DNA MUTATIONS

• Definition
- Abnormal, stable variation in a gene (base
sequence), leading to production of altered
amino acid sequence, leading to:
i) Abnormal, functionless protein
ii) Proteins being made at the wrong time
iii) Proteins being made in the wrong cell type
iv) Too little protein synthesized
v) Too much '' ''
 Germ-line mutations:
- Inherited, hence cause diseases that run in
families e.g. Albinism, diabetes, haemophilia etc

Abnormal, stable variation in gene

coding for one of the enzymes
involved in melanin production

Abnormal enzyme with altered

amino acid sequence

Failure to form melanin

 Types of DNA mutations
a) Spontaneous mutations
- Changes in DNA sequences that occur during
i) normal genetic & ii) metabolic functions of a
cell
- Occur randomly and are not induced by
mutagens

b) Induced mutations
- Changes in DNA sequences that occur due to
exposure of DNA to physical or chemical agent
(mutagen)
 Spontaneous mutations caused by
normal genetic functions of a cell

• Occur during incorporation of dNTPs into daughter

strand by DNA pol III during DNA replication

• How do these mutations occur?

i) Deletion of one or more bases
ii) Addition/insertion of one or more base pairs
iii) Substitution of one or more base pairs
 Deletion of one or more bases

New strand Direction of DNA synthesis

5'
C G T T T
G C A A A A A C G T A C
3' 5' Parent strand

Slippage in parent strand

5'
C G T T T
G C A A A C G T A C
3' 5'
AA

Conclusion
Slippage in parent strand
leads to deletion, in
C G T T T G C A T G newly synthesized DNA
G C A A A C G T A C strand in this case two Ts
3' 5'
AA
Addition/insertion of one or more bases

5'
C
G C A A A A A C G T A C
3' 5' Parent strand

Slippage in new DNA strand

GT
5'
C T T T T T G C A T G
G C A A A A A C G T A C
3'
Conclusion
Slippage in new DNA
strand leads to
GT addition/insertion in
5' C
newly synthesized
T T T T T G C A T G
DNA strand in this
G C A A A A A C G T A C case a T
3'
 Substitution of one or more bases

●Most common type of spontaneous mutation

● Caused by tautomerization (re-arrangement in

nitrogen base) to allow:
i) Purine to be substituted by another purine
ii) Pyrimidine to be substituted by another
pyrimidine
 Example
-Common keto-state of 5-bromo uracil (5Bu) is
an analogue of T
Hence, 5Bu can H2-bond with A (See fig)
Analogue of Analogue of
thymine cytosine
Tautomeric shift

Conclusion:
Adenine has been substituted with Guanine
 Spontaneous mutations caused by
metabolic functions of the cell

a) Hydrolysis of N-glycosidic bonds can bring about

removal of purines (A and/or G)
Guanine N-glycosidic bond

P H2O P
Apurinic site
S S
P Guanine
P P

Conclusion:
Guanine has been removed (deletion)
 Adenine N-glycosidic bond

P H2O P
S S S Apurinic site
Adenine
P P

Conclusion:
Adenine has been removed (Deletion)
ii) Hydrolysis of purine and pyrimidine bases leads
to removal of their amine groups (De-amination),
further leading to base modifications followed by
substitutions
Examples:
● Cytosine
Cytosine Uracil
P H 2O P

S S
NH2
P P

Modified cytosine (Uracil) base pairs with Adenine.

G has been replaced with A (substitution)
● Adenine

Adenine Hypoxanthine

P H2O P

S S
NH2
P P

Modified adenine (hypoxanthine ) base pairs

with cytosine
T has been replaced with C (substitution)
● Guanine
Guanine Xanthine

