MUTATIONS AND DNA REPAIR MECHANISMS

Mrs. OFELIA SOLANO SALUDAR

Department of Natural Sciences
University of St. La Salle
Bacolod City
Damage to DNA occurs spontaneously.
Under normal conditions, spontaneous hydrolysis
of DNA leads to depurination (breaking the
glycosidic bond between the deoxyribose and the
purine) or deamination (the loss of the amino
group from A, C or G).
This damage can result in the inclusion of an
incorrect nucleotide to produce a mutation.
Damage to DNA occurs in response to mutagens
(either chemical or radiation).
Mutagenic chemicals include
    1) base analogues (similar in structure to the
normal bases and can become incorporated into
DNA);
    2) base-modifying agents (which can change a
base) and
    3) intercalating agents (cause insertions and
deletions).
Ultraviolet (UV) radiation (sunlight) can cause
pyrimidine dimer formation (such as covalently
linked thymines) block replication and
transcription.
Ionizing radiation (such as X-rays) knock electrons
Excision repair mechanisms remove abnormal
nucleotides to correct mutations.
Base excision repair mechanisms first remove a
damaged base then causes excision of the
remaining sugar-phosphate unit
Pyrimidine dimers and other bulky lesions are
removed through nucleotide excision repair
(NER).
NER causes two nicks which leads to the
removal of a stretch of damaged single-
stranded DNA (12 in E. coli and 29 in humans).

Mismatch repair corrects mutations of non-

complementary bases that become included in
DNA during replication that are not fixed by
proof-reading.
The original strand is recognized as such by the
action of DNA methylases (the old DNA strand
is methylated).
Mismatch repair endonucleases cut the sugar
phosphate backbone (a nick) and an
exonuclease removes the incorrect nucleotides
from the nicked strand.
 KEY POINTS
Mutations can occur in a number of ways:
1. Errors can occur during DNA replication, DNA
repair, or DNA recombination which can lead to
base-pair substitutions, insertions, or deletions, as
well as mutations affecting longer stretches of DNA.
2. Mutagens are chemical or physical agents that
interact with DNA to cause mutations.
3. Physical agents include high-energy radiation like X-
rays and ultraviolet light.
4. Some errors can be corrected by direct repair, while
others are repaired by more complex mechanisms.
 MUTATIONS are changes in the genetic material of a cell
(or virus).
 Some are large-scale mutations in which long segments of
DNA are affected (example: translocations, duplications,
and inversions).
 A chemical change in just one base pair of a gene causes a
spontaneous or point mutation.
 A base-pair substitution is a point mutation that results in
replacement of a pair of complimentary nucleotides with
another nucleotide pair.
 Some base-pair substitutions have little or no impact on
protein function.
 If these occur in gametes or gamete-producing cells, they
may be transmitted to future generations and cause novel
traits or defects.
 Silent /synonymous mutations changes a codon but does not alter
the amino acid encoded. Alterations of nucleotides still indicate the
same amino acids because of redundancy in the genetic code. Such
mutations may still have effects on mRNA stability.
 Nonsynonymous mutations result in an altered sequence in a
polypeptide or functional RNA: one or more components of the
sequence are altered or eliminated, or an additional sequence is
inserted into the product.

