

Physical mutagens
High-energy ionizing radiation: X-rays and g-rays
strand breaks and base/sugar destruction
Nonionizing radiation : UV light pyrimidine dimers

Chemical mutagens
Base analogs: direct mutagenesis
Nitrous acid: deaminates C to produce U
Alkylating agents
Lesions-indirect mutagenesis
Intercalating agents
Base analogs: derivatives of the normal bases
incorporated in DNA, altering base pairing properties.

Nitrous acid: deaminates C to produce U, resulting in GC


AU
1 Mutaagenesis

Mutagenesis
The molecular process
in which the mutation is generated.

Note: the great majority of lesions introduced by


chemical and physical mutagens are repaired by
one or more of the error-free DNA repair
mechanisms before the lesions is encounter by a
replication fork
1 Mutaagenesis

Direct mutagenesis

The stable, unrepaired base with


altered base pairing properties in the
DNA is fixed to a mutation during DNA
replication.
1 Mutaagenesis
OH AGCTTCCTA
Br TCGAAGGAT
H
:G 1. Base analog
O incorporation
AGCTBCCTA
enol form TCGAAGGAT
2. 1st round
of replication
AGCTTCCTA AGCTBCCTA
Br TCGAAGGAT TCGAGGGAT
H
:A 3. 2nd round
O of replication
AGCTBCCTA AGCTCCCTA
Keto form
TCGAAGGAT TCGAGGGAT
5-BrU
ATGC transition
1 Mutaagenesis

Indirect mutagenesis
The mutation is introduced as a result
of an error-prone repair.
Translesion DNA synthesis to
maintain the DNA integrity but not
the sequence accuracy: when damage
occurs immediately ahead of an advancing
fork, which is unsuitable for recombination
repair
the daughter strand is synthesized
regardless of the the base identity of the
damaged sites of the parental DNA.
1 Mutaagenesis
E. coli translession replication: SOS
response: Higher levels of DNA damage
effectively inhibit DNA replication and trigger a
stress response in the cell, involving a regulated
increase (induction) in the levels of a number of
proteins. This is called the SOS response.

1. Some of the induced proteins, such as the UvrA and


UvrB proteins, have roles in normal DNA repair
pathways.
2. A number of the induced proteins, however, are part
of a specialized replication system that can
REPLICATE PAST the DNA lesions that block
DNA polymerase III. back
Proper base pairing is often impossible and not
strictly required at the site of a lesion because
of the SOS response proteins, this translesion
replication is error-prone.

The resulting increase in mutagenesis does


not contradict the general principle that replication
accuracy is important (the resulting mutations
actually kill many cells). This is the biological price
that is paid, however, to overcome the general
barrier to replication and permit at least a few
mutant cells to survive.
DNA damage, repair and mutagenesis

DNA damage and repair


Mutagen chemical reactivity of the
bases

minor or DNA damage Extensive, right


moderate (lesions) before Replication
Fork (not repairable)
Error-free Direct
mutagenesis Indirect
Repairing mutagenesis

Completely repaired mutations