1. Mutation
Raj Krishna Dangol
Department of Biochemistry
Lumbini Medical College

2. Mutation
A mutation is defined as a change in nucleotide
sequence of DNA
Mutagens are substances which can induce
mutations. These can be chemicals, radiations or
viruses
The changes that occur in DNA on mutation are
reflected in replication, transcription and
translation
Statistically, out of every 106 cell divisions, one
mutation takes place

3. Types of mutations
Mutation
Point
mutation
Transition Transversions
Frame shift
Mutation
Insertions Deletions

4. Point mutation
Replacement or change in a single base
Two types
Transition : replacement of a purine by
another purine (A to G or G to A)or pyrimidine
by pyrimidine (T to C or C to T)
Transversion : replacement of a purine by
pyrimidine (A to C) or pyrimidine by Purine (T
to G)

5. Deletion
Large gene deletions e.g. alpha thalassemia
(entire gene) or homophilia (partial)
Deletion of a codon, e.g. cystic fibrosis (one
amino acid, 508th phenyl alanine is missing in
the CFTR gene
Deletion of single base, which will rise to
frame shift effect

6. Insertion
Single base additions, leading to frame-shift
effect
Trinucleotide expansions, e.g. in Huntington’s
chorea, CAG trinucleotides are repeated 30 to
300 times. This leads to a polyglutamine
repeat in the protein
Duplications. E.g.in Duchenne Muscular
Dystrophy (DMD), the gene is duplicated

7. Effect of mutation
Point mutation may lead to
– Silent Mutation
– Mis-sense Mutation
• Acceptable
• Partially acceptable
• Unacceptable
– Non-sense
Insertion or deletion of single base leads to
– Frame-shift Mutation

8. Silent Mutation
A point mutation may change the codon for
one amino acid to synonym for the same
amino acid
Mutation is silent and has no effect on the
phenotype
E.g. CUA is mutated to CUC; both code for
leucine, and so this mutation has no effect

9. Mis-sense but Acceptable Mutation
A change in amino acid may be produced in
the protein; but with no functional
consequences
Acceptable mutation
HbA-β chain 67 Val
Hb(Sidney)-β chain 67 Ala
GUU
GCU

10. Mis-sense; Partially Acceptable
Mutation
o The amino acid substitution affects the functional
properties of the protein
o HbS has abnormal electrophoretic mobility and
subnormal function, leading to sickle-cell anemia
Partially Acceptable mutation
HbA-β chain 6 Glu
HbS-β chain 6 Val
GAG
GUG

11. Mis-sense; Unacceptable Mutation
o The single amino acid substitution alters the
properties of the protein to such an extent
that it becomes nonfunctional and the
condition is incompatible with normal life
Unacceptable mutation
HbA-α chain 58 His
HbM(Boston)-α chain 58 Tyr
CAU or CAC
UAU or UAC

12. Nonsense; Terminator Codon
Mutation
The codons with the altered base may become
one of the three termination codon (UAA, UAG or
UGA) called as “nonsense codon”.
This leads to premature termination of the
protein, and so functional activity may be
destroyed. E.g. beta-thalassemia
A terminator codon is altered into a coding codon
(UAA to CAA), resulting in elongation of the
protein to produce “run on polypeptide” (Hb
Constant spring)

13. Frame-shift Mutation
This is due to addition or deletion of bases.
From that point onwards, the reading frame
shifts. A “garbled” (completely irrelevant)
protein, with altered amino acid sequence is
produced.

15. Not only the sequence of amino acids distal to
the addition or deletion is garbled, there may
appear a nonsense (chain termination or run-
on-polypeptide) that are non-functional

16. Manifestations of Mutations
Lethal Mutations
 The alteration is incompatible with life of the cell
or the organism
 E.g. mutation producing alpha-4 Hb is lethal, and
so the embryo dies
Silent Mutations
 Alteration at an insignificant region of a protein
may not have any functional effect

17. Beneficial Mutations
 Beneficial spontaneous mutations are the basis of
evolution
 Such beneficial mutants are artificially selected in
agriculture.
 E.g. normal maize is deficient in tryptophan.
Tryptophan-rich maize varieties are now available
for cultivation
Carcinogenic Effect
 The mutation may not be lethal, but may alter the
regulatory mechanism.
 Such a mutation in a somatic cell may result in
uncontrolled cell division leading to cancer