 Transversions (blue): replacement of a

purine by a pyrimidine or that of a
pyrimidine by a purine.
 Transitions – (black ): replacement of one
purine by the other or that of one
pyrimidine by the other.
 Missense mutations
are those that still code
for an amino acid but
change the indicated
amino acid.
 Nonsense mutations
change an amino acid
codon into a stop
codon, nearly always
leading to a
nonfunctional protein.
 Insertions and deletions are
additions or losses of nucleotide
pairs in a gene.
 These have a disastrous effect on
the resulting protein more often
than substitutions do.
 Unless these mutations occur in
multiples of three, they cause a
frameshift mutation.
 All the nucleotides downstream
of the deletion or insertion will
be improperly grouped into
codons.
 The result will be extensive
missense, ending sooner or later
in nonsense - premature
termination.
Mutation class Type of mutation Incidence
Base Comparatively common type of mutation in coding DNA but
All types
substitutions also common in noncoding DNA
Transitions and Transitions are more common than transversions, especially in
transversions mitochondrial DNA
Synonymous and Synonymous substitutions are more common than
nonsynonymous nonsynonymous substitutions in coding DNA; conservative
substitutions substitutions are more common than non-conservative
Gene conversion-like events Rare except at certain tandemly repeated loci or clustered
(multiple base substitution) repeats
Very common in noncoding DNA but rare in coding DNA
Insertions One or a few nucleotides
where they produce frameshifts
Rare but can contribute to several disorders, especially
Triplet repeat expansions
neurological disorders
Rare; can occasionally get large-scale tandem duplications, and
Other large insertions
also insertions of transposable elements
Very common in noncoding DNA but rare in coding DNA
Deletions One or a few nucleotides
where they produce frameshifts
Rare, but often occur at regions containing tandem repeats or
Larger deletions
between interspersed repeats
Rare as constitutional mutations, but can often be pathogenic.
Chromosomal
Numerical and structural Much more common as somatic mutations and often found in
abnormalities
tumor cells
DNA suffers a wide range of mutations which can be repaired by
direct or complex mechanisms:
1. Purine bases are lost by spontaneous fission of the base-sugar link.
2. Cytosines, and occasionally adenines, spontaneously deaminate to
produce uracil and hypoxanthine respectively.
3. Many chemicals, for example alkylating agents, form adducts with
DNA bases.
4. Ultraviolet light causes adjacent thymines to form a stable chemical
dimer.
5. Ionizing radiation causes single or double-strand breaks.
6. Reactive oxygen species in the cell attack purine and pyrimidine
rings.
7. Mistakes in DNA replication result in incorporation of a mismatched
base.
8. Mistakes in replication or recombination leave strand breaks in
DNA.
 Spontaneous DNA damage: (A) depurination (loss of purine bases) resulting
from cleavage of the bond between the purine bases and deoxyribose, leaving an
apurinic (AP) site in DNA and (B) deamination (converts cytosine to uracil;
adenine to hypoxanthine)
 Alkylation is the addition of methyl or ethyl groups to various
positions on the DNA bases. Example: alkylation of guanine by
ethylmethane sulfonate (EMS). At the left is a normal G-C base
pair. Note the free O6 oxygen (red) on the guanine. EMS donates
an ethyl group (blue) to the O6 oxygen, creating O6-ethylguanine
(right), which base-pairs with thymine instead of cytosine.
This lesion can be repaired
by an enzyme (O6-
methylguanine
methyltransferase) that
transfers the methyl group
from O6-methylguanine to
a cysteine residue in its
active site, and the original
guanine is restored. This
direct repair reaction is
widespread in both
prokaryotes and
eukaryotes, including
humans.
Ultraviolet radiation (UV) from the sun is carcinogenic and is a
principal cause of skin cancer.
 Of the 3 types of ultraviolet radiation (UV) from the sun: UVA
(wavelength 320–380 nm), UVB (wavelength 290–320 nm),
and UVC (wavelength 200–290 nm, UVB is the most effective
carcinogen because it causes UV photoproducts.
 Cyclobutane pyrimidine dimers are responsible for at least 80%
of UVB-induced mutations. The precise class of mutations
resulting from pyrimidine dimers is a unique molecular
signature of skin cancer.
 UVA indirectly damages DNA via free radical-mediated
damage. Water is fragmented by UVA, generating electron-
seeking ROS that cause DNA damage (transversions are
characteristic of UVA damage).
 Thymine dimers: most common type of DNA damage caused by
UV irradiation. (a) UV light cross-links the two thymine bases on
the top strand. This distorts the DNA so that these two bases no
longer pair with their adenine partners. (b) The two bonds joining
the two thymines form a 4-membered cyclobutane ring (red).
 Direct repair of thymine dimers. UV-
induced thymine dimers can be
repaired by photoreactivation. The
enzyme (photolyase) absorbs visible
light and binds to damaged DNA. The
enzyme breaks the dimer, and finally
dissociates from the repaired DNA.
Repair of pyrimidine dimers by
photoreactivation is common to
prokaryotic and eukaryotic cells,
including E. coli, yeasts, and some
species of plants and animals.
Photoreactivation is not universal;
many species (including humans) lack
this mechanism of DNA repair.