P H2O P

S S
NH2
P P

Modified Guanine (xanthine ) base pairs with

Cytosine – no problem!
 Repair of DNA with AP sites

Step 1
AP endonuclease breaks
Base phosphodiester bonds Base
P within AP site P
S S
AP endonuclease
P
AP site
S
P P
Base Base
P
S S
P P
S
Section of DNA with Section of DNA
damaged AP site Section of DNA with AP site released without AP site
 Base Step 2 Base
P DNA polymerase I incorporates P
correct dNTP (dATP or dGTP)
S Step 3 S
DNA ligase seals gap thro formation P A or G
of phosphodiester bond
S
P Base Base P Base
S S
P P
Repair of DNA whose purine & pyrimidine
bases have been modified through de-
amination
● Base Excision Repair (BER) mechanism
Damaged base due to
de-amination of C, A or G
Base
Base
Step 1 P
P DNA glycosidase S
S catalyse removal of
damaged base P
P U, HX or X
S
S
P Base
P Base U, HX or X
S S
P P
 Step 2
AP endonuclease
cleaves these
Base phosphodiester bonds
P Base
P
S AP
endonuclease
S
P

S
P Base P Base
S P
P S
S P

Section of DNA with Section of DNA

AP site released without AP site
damaged AP site
 Base Base
Step 3
P i) DNA pol I incorporates
P
S correct nucleotide dATP S
or dGTP
P Correct base
ii) DNA ligase seals final gap
S
P Base P Base
S S
P P
iii) Reactive intermediates from metabolic
processes
• ROS (e.g. O2- , H2O2 and OH) oxidize Guanine
●

to 8-oxoguanine (8-OG)

O2-, H2O2 or ●OH

8-oxoguanine
Guanine (modified Guanine)

Modified Guanine (8-OG) then base pairs with

Adenine rather than Cytosine (Substitution)
Repair of DNA mutations caused by ROS

i) MthI enzyme removes pre-formed 8-OG to

be incorporated into replicating DNA

ii) Mut M and 8-OG Mut Y DNA glycosylases

remove 8-OG already incorporated in DNA
Damaged base may also be removed by base
excision repair mechanism
 Induced Mutations

● Changes in DNA sequences that occur

due to exposure of DNA to physical or
chemical agent (mutagen)

● Examples of mutagens and how they

bring about mutations
i) Nitrous acid (De-aminating agent)
– De-aminates A, G & C, leading to incorrect
base-pairing in DNA (substitution)
Base Deamination New base New base Substitution
pairs
with?
Hypoxanthine
Adenine NH2 (Modified A) Cytosine 'T' substituted
with 'C'
Xanthine
Guanine NH2
(Modified G) Cytosine No problem!

Uracil Adenine
Cytosine (Modified C) 'G' substituted
NH2 with 'A'
ii) Alkylating agents
● Add methyl group to Guanine
● Include:
- Dimethyl sulphate (DMS)
- Ethyl methylene sulphate (EMS)
- N-methyl-N’-nitro-N-nitrosoguanidine
- Dimethyl nitrosamine
-DMS

●Applications
- Methylation of phenols, amines & thiols
• - Adds CH3- group to G, leading to formation
of quartenary nitrogen (N+) that de-stabilizes
glycosidic bond and release of G (Deletion)
- N-methyl-N’-nitro-N- nitrosoguanidine

Applications
- No known commercial use, it is a research
chemical used as an experimental mutagen
● Methylates guanine to form O6-methyl
guanine which pairs with Thymine
● Thus, C is substituted with T

Alkylation with N-methyl-

N’Nitro-N-nitrosoguanine

CH3

O-6 methylguanine
- Dimethyl nitrosamine
● Chemical structure
● Applications
- Industrial solvent
- Chemical intermediate in the production of
1,1-dimethylhydrazine
- Nematocide
- Lubricant additive
- Antioxidant
- Softener for copolymers
- Tobacco & tobacco smoke
- Alcoholic beverages
- Cure food products
 ● DMN methylates bases (modified bases)
in DNA leading to substitutions, which in
turn leads to mis-sense type of mutations

● Example: β-Hb
1 2 3 4 5 6 7 8 9 10

Normal β-Hb TAC CAC GTG GAC TGA GGA CTC CTC TTC AGA CGG
gene
Transcription

mRNA
AUG GUG CAC CUG ACU CCU GAG GAG AAG UCU GCC
Translation
Normal β-Hb Met-Val - His -Leu –Thr-Pro -Glu –Glu -Lys -Ser- Ala
protein
 Dimethyl nitrosamine
methylates T in normal β-Hb
gene and in process substitutes
it with an A

1 2 3 4 5 6 7 8 9 10

Normal β-Hb TAC CAC GTG GAC TGA GGA C A C CTC TTC AGA CGG

Transcription

mRNA
AUG GUG CAC CUG ACU CCU GUG GAG AAG UCU GCC
Translation

Met – Val- His- Leu –Thr –Pro –Val –Glu –Lys –Ser -Ala

Missense mutation in abnormal β-Hb caused by deletion in

normal β-Hb gene
 Repair
● O6-Alkylguanine alkyltransferase transfers alkyl
(CH3- or CH3CH2-) groups from modified
guanine (e.g O6-methyl guanine and O6 ethyl
guanine) to cysteine amino acid residues in its
active site
● Enzyme functions only once upon its
alkyaltion
- Hydroxylamine
●Powerful mutagen
● Works in vitro, since normal cell
components can scavenge and destroy it
● Modifies cytosine, and modified cytosine
base-pairs with adenine instead of
guanine (Substitution)
cytosine
- Ionizing radiations
● Very effective in inducing radiations
● Include x-ray and UV radiations

x-ray radiations
Cause single strand chain breaks in DNA
chain, leading to loss of bases (Deletion)
UV radiations

C G T T T G C A T G
G C A A A C G T A C

C G T T C A T G Gap created due

G C A A A C G T A C
to loss of bases
UV radiations
● Exposure of DNA to UV light (260nm) leads to
formation of pyrimidine dimers

● DNA pol III cannot replicate through dimers,

hence DNA synthesis is skipped at this point,
leading to deletion
i) Thymine dimers
● Two adjacent T residues in a DNA molecule
that are linked via their C-5 and C-6 atoms ,
respectively
Thymine Thymine

5' 3' DNA backbone

UV light (260nm)

Thymine dimer

5' 3' DNA backbone

ii) 6-4 photo product
● Special type of pyrimidine dimer linked via
C-6 of 5’ pyrimidine ( C or T) and C-4 of 3’
pyrimidine (usually C, but occasionally T)
Thymine Thymine

DNA backbone

UV light (260nm)

DNA backbone
 Repair- Nucleotide excision repair (NER) mechanism

● Repairs bulky helix distorting lesions

Lesion caused
by thymine
5' TT 3' dimer
A
A
3' B 5'

ATP
Step 1
Uvr A, A, B trimer ADP +Pi
scans DNA

5' TT 3'
A
A
3' B 5'
 Step 2
On reaching the dimer, Uvr A subunit ATP
recognizes the distortion/lesion and
ATP hydrolysis occurs, leading to ADP +Pi
bending of DNA at this site

TT
3'
5'
B 5'
A
3' A

Step 3 C
uvr A leaves and uvrC comes in and binds to
the uvr B monomer to form uvrBC dimer.
uvr B cleaves/nick a phosphodiester bond 4 A A Nick created
or 5 nucleotides downstream of the damage by uvrB

5' TT 3'

3' C B 5'
 Step 4
uvrBC complex moves upstream
DNA, and uvrC cleaves a
phosphodiester bond 8 nucleotides
Nick created by uvrC upstream of the DNA damage to
Excised create a 12 nucleotide excised
fragment fragment

5' TT 3'

3' C B 5'

Step 5 D
a) Uvr BC complex dissociates from DNA as
individual proteins
b) Uvr D (helicase) comes in and removes
excised fragment by breaking H2-bonds C + B
between complementary bases

5' TT 3'
D
3' 5'
 DNA polymerase I

TT Excised DNA
fragment

Direction of
synthesis (repair)
5' 3'

3' Step 6
5'
a) DNA polymerase I repairs
damage by incorporating correct
nucleotides in the excised
fragment using undamaged DNA
strand as template
b) This enzyme cannot seal final
Enzyme dissociates phosphodiester bond, hence
leaves a nick/gap. It then
Nick dissociates from DNA
5' 3'

3' 5'
 DNA ligase

5' 3'
Step 7
DNA ligase seals the final
3' 5' phosphodiester bond

Enzyme dissociates

5' 3'

3' 5'
Repaired DNA
 Photoreactivation
• Process that repairs damaged DNA due to
exposure to UV light (pyrimidine dimers)
• Requires:
i) Photo-reactivating (DNA photolyase) enzymes
ii) Visible light 370nm
The process:
i) Enzyme binds specifically to pyrimidine
dimers in DNA (370nm)

ii) Enzyme breaks bonds linking the dimers

iii) Enzyme dissociates (dark)

- Intercalating agents (IA)
● Are flat hydrophobic, usually aromatic molecules
● Inset/intercalate between stacked bases in
duplex DNA, hence separating them physically
● Examples of intercalating agents

Benzo(a)pyrene Acridine Ethidium bromide

● When such chains are replicated, they block DNA
pol III which then cannot bring in complementary
dNTP(s) which in turn leads to deletion

● Effect on protein sequence is frame-shift mutation

● Example: Consider β-haemoglobin

 1 2 3 4 5 6 7 8 9 10 11

Normal β-Hb gene TAC CAC GTG GAC TGA GGA CTC CTC TTC AGA CGG
Transcription

mRNA AUG GUG CAC CUG ACU CCU GAG GAG AAG UCU GCC
Translation

Normal β-Hb protein Val His Leu Thr Pro Glu Glu Lys Ser Ala
 IA inserts btw A &
complementary base T in ds
DNA, leading to blocking of A
DELETION
1 2 3 4 5 6 7 8 9 10

Normal β-Hb gene TAC CAC GTG GAC TGA GGA CTC CTC TTC AGA CGG

Transcription

mRNA AUG GUG CAC CUG ACC CUG AGG AGA AGU CUG CC
Translation

Normal β-Hb Met Val His Leu Thr Leu Arg Arg Ser Leu
protein

Frame-shift mutation in abnormal β-Hb caused by

deletion in normal β-Hb gene
 Effect on protein sequence
Frameshift mutation:
Mutation caused by insertion or deletion of a
number of nucleotides that is not divisible by
three from a DNA sequence. Due to the triplet
nature of gene expression by codons, the insertion
or deletion can disrupt the reading frame, or the
grouping of the codons, resulting in a completely
different translation from the original.
• The earlier in the sequence the deletion or
insertion occurs, the more altered the protein
produced is.
ii) In-frame mutation: Mutation involving
insertion(s) or deletion(s) that is/are divisible
by three

iii) Nonsense mutation is a point mutation in a

sequence of DNA that results in a premature
stop codon, or a nonsense codon in the
transcribed mRNA, and possibly a truncated,
and often nonfunctional protein product.
iv) Missense mutations or nonsynonymous
mutations are types of point mutations where
a single nucleotide is changed to cause
substitution of a different amino acid. This in
turn can render the resulting protein
nonfunctional. Such mutations are responsible
for diseases such as Epidermolysis bullosa,
sickle-cell disease, and SOD1 mediated ALS
(Boillée 2006)
v) Neutral mutation is a mutation that occurs in
an amino acid codon which results in the use
of a different, but chemically similar, amino
acid. The similarity between the two is enough
that little or no change is often rendered in
the protein. For example, a change from AAA
to AGA will encode arginine, a chemically
similar molecule to the intended lysine.
vi) Silent mutations
● Mutations that may occur in exons or introns of
gene(s), and do not result in a change to the
amino acid sequence of a protein concerned
● Silent mutations occur because of the
degenerate nature of the genetic code

vii) Synonymous mutations

● Mutations that only occur within exons
● lethal
 By effect on fitness
• Mutations are either harmful, beneficial or neutral

i) A harmful/deleterious mutation
Has a negative effect on the phenotype, and
thus decreases the fitness of the organism

ii) A beneficial/advantageous mutation

Increases fitness of the organism, or which
promotes traits that are desirable
iii) A neutral mutation
- Has no harmful or beneficial effect on organisms
- Occur at a steady rate, forming the basis for
the molecular clock
iv) A nearly neutral mutation
- May be slightly deleterious or advantageous,
although most nearly neutral mutations are
slightly deleterious
• Attempts have been made to infer the distribution
of fitness effects using mutagenesis experiments