18. DNA damage and DNA Repair
DNA is replicated with great fidelity (accuracy).
However, DNA can be damaged by variety of
causes resulting in several distinct types of
lesions
Various physical and chemical agents produce
base alterations; these are to be appropriately
corrected immediately
The DNA polymerase has 3’ to 5’ exonuclease
activity. Hence any mispaired nucleotide added is
immediately removed

19. • Cause of DNA damage
 Misincorporation of deoxynucleotides during
replication
 By spontaneous deamination of bases during normal
genetic functions
 From x-radiation that cause “nicks” in the DNA
 From UV irradiation that causes thymine dimer
formation
 From various chemicals that interact with DNA e.g.
ozone (produced by lightning), hydrazines (present in
edible mushrooms), allylisothiocynates, aflatoxin (mold
growing on peanuts and grains), alkylating agents
(busulphan, cyclophosphamide) and free radicals
(oxidative stress)

21. Types of damage to DNA
1. Single-base alteration
a. Depurination
b. Deamination of cytosine to uracil
c. Deamination of adenine to hypoxanthine
d. Alkylation of base
e. Insertion or deletion of nucleotide
f. Base-analog incorporation

22. 2. Two-base alteration
a. UV light-induced thymine-thymine (pyrimidine) dimer
b. Bifunctional alkylating agent cross-linkage
3. Chain breaks
a. Ionizing radiation
b. Radioactive disintegration of backbone element
c. Oxidative free radical formation
4. Cross-linkage
a. Between bases in same or opposite strands
b. Between DNA and protein molecules (e.g. histones)

23. Mechanism of DNA Repair
the maintenance of the integrity of DNA is
very important in order to provide correct
genetic information.
The integrity of DNA after DNA replication is
maintained by the presence of specific DNA
repair system
There are several DNA repair system

24. Mechanism of DNA Repair
Mechanism Problem Repair
Mismatch repair Copying errors (single base
or two- to five-base
unpaired loops
Methyl-directed strand
cutting, exonuclease
digestion, and replacement
Base excision-repair Spontaneous, chemical, or
radiation damage to a single
base
Base removal by N-
glycosylase, abasic sugar
removal, replacement
Nucleotide excision-repair Spontaneous, chemical, or
radiation damage to a DNA
segment
Removal of an approximately
30-nucleotide oligomer and
replacement
Double-strand break
repair
Ionizing radiation,
chemotherapy, oxidative
free radicals
Synapsis, unwinding,
alignment, ligation

25. General Mechanism
Recognition of altered base
Removal of altered base along with a few
bases around that area.
A small segment of DNA with correct base
sequence is then synthesized by DNA
polymerase beta.
Then the gap or nick is sealed by DNA ligase

26. Mismatch Repair
Mismatching of bases can occur during DNA
synthesis since proof reading is not 100%
accurate
Repair enzymes:- mismatch repair protein
complexes(MutS, MutC and MutL in Ecoli and
MSh and MLH in humans), exonucleases, DNA
polymerases and DNA ligases are involved in
mismatch repair

27. Repair process
Specific proteins scan the newly synthesized DNA, using adenine
methylation within a GATC sequence as the point of reference
The template strand is methylated, and the newly synthesized strand
is not.
This difference allows the repair enzymes to identify the strand that
contains the errant nucleotide which requires replacement.
If a mismatch or small loop is found a GATC endonuclease cuts the
strand bearing the mutation at a site corresponding to the GATC.
Exonuclease digest this strand from the GATC through the mutation,
thus removing he faulty DNA
DNA polymerase fills the gap
The last phosphodiester linkage is closed by DNA ligase

29. Base Excision Repair
Involves repair of alkylated bases, repair of
deaminated bases and repair of depurination
Repair enzymes:- DNA glycosylates, AP
endonucleases, helicases, excision nuclease,
DNA polymerase and DNA ligase

30. Repair of deamination
Cytosine spontaneously deaminates to form uracil
Uracil is recognized by uracil DNA glycosidase and uracil
is excised
Creation of AP (either apurine or apyrimidine) site
consisting of only deoxyribose phosphate backbone
AP endonuclease nicks the deoxyribose phosphate
backbone
Excision nuclease removes the AP site and several
nucleotides
DNA polymerase fills the gaps
DNA ligase seals the phosphodiesterase bond

