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With the aid of examples, describe how mutations may arise. Describe the mechanisms that
prevent these mutations from accumulating in our genome.
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Essay plan
Chromosome mutation vs gene mutation (somatic & germline)
Point mutations. Mutations affecting amino acid sequence; point mutations & frameshifts.
Spontaneous mutations vs induced mutations (chemicals and radiation)
Repair mechanisms; direct repair of DNA damage; excision of base pairs; post replication
repair; SOS response.
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A mutation is a process by which a DNA base-pair change or a chromosome change occurs.
Thus mutations can be:
1. Chromosome mutation/aberrations
2. Gene mutations

A gene mutation can be defined as a stable change in the DNA structure of a gene which may
be expressed as a phenotypic change in the corresponding organism. A gene mutation can:
a. Somatic mutation this occurs when a mutant cell gives rise to somatic cells resulting in
mutant spots/areas, but the mutant characteristic is not passed to successive generations.
b. Germline mutation this occurs when the mutation occurs in a germline of a sexually
reproducing organism, and may be transmitted by the gametes to the next generation.
Thus, a somatic mutation affects the individual in which it happens while a germline mutation
affects individuals of subsequent generations.

Mutations often result in loss of normal function of the affected allele. If the loss of function is full
it is a null mutation, if loss of function is partial it is a leaky mutation. Loss of function
mutations are generally recessive. On the other hand, gain of function mutations are dominant.


Point mutations

Point mutations are single base substitutions. These affect the base sequences but not the
overall DNA structure. Point substitutions can be categorised as follows:
i. Transition involves replacement of a purine base with another purine base, or
replacement of a pyrimidine base to another pyrimidine base.
ii. Transversion involves replacement of a purine with a pyrimidine and vice versa.
Transition mutations are much more common than transversion.





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Point mutations may also be categorised according to function / the effect they have on amino
acids:

i. Missense mutation: is a base pair change in the DNA which causes a change in the
mRNA codon so that a different amino acid is inserted in the polypeptide chain. This will
code for a different amino acid. E.g. Sickle cell anaemia is a disease caused by a single
base pair change that converts a glutamate (GAG) to a valine (GTG) in the sixth position
of the -globin.
ii. Nonsense mutation: is a base pair change that changes a codon in the mRNA that codes
for an amino acid into a stop codon (AUG, UUA, UGA).
iii. Neutral mutation: is a base pair change in a gene that changes a codon in the mRNA such
that the resulting amino acid substitution produces no detectable change in the function of
the protein e.g. arginine (AGA) to lysine (AAA).
iv. Silent mutation: is a base mutation that alters a codon in mRNA such that the same amino
acid is inserted in the protein e.g. AAA and AAG both code for lysine. This like neutral
mutation also results in no functional change of the protein.

Gene mutations affecting the amino acid sequence can also be frameshift mutations. These
result from the deletion or addition of one or more base pairs in the gene. This shifts the mRNA
reading frame so the incorrect amino acids are incorporated into the protein after the mutation.
This usually results in a non-functional protein.


Causes of mutations

A. Spontaneous mutations occur naturally.

These may occur from a number of events:
1. Error in DNA replication
2. Spontaneous chemical changes in DNA
The can occur due to thermal fluctuations or reactions with reactive oxygen species. As a result
purines and pyrimidines along with the nucleotides of which they form part, undergo a number
of spontaneous alterations in their covalent structure. The rate of these spontaneous mutations
is very low, but can be physiologically significant due to the cells very low tolerance for
alterations in its genetic code. Evidence shows that there is a direct link between the
accumulation of mutations and the processes of aging and carcinogenesis.

The most common type of spontaneous DNA changes are:
i. Depurination reactions
In depurination, a purine is lost by hydrolysis of the N--glycosyl bond. The deoxyribose
that remains after depurination is readily converted from -furanose to an aldehyde form.
This type of loss of the base from the DNA molecule is much more common for purines
than pyrimidines. As much as 10,000 purines per cell are lost from DNA every 24 hours,
under typical cellular conditions.

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ii. Deamination reactions
Several nucleotide bases spontaneously lose their exocyclic amino groups. An example of
such an event is the deamination of cytosine in DNA to uracil. This occurs about 100 times
a day in a mammalian cell. Adenine can also be deaminated to guanine but this occurs
100X slower.


B. Induced caused by exposure to mutagens.

1. From chemical compounds
Mutations caused by chemical compounds may lead to cancers. All carcinogens act by causing
changes in the genome of the cell. Chemical carcinogens are responsible for most of the cancer
deaths in the western world mainly due to tobacco smoke and diet. Chemical carcinogens can
be:
i. Direct acting carcinogens these act directly on the genome.
ii. Procarcinogens act on the genome indirectly; these are converted metabolically by
cellular enzymes to ultimate carcinogens that bind to DNA and cause mutations. E.gs of
procarcinoges include;
Polycyclic aromatic hydrocarbons; in food, exhaust fumes, cigarette smoke and coal-
tar derivatives.
Aromatic amines; used in chemical and rubber industries.
Azo dyes (azo include an N=N).
Natural metabolites such as aflatoxin produced by fungal contamination of food.
Nitrosamines; meat preservatives.
Saccharin; artificial sweetener.
The enzymes that activate carcinogens are often members of the cytochrome P450 family.

Chemical carcinogens are further classified as initiators and promoters:
i. Initiators these are chemicals that cause mutations e.g. asbestos and Benzo(a)pyrene
in tobacco smoke.
ii. Promoters these are chemicals that act on cells already affected by initiators. They
promote or increase the rate of cell growth. This is accomplished by the induction of
protein kinase C, an enzyme that stimulates growth. E.g. oestrogen and arsenic.

Note on Benzo(a)pyrene:
Benzo(a)pyrene is not carcinogenic until it is oxidized within cells. After oxidation it can bind
covalently with guanine residues in the DNA, interrupting hydrogen bonding in G-C base pairs
and thus produce distortions of the helix.

Chemical compound that induce mutations can be:
A. Base analogues
Example: 5-bromouracil (5BU) is a brominated derivative of uracil that acts as a base analogue
mainly used as an experimental mutagen. 5BU stearically resembles thymine in DNA and
exists in 3 tautomeric forms that have different base pairing properties. The keto form is

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complementary to adenine, while the enol and ion forms are complementary to guanine. This
means that 5BU can be present in DNA either opposite to adenine or guanine. The 3 tautomeric
forms frequently interchange, inducing point mutations via base substitutions. The base pair will
change from an A-T to a G-C or from a G-C to an A-T after a number of replications cycles.

B. Base modifying agents
DNA may also be damaged by reactive chemicals introduced in the environment as products of
industrial activity. Such products may not be injurious per se, but may be metabolized by cells
into forms than are. 2 prominent classes of such agents are:
Deaminating agents e.g. nitrous acid or compounds that can be metabolized into nitrous
acid or nitrites.
Alkylating agents
Nitrous acid is a potent accelerator of the deamination of bases. Nitrous acid is used as a
preservative in processed foods to prevent the growth of toxic bacteria such as Clostridium
botulinum (causes botulism). Used in such small amounts they to not increase cancer risk
significantly and thus make only a minor contribution to the overall levels of DNA damage.

C. Intercalating agents
DNA intercalating agent is the reversible inclusion of a molecule into DNA. Intercalating
mutagens e.g. ethidium bromide (commonly used as a fluorescent flag in molecular biology e.g.
to stain DNA in gel electrophoresis) act by inserting themselves between adjacent bases in one
or both DNA strands. This would stretch the double helix, thus and extra base must be inserted
in the DNA strand opposite the intercalating agent. After one or more rounds of replication, the
intercalating agent is lost and a frameshift mutation results due to the insertion of a base pair.


2. From radiation, which can be ionizing, or non-ionizing.
High energy radiation causes damage largely by production of free radicals which result in
oxidation of DNA bases. Effects can be severe, including single or even double strand breaks.

A. Ionizing radiation
Collision of ionizing radiation e.g. X-Ray and gamma rays, with atoms in its path gives rise to
ions and reactive chemical radicals that can break chemical bonds including those in DNA, thus
inducing chromosome breakage, chromosome rearrangements and point mutations in DNA.
Ionizing radiation is the main cause of gross chromosomal mutations in humans. High dosages
of ionizing radiation kills cells e.g. used in the treatment of some forms of cancer. Low dosages
result in the accumulation of point mutations, the effects of which are cumulative. Thus the
benefits of X-rays as a diagnostic procedure should be weighed carefully against the risks of
exposure to X-ray radiation.

B. Non-ionizing radiation (UV light; 200-300nm)
UV rays have insufficient energy to cause ionizations but in high enough dosages can still kill
cells. UV light is used as a sterilization agent in some applications. The sun is also a powerful
source of UV radiation, but much of the UV is screened out by the ozone in the atmosphere. UV

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irradiation of DNA can result in the formation of thymine-thymine dimers between adjacent
pyrimidines on the same DNA strand. This causes a bulge in the DNA strand and disrupts the
normal pairing of thymine with the corresponding A on the opposite strand.

2% of cancer deaths are caused by radiation, e.g. aggressive melanoma skin cancers. The UV
light reaching the earth consists of 2 forms; UVA (320-400nm) and UVB (290-320nm). The
ambient level of UVA is 2 to 3 the order of magnitude that of UVB. Both UVA and UVB play a
role in carcinogenesis. Sunburn is mainly caused by UVB, which also causes skin cancer
because the radiation in the wavelength range of UVB (290-320nm) is mutagenic. UVA plays a
role in skin cancer by acting to increase the carcinogenic effects of UVB.


DNA repair mechanisms

It is very important for the cell to maintain the integrity of the information in DNA. Thus when
DNA becomes damaged, a number of repair mechanisms exist. DNA repair is possible largely
because the DNA molecule consists of 2 complementary strands. DNA damage in one strand
can be removed and accurately replaced by using the undamaged complementary strand as a
template. As a result of DNA repair, fewer than one lesion in 1000 becomes a mutation. The
number and diversity of repair systems reflect both the importance of DNA repair to cell survival
and the diverse sources of DNA damage.

The repair mechanisms are classified in the following way:
A. Direct correction/reversal of DNA damage
1. Correction by DNA polymerase
2. Photoreactivation of UV-induced pyrimidine dimers
3. Repair by alkylation damage
B. Repair involving excision of base pairs
1. Excision repair; nucleotide excision repair, base excision repair (by glycosylases)
2. Mismatch repair
3. Post-replication repair
4. SOS response


Direct correction of DNA damage

1. Repair by DNA polymerase:
The incorrect nucleotide is removed by the 3-to-5 exonuclease activity of DNA polymerase III
which reverses along the template strand to repair DNA. DNA polymerase III then moves
forward again resuming its 5-to-3 DNA synthesis activity. Due to this property the frequency of
base-pair substitutions varies from 10
-7
to 10
-10
. Proofreading occurs also in eukaryotes,
although the main DNA polymerase involved lacks the 3-to-5 exonuclease activity. This
property is found in other proteins that are present in the replication fork.


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2. Photoreactivation of UV-induced pyrimidine dimers
This occur in prokaryotes and lower eukaryotes. The dimers are reverted directly to their original
form by exposure to visible light (320-370nm). Photoreactivation is catalyzed by an enzyme
photolyase which when activated by a photon of light splits the dimer apart.

3. Repair by alkylation damage
Guanine is susceptible to alkylation by chemical mutagens such as dimethylsulphate. The
alkylated guanine now base pairs with thymine, resulting in a change from GC to AT when the
DNA is replicated. An enzyme O
6
-methyl-guanine methyl transferase recognizes the O
6
-methyl-
guanine in the DNA and removes the methyl group to repair the alkylated guanine.


Repair involving excision of base pairs
These pathways all comprise of a) damage recognition, b) damage excision, c) DNA repair
synthesis and d) DNA ligation.

1. Nucleotide excision repair (NER pathway)
This removes bulky lesions caused by e.g. UV and benzo(a)pyrine. Nucleotide excision repair in
E. coli and human DNA are analogous. The enzyme complex that excises the damaged
nucleotides is ABC excinuclease. In E. coli DNA polymerase I and ligase re-synthesize and
relegate the DNA molecule. In humans, the resulting gap is filled by DNA polymerase . Genetic
deficiencies in nucleotide-excision repair in humans give rise to a variety of serious disease e.g.
xeroderma pigmentosa.

2. Base excision repair (BER pathway)
Chemically modified bases can be removed by enzymes called DNA glycosylases. These
enzymes cleave the glycosidic bond of a specific type of altered nucleotide. Excision of the base
leaves a deoxyribose without a base (apurinic/apyrimidinic site - AP). The enzyme AP
endonucleases nicks the phosphodiester backbone at the depurinized site and excises the
sugar-phosphate backbone. DNA polymerase I and ligase re-synthesize and seal the DNA. For
example, uracil does not normally occur in DNA but can sometimes occur from a spontaneous
deamination of cytosine. Uracil is excised by uracil N-glycosylase and replaced by C through
base excision repair.

3. Mismatch repair mechanisms
Neither of the bases in a mismatch is damaged, thus repair enzymes must be able to determine
which base of the mismatched pair to correct. In E. coli, 3 proteins are involved; MutS, MutL and
MutH. This mismatch-repair enzyme complex acts during replication when an incorrect but
normal base is incorporated into the growing chain. In E. coli, parental DNA strands contain
methyl groups on adenine bases in specific sequences. During replication, the newly
synthesized strands are not immediately methylated. Before methylation occurs, the proteins
involved in mismatch repair can distinguish parental from newly synthesized strands. A region of
the new, un-methylated strand, containing the mismatched base, is removed and replaced.
Eukaryotic cells have proteins structurally and functionally analogous to the bacterial proteins

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involved in mismatch repair. Inherited cancer-susceptibility syndromes e.g. hereditary
nonpolyposis colon cancer (HNPCC) is associated with defects in mismatch repair.


Post-replication repair
Most DNA lesions in E. coli are repaired prior to replication. However, if an unrepaired lesion is
encountered by the replication complex near the replication fork, replication is blocked.
Eventually replication resumes past the site of the lesion with the polymerase skipping over the
damaged bases. The new daughter strand would then be missing a base opposite the damaged
base. This genetic lesion cannot be eliminated by excision repair since this requires an intact
complementary strand. The lesion is corrected by exchanging the corresponding part from the
sister DNA strand (the complementary strand of the parent DNA) by the process of
recombination. The gap is now in the sister DNA strand, but since it is complementary to normal
DNA, the gap is filled as normal.


SOS repair (only in bacteria)
In E. coli unrepaired DNA damage induces a complex system the SOS response. The SOS
response operates to allow the cell to survive otherwise lethal events, although at the expense
of generating new mutations. This response involves 2 proteins; LexA and RecA.
When there is no DNA damage, LexA acts as a repressor and inhibits the expression of about
17 proteins involved in repair. When there is DNA damage, RecA protein becomes activated
and stimulates LexA to cleave (making it inactive); this relieves the repression from the repair
genes.