17
Ionizing radiation- high-energy radiation capable of producing
ionization in substances through which it passes, e.g. x-rays, alpha and
beta rays, and neutrons from a nuclear reaction.
 It can directly ionize atoms comprising DNA, or indirectly by the
interaction with water molecules (radiolysis) that generate
dangerous reactive oxygen species (ROS): the hydroxyl radical (–
OH), hydrogen peroxide (H2O2), and the superoxide radical (O–2).
 A free radical reacts very strongly with other molecules as it seeks to
restore a stable configuration of electrons. A free radical may drift
about up to 1010 longer than the time needed for the initial ionization,
increasing the chance of it disrupting DNA and cause mutations.
 Oxidation of DNA is one of the main causes of mutation, and
explains why free radicals are such potent carcinogens.
 Oxidation can produce oxidized bases, e.g., adenine mispairs
with 8-oxoguanine during replication leading to a G→T
transversion mutation.
 The -OH radical removes electrons from any molecule in its
path, turning that molecule into a free radical and so
propagating a chain reaction.
 H2O2 is more dangerous to DNA than the -OH radical. Its
slower reactivity gives it time to travel into the nucleus of a
cell, where it is free to wreak havoc upon DNA.
 The superoxide radical is not very reactive but acts more as a
catalyst for the generation of the other ROS intermediates.
 CHEMICAL MUTAGENS/ CARCINOGENS :
 The common mechanism of action is that an electrophilic (electron-
deficient) form reacts with nucleophilic sites (sites that can donate
electrons) in the purine and pyrimidine rings of nucleic acids.
 Some chemicals are base analogues that may be substituted into DNA,
and pairs incorrectly during DNA replication.
 Other mutagens interfere with DNA replication by inserting into DNA
and distorting the double helix.
 Still others cause chemical changes in bases (DNA adducts) that change
their pairing properties.

Carcinogens can be segregated into 10 groups:

polycyclic aromatic hydrocarbons carbamates
halogenated compounds aromatic amines nitrosamines and
nitrosamides azo dyes
hyrazo and azoxy compounds natural products
inorganic carcinogens
miscellaneous compounds (alkylating agents,
aldehydes, phenolics)
Heterocyclic amines (HCAs) are carcinogens
produced by cooking meat, formed from
heating amino acids and proteins. About 20
HCAs have been identified. Three examples,
Phe-P-1, IQ, and Mel Q, are shown.

These are examples of carcinogens to

which we are exposed daily and which
are produced in our own kitchens!
Oven roasting, marinading, and coating
food with breadcrumbs before frying
are modifications that may reduce the
formation of HCAs.
 Nitrosamines and nitrosamides are
found in tobacco or are formed when
preservative nitrites react with amines in
fish and meats during smoking. Their
principal carcinogenic product is
alkylated O6 guanine derivatives.

(a) An example of nitrosamines: alkylnitrosoureas.