31. Repair of depurination
Depurination occurs by breaking of N-glycosyl
bond between the purine and deoxyribose
AP (apurinic site) endonucleases recognizes the
site of missing purines and nicks the deoxyribose
sugar phosphate
Phosphodiesterase excises the deoxyribose
phosphate
DNA polymerase replace the purine nucleotide
DNA ligase seals the phosphodiester bond

32. Nucleotide Excision Repair
 It repairs covalent bonding between adjacent thymine bases
or adjacent thymine-cytosine bases caused by ultravoilet
light. This produces thymine dimers or thymine-cytosine
cross links. Both of these alterations produce distortions of
DNA helix
 Enzymes:- excinuclease, DNA polymerase and DNA ligase. At
least 18 different proteins are involved in nucleotide
excision repair. Proteins encoded by 7 genes related to
xeroderma pigmentosum (XPA to XPG) are involved in
nucleotide excision repair. Cockayne syndrome related
genes (CSA or CSB) are involved in transcription coupled
DNA repair

33. Excinuclease detects the distortion of the
helix, nicks the damaged strand on both sides
of the lesion and removes the nucleotides
DNA polymerase fills in the gap,
using the undamaged strand as
template
DNA ligase seals the
phosphodiester bond

34. • In transcription coupled repair, RNA
polymerase is made to transverse back from
the site of lesion followed by correction of the
lesion

35. Double Strand Break Repair
It is usually caused by ionizing radiation,
oxidative stress and chemicals such as
bleomycin
It can also occur during immunoglobulin gene
arrangement
Enzymes:- Ku protein with helicase activity, DNA
dependent protein kinase, exonuclease and
ligase

36. Ku protein binds to both ends of DNA double stranded
DNA segments
Recruit DNA dependent protein kinase
DNA dependent protein kinase approximates the two
separated strands and activate Ku protein
Activated Ku protein has helicase activity and unwinds
the two ends of DNA
Approximated DNA segments form the base pairing
Extra nucleotides are removed by exonuclease
Gaps are filled by ligase

37. Clinical aspect
• Xeroderma Pigmentosum
(greek xeros – dry + derma- skin)
– Defect: nucleotide excision repair; caused by the
defect in the removal of pyrimidine dimer caused
by the defective excinuclease, mutation in XPA
gene
– Features: hypersensitivity to sunlight (UV
radiation) leading to the development of skin
lesions and skin cancer

38. • Ataxia Telangiectasia
– Defect in gene involved in DNA repair and cell
cycle
– Characterized by hypersensitivity to ionizing
radiation, cerebellar ataxia, oculocutaneous
telangiectasia and immunodeficiency. These
patients are susceptible for the development of
lymphomas

39. • Fanconi’s Anemia
– Defect in double strand break repair
– Feature: hypersensitivity to DNA cross linking
agents, bone marrow failure (aplastic anemia) and
leukemia
• Bloom’s syndrome
– Defect in double strand break repair, defective
helicase
– Feature: susceptibility to ultraviolet radiation, and
the development of leukemia

40. • Hereditary Nonpolyposis Colorectal Cancer
(HNPCC)
– Defect in mismatch repair, defective HNPCC
genes, 50-60% of HNPCC is associated with
mutation on hMSH2, hMLH1 is associated with
most of other cases
– Features: condition accounts for about 15% of
colon cancers, early development of tumors
– Identification of the genes responsible for HNPCC
permit the early detection of the condition

41. • Cockayne Syndrome
– Defect in preferential repair of he transcribed
strand, mutation in proteins CSA and CSB
– Features: neurological degeneration and growth
retardation
• Warner’s syndrome
– Inherited defect in excision repair of DNA,
defective helicase
– Characterized by accelerated aging

42. References
Harper’s Illustrated Biochemistry, 28th edition
Biochemistry by Voet and Voet, 4th edition
Medical Biochemistry, AR Aroora
Text Book of Biochemistry, DM Vasudevan
Text Book of Medical Biochemistry, MN
Chatterjea
Biochemistry, U Satyanarayana