MUTATIONS

Changes in DNA that affect genetic

information or inherited alterations in
the sequence of nucleotides in DNA.
Mutations alter the nucleotide
sequence of DNA
Mutations are a random process
IMPORTANCE OF MUTATIONS

•Account for evolutionary

changes in m/o. (antibiotic
resistance)
•Account for alterations that
produce different strains.
•Account for adaptations to new
Environment.
•Account for change in genotype
but may or may not change the
phenotype. UUU—UUC both
code for phenylalanine.
 Effect of mutations on M/O
Alter nutritional requirements –
Auxotrophs are the org. That require special substances in
their medium in order to grow which are not required by
their unmutated forms called prototrophs or wild type
bacteria which can grow on a simple growth medium
•Inability to withstand certain growth conditions e.g
temperature
•Become resistant to antibiotics or other inhibitors
•Alterations in colony morphology, colony color,
spore formation, capsule production, pigment
production etc.
REPLICA PLATING TECHNIQUE TO IDENTIFY MUTANTS
• In this procedure, the mutagenized
culture is plated out to obtain
single colonies on a nutrient
medium on which mutants and
parents will grow.

• After incubation, the colonies are

replicated, using a sterile velvet
pad, onto a minimal agar plate and
then a similar plate to which the
appropriate supplement (in this
case, histidine, since we are
looking specifically for histidine
auxotrophs) has been added.

• Histidine requiring auxotrophs will

be unable to grow on the first
plate, but will grow on the second
one.

• Thus, mutant colonies can be

identified with localization of
colonies that have failed to grow in
second plate in comparison with
 Replica plating is used to detect
1. Mutants that are resistant to antibiotics, or to
specific bacteriophages, toxic chemicals or any
other agents that are usually lethal or inhibitory to
the non mutated parent cell;
2. Auxotrophs, i.e. mutants that require some
additional growth factor, such as an amino acid;
3. Mutants that are unable to use a particular
growth substrate (usually a sugar).
Mutations can be classified in number of
ways based on –

•The molecular nature of the defect

•The nature of phenotypic effect and
•Causes of the mutations.
Mutations based on the molecular nature of the
defect i.e effect on DNA sequence (point
mutations)are:
1. Base substitution- one base is replaced by the
other. Causes change in a single codon, which may or
maynot change the amino acid sequence in a protein.
2 Types-
2. Insertions and deletions – one or more base pairs are
added or deleted.
Shifts the translational reading frame of codons in mRNA.
Hence, called frame shift mutations resulting in the
formation of Non Functional proteins.

C
 2. Classification on the basis of their
phenotypic effects :
•Same sense
mutations
• Mis-sense mutations- causes a change in the
codon to the one which specifies different a.a.
Non-sense mutations- that creates one of the stop codons causes
premature termination of the translation process
 On the basis of Causes of Mutations
•
☠
Spontaneous mutations are changes in DNA
structure that result from abnormalities in biological
processes. A distinguishing feature of spontaneous
mutations is that their underlying cause originates
within the cell.
• Induced mutations- The cause of induced mutations
originates outside the cell. Induced mutations are
produced by environmental agents, either chemical
or physical, that enter the cell and lead to changes
in DNA structure. Agents known to alter the
structure of DNA which lead to mutations are called
mutagens.
 Spontaneous mutations:
Spontaneous mutations arise from a variety of
Sources:
• Replication errors- Errors in DNA replication,
Errors in proofreading
• Spontaneous lesions- Due to abnormalities in
biological processes in cell

• Errors in replication
DNA polymerase can make a mistake during DNA
replication by putting the wrong base in a newly
synthesized daughter strand.
e.g HIV have relatively high rates of spontaneous
mutations.
 Spontaneous lesions
• Naturally occurring damage to the DNA -
normal metabolic processes may produce
chemicals within the cell that can react
directly with the DNA and alter its structure.
Two of the most frequent spontaneous lesions
result from
Depurination and
Deamination.
 ☠ DEPURINATION
• The most common type of chemical change that
occurs naturally is depurination, which involves
the removal of a purine (adenine or guanine) from
the DNA.
• The covalent bond between deoxyribose and a
purine base is somewhat unstable and occasionally
undergoes a spontaneous reaction with water that
releases the base from the sugar, thereby creating
an apurinic site.
CT CT C

GA G G
 SPONTANEOUS MUTATIONS-
Depurination
– Most common type of naturally occurring
chemical change- Reaction with water
removes a purine (A or G) from the DNA
• “Apurinic site”
- Rise in temp., drop in pH and
chemical mutagens are
also responsible for apurination.