(b) A potential carcinogenic product of nitrosamines: O6 adduct of guanine.
Guanine is shown for comparison.
 Nitrous acid treatment of DNA results in the conversion of
adenine into hypoxanthine, which pairs with cytosine, inducing a
transition from A-T to G-C.
Acridine dyes induce frameshift mutations by intercalating into
the DNA, leading to the incorporation of an additional base on
the opposite strand.
Aflatoxin Reaction. The compound, produced by molds that grow
on peanuts, is activated by cytochrome
P450 to form a highly reactive species that modifies bases such as
guanine in DNA, leading to mutations.
 Base Pair with Mutagenic Tautomer. The
bases of DNA can exist in rare tautomeric
forms. The imino tautomer of adenine can
pair with cytosine, eventually leading to a
transition from A-T to G-C.
(Tautomerization is the interconversion of
two isomers that differ only in the position
of protons and often, double bonds).
Base Pair with 5-Bromouracil.
This base analog of thymine has a
higher tendency to form an enol
tautomer than does thymine itself.
The pairing of the enol tautomer of
5-bromouracil with guanine will
lead to a transition from T-A to C-
G.
 (a) Polycyclic aromatic amines
(b) Metabolic activation of BP (Benzopyrene)
Benzopyrene ( found in cigarette smoke) reacts with DNA bases, resulting
in the addition of large bulky chemical groups to the DNA molecule.
Locations of these adducts matched the distribution of p53 gene mutations
in lung tumors from smokers (Science,1996).
Each day the DNA of a human cell loses about 5,000 purines, and about
100 cytosines spontaneously deaminate to uracil. Damage to DNA can
block replication or transcription, and can result in a high frequency of
mutations—consequences that are unacceptable from the standpoint of
cell reproduction.
Major DNA repairing mechanisms: base excision,
nucleotide excision and mismatch repair. 
 Base excision repair of a GT mismatch.
A DNA glycosylase specific for G-T mismatches,
usually formed by deamination of 5-methyl C
residues, flips the thymine base out of the helix and
then cuts it away from the sugar-phosphate DNA
backbone (1), leaving just the deoxyribose (black
dot). An endonuclease specific for the resultant
baseless site then cuts the DNA backbone (2), and
the deoxyribose phosphate is removed by an
endonuclease associated with DNA polymerase
(3). The gap is then filled in by DNA Pol and
sealed by DNA ligase (4), restoring the original G-
C base pair.
 Base excision repair in
E.coli
DNA's bases may be modified
by deamination or
alkylation. The position of the
modified (damaged) base is
called the "abasic site" or "AP
site". DNA glycosylase can
recognize the AP site and
remove its base.  Then, the AP
endonuclease removes the AP
site and neighboring
nucleotides.  The gap is filled
by DNA polymerase I and
DNA ligase. 
 Nucleotide excision
repair (NER) in E. coli.
Proteins UvrA, UvrB, and
UvrC are involved in
removing the damaged
nucleotides (e.g., the dimer
induced by UV light).  The
gap is then filled by DNA
polymerase I and DNA ligase. 
In yeast,  the proteins similar
to Uvr's are named RADxx
(radiation), such as RAD3,
RAD10, etc.
Nucleotide excision repair in human
cells. A DNA lesion that causes
distortion of the double helix, such as a
thymine dimer, is initially recognized by a
complex of the XP-C (Xeroderma
pigmentosum C protein) and 23B proteins
(1). This complex then recruits
transcription factor TFIIH, whose helicase
subunits, powered by ATP hydrolysis,
partially unwind the double helix. XP-G
and RPA proteins then bind to the
complex and further unwind and stabilize
the helix until a bubble of ≈25 bases is
formed (2). Then XP-G (now acting as an
endonuclease) and XP-F, a 2nd
endonuclease, cut the damaged strand at
points 24–32 bases apart on each side of
the lesion (3).
This releases the DNA fragment with the damaged bases, which
is degraded to mononucleotides.