(A glycosidic bond forms by a condensation

reaction, which means that one water
molecule is produced during formation of a
glycoside. The reverse reaction, the
breakage of a glycosidic bond, is
a hydrolysis reaction) 20
 SPONTANEOUS MUTATIONS
– Generally recognized by
DNA repair enzymes
– Mutation may result if
repair system fail.
• Because a complementary
base is not present to
specify the incoming base
for the new strand, any of
the four bases are added
to the new strand in the
region that is opposite the
apurinic site. This may
lead to a new mutation.
21
SPONTANEOUS MUTATIONS- Deamination
of Cytosine-
• occurs at a rate of about 100 bases per cell per
day
– Other bases are not readily deaminated
– Deamination involves Removal of an amino group
from the cytosine base
• The deamination of cytosine yields uracil.
• DNA repair enzymes generally remove this base, recognized
as an inappropriate base
– Mutation may result if repair system fails
• Uracil hydrogen bonds with A not G

23
One of the repair enzymes in the cell, uracil-DNA glycosylase,
recognizes the uracil residues in the DNA that arise from deamination
of cytosine and excises them, leaving a gap that is subsequently filled
by DNA polymerase enzyme.
• When that amino group is lost, a uracil (U) base is
formed, and a normal C-G DNA base pair is changed
to a premutagenic U-G base pair (uracil is not a
normal part of DNA).
• The U-G base pair is called premutagenic because if it
is not repaired before DNA replication, a mutation
will result.
• Unrepaired uracil pairs with adenine in replication,
resulting in the conversion of a G–C pair into
an A–T pair (a GC → AT transition).
• It is very important for the cell to repair DNA damage
before DNA replication commences, in order to avoid
mutation
 SPONTANEOUS MUTATIONS-
Tautomeric shift
a temporary change in
base structure called a
tautomeric shift.
– Common, stable form of
T and G is the keto form
• Rarely Interconvert to an
enol form
– Common, stable form of
A and C is the amino
form
• Rarely Interconvert to an
imino form the H atoms can move from one position in a
base to another position. 26
 Tautomeric shift
Normal base pairing
– Enol and imino forms do
not conform to normal
base-pairing rules

• In imino form, A base

pairs with C

• In enol form, T base pair

with G

• AC and GT base pairs are

formed 27
 Tautomeric shifts
• Tautomeric shifts
immediately prior to DNA
replication can cause
mutations.
• When DNA is in a
double-stranded
condition, the base pairing
usually holds the bases in
their more stable forms.
• After the strands unwind,
however, a tautomeric
shift may occur.
• Resulting mismatch
could be repaired
• Mutation may result if
repair system fails In imino
form, A base
pairs with C

28
 Deamination of modified cytosines
• DNA of both bacteria and eukaryotes contains small
amounts of the modified base 5-methylcytosine
(Adenine or Cytosine methylation is part of the
restriction modification system of many bacteria.
• A methylase is the enzyme that recognizes a specific
sequence and methylates one of the bases in or near that
sequence).

30
 Deamination of the 5-methyl cytosine
produces thymine

The deamination of 5-methylcytosine

generates thymine,
which is not recognized by the DNA repair
enzymes as incorrect nucleotide and thus is not
repaired.
 INDUCED
MUTATIONS
• Enormous array of
agents can act as
Types of Mutagens
mutagens that
permanently alter the
structure of DNA.
• Deliberately induced
mutations have played,
and continue to play, an
important role in the
study of mutations.
• Used to increase the
frequency of mutation
so that a significant
number of organisms
have mutations in the
gene being studied.
CHEMICAL MUTAGENS
(a) BASE ANALOGS- e.g 5-BU has bromine instead of methyl
gp of thymine.
• Compounds such as 5-bromouracil
(5BU) and 2-aminopurine are base
analogues that become incorporated into
daughter strands during DNA
replication.
• 5BU is a thymine analogue that can be
incorporated into DNA instead of
thymine by DNA Polymerase III during
replication.
• Like thymine, 5BU base-pairs with
adenine.
• However, at a relatively high rate, 5BU
undergoes a tautomeric shift and base-pairs
with guanine
 When this occurs during DNA replication, 5BU causes a mutation in
which a TA base pair is changed to a 5BU-G base pair. This is a transition,
because the adenine has been changed to a guanine, both of which are
purines. During the next round of DNA replication, the template strand
containing the guanine base produces a GC base pair. In this way, 5BU can
promote a change of an AT base pair into a GC base pair.

k e

e k

Compounds like 5BU are sometimes used in chemotherapy for cancer. The rationale is that these compounds are incorporated
only into the DNA of actively replicating cells such as cancer cells. When incorporated, these compounds tend to cause many
mutations in the cells, leading to the death of cancer cells.
Unfortunately, other actively dividing cells, such as those in the skin and the lining of the digestive tract, also incorporate 5BU, which
leads to unwanted side effects of chemotherapy, such as hair loss and a diminished appetite.
 Another Base Analog is