Finally the gap is filled by DNA polymerase exactly as in DNA

replication, and the remaining nick is sealed by DNA ligase (4 )
 Mismatch repair in E. coli
The mismatch repair system detects
and excises mismatched bases in
newly replicated DNA, which is
distinguished from the parental
strand because it has not yet been
methylated. MutS binds to the
mismatched base, followed by
MutL. The binding of MutL
activates MutH, which cleaves the
unmodified strand opposite a site of
methylation. MutS and MutL,
together with helicase II, SSB
proteins, and an exonuclease, then
excise the portion of the unmodified
strand that contains the mismatch.
The gap is then filled by DNA
polymerase and sealed by ligase.
 Mismatch repair in eukaryotes may be similar to that
in E. coli.  Homologs of MutS and MutL have been
identified in yeast, mammals, and other eukaryotes. 
MSH1 to MSH5 are homologous to MutS; MLH1,
PMS1 and PMS2 are homologous to MutL. 
 Mutations of MSH2, PMS1 and PMS2 are related to
colon cancer.
 In eukaryotes, the mechanism to distinguish the
template strand from the new strand is still unclear.
 Mismatch excision repair of
newly replicated DNA in human
cells. A complex of the MSH2 and
MSH6 proteins binds to a mispaired
segment of DNA such as to distinguish
between the template and newly
synthesized daughter strands (1). This
triggers binding of the MLH1
endonuclease, as well as other proteins
such as PMS2, which has been
implicated in onco-genesis through
mismatch-repair mutations. A DNA
helicase unwinds the helix and the
daughter strand is cut; an exonuclease
then removes several nucleotides,
including the mismatched base (2).
Finally, as with base excision repair, the
gap is then filled in by a DNA
polymerase (Pol, in this case) and
sealed by DNA ligase (3 ).
  Postreplication repair.
The presence of a thymine dimer
blocks replication, but DNA
polymerase can bypass the lesion and
reinitiate replication at a new site
downstream of the dimer. The result
is a gap opposite the dimer in the
newly synthesized DNA strand. In
recombinational repair, this gap is
filled by recombination with the
undamaged parental strand. Although
this leaves a gap in the previously
intact parental strand, the gap can be
filled by the actions of polymerase
and ligase, using the intact daughter
strand as a template. Two intact DNA
molecules are thus formed, and the
remaining thymine dimer eventually
can be removed by excision repair.
When DNA damage is unrepaired: If the replication fork encounters an unrepaired lesion
or strand break, replication generally halts and the fork may collapse. A lesion is left behind in
an unreplicated, single-stranded segment of the DNA; a strand break becomes a double-strand
break.
There are two possible
avenues for repair:
recombinational DNA
repair or, when lesions are
unusually numerous, error-
prone repair. The latter
involves DNA polymerase
V, encoded by the umuC
and umuD genes that can
inaccurately replicate over
many types of lesions. The
repair mechanism is
referred to as error-prone
because mutations often
result.
 Error-
prone
(SOS)
repair

UV light activates the

RecA co-protease, which
stimulates the LexA
protein (purple) to cleave
itself, releasing it from
the umuDC operon. This
results in synthesis of
UmuC and UmuD
proteins, which somehow
allow DNA synthesis
across from a thymine
dimer, even though
mistakes (blue) will be
made.
Repair of double-strand breaks by
homologous recombination. The
black and red DNAs represent the
homologous sequences on sister
chromatids. (1) A double-strand DNA
break forms in the chromatids. (2) The
double-strand break activates the ATM
kinase; this leads to activation of a set
of exonucleases that remove
nucleotides at the break from the 3’
and 5’ ends of both broken strands,
ultimately creating single stranded 3’
ends. In a process that is dependent on
the BRCA1 and BRCA2 proteins, as
well as others, the Rad51 protein
(green ovals) polymerizes on single-
stranded DNA with a free 3’ end to
form a nucleoprotein filament.
(3): Aided by yet other proteins,
one Rad51 nucleoprotein
filament searches for the
homologous duplex DNA
sequence on the sister
chromatid, then invades the
duplex to form a joint molecule
in which the single stranded 3’
end is base-paired to the
complementary strand on the
homologous DNA strand. (4)
The replicative DNA
polymerases elongate this 3’
end of the damaged DNA
(green strand), templated by the
complementary sequences in
the undamaged homologous
DNA segment.
 (5) Next this repaired 3’
end of the damaged DNA
pairs with the single
stranded 3’ end of the other
damaged strand. (6) Any
remaining gaps are filled
in by DNA polymerase and
ligase (light green),
regenerating a wild-type
double helix in which an
entire segment (dark and
light green) has been
regenerated from the
homologous segment of
the sister chromatid.
 Repair of double-strand breaks by
end-joining.
In general, nucleotide sequences are butted
together that were not apposed in the
unbroken DNA. These DNA ends are
usually from the same chromosome locus,
and when linked together, several base
pairs are lost. Occasionally, ends from
different chromosomes are accidentally
joined together. A complex of two proteins,
Ku and DNA-dependent protein kinase,
binds to the ends of a double-strand break
(1). After formation of a synapse, the ends
are further processed by nucleases,
resulting in removal of a few bases (2), and
the two double-stranded molecules are
ligated together (3). As a result, the double-
strand break is repaired, but several base
pairs at the site of the break are removed.
 If DNA can repair itself,
Go ahead, indulge yourself and
enjoy life’s pleasures!
After all, life is short …

But DNA can only do so much for itself…

Abusing its potentials can cause YOU and
your future generations
major, major problems!