2- aminopurine- an
analog of Adenine
In Stable state pairs with
Thymine
In Rare state pairs with
Cytosine Induces AT-GC
transition
 BASE MODIFYING AGENTS
Chemicals that modify the chemical
structure and properties of bases-
• Deaminating agents e.g nitrous acid
• Alkylating agents- Ethyl methane sulfonate
(EMS) or MMS
• Hydroxylating agent- adds OH gp. to cytosine
• Intercalating agents
1. Deaminating agents e.g nitrous acid –
removes amino gp. from the bases
 HNO2
Nitrous acid

Converts cytosine to uracil

CG-to-AT transition

Uracil hydrogen bonds with A not G

HNO2
Nitrous acid

AT-to-GC transition

Similarly deamination of adenine creates the base

hypoxanthine, which will base-pair with cytosine.
(2) Alkylating agents- Ethyl methane sulfonate (EMS) or
MMS- introduces alkyl gp. onto the bases.

During alkylation, methyl or ethyl groups are covalently attached to the bases. Examples of alkylating agents include
nitrogen mustard (a type of mustard gas) which was used as a chemical weapon during World War I. Such agents
damage the skin, eyes, mucous membranes, lungs etc.
3. Hydroxylating agent- adds OH gp. to cytosine
Which converts cytosine to Hydroxyl amine- base pair with Adenine
Instead of Guanine

Causes CG-to-AT transition

 4. Intercalating agents
Acridines, ethidium bromide, proflavin are
positively charged molecules mimics base
• May be slipped (inserted) themselves in
(intercalate) between the nitrogen bases.
• Point deletion or insertions lead to frame shift
mutaions.
 5’ATCAGTTACT 3’
• If the intercalating 3’TAGTCAATGA 5’
agent inserts itself 10 bp
between adjacent
base pairs of the DNA
template strand. 10 bp
• An extra base
(chosen at random;
G in the fig.) is
inserted into the new
DNA strand opposite
the intercalating
agent.
• After one more round
of replication, during 11 bp
which the intercalating
agent is lost, the
overall result is a
base-pair addition
mutation. (C–G is Result: Frame shift mutation due to
added)
insertion of one base pair
 5’ATCAGTTAC
3’TAGTCAATG
• If the intercalating 10 bp
agent inserts itself
into the new
growing DNA
strand in place of a Instead of A, intercalating
agent has been inserted
base.
• When that DNA
double helix
replicates after the
intercalating agent is 9 bp
lost, the result is a
base-pair deletion
mutation. (T–A is
Lost).
Result: The repair system removes T
after the lost of the intercalating agent,
the result is a base-pair deletion.
• May be slipped (inserted) themselves in
(intercalate) between the nitrogen bases. An
extra base is inserted into the new DNA strand
opposite the intercalating agent. After DNA
repair, it is removed and the overall result is a
base pair addition mutation.
• If the intercalating agent inserts itself into the
new DNA strand in place of a base, then when
that DNA replicates after the removal of
intercalating agent, the result is a base-pair
deletion.
 PHYSICAL AGENTS

• Radiation of short wavelength and high energy,

known as ionizing radiation, can alter DNA
structure.
• Includes X-rays and gamma rays. Ionizing radiation
can penetrate deeply into the cells, where it
produces chemically reactive molecules known as
free radicals.
Ionizing radiations Penetrate deeply into the cells-
produces chemically reactive molecules called free
radicals.

•Exposure to high doses of Ionizing radiations

•Base deletions

•Oxidized bases
•Single nicks in DNA strands
•Cross-linking, and even chromosomal
breaks.
X-rays are one of the most
common types of ionizing
radiation.
At the molecular level, the lethal
dose of X-ray (350-500 rems)
breaks the phosphodiester
bonds between the DNA and
thus results in strand
breakages.
• For his pioneering work in this area in the
1930s, Hermann Joseph Muller received the
1946 Nobel Prize in Physiology or Medicine for
“the discovery of the production of mutations
by means of X-ray irradiation.
• The X-ray is a form of ionizing radiation that
has been used to induce mutations in
laboratory experiments.
• Ionizing radiation is the leading cause of gross
chromosomal mutations in humans. High
dosages of ionizing radiation kill cells—hence
their use in treating some forms of cancer.
• Nonionizing radiation- UV light
• contains less energy, and so it penetrates only the
surface of an organism, such as the skin.
• UV light is known to cause DNA mutations.
• UV light causes mutations by increasing the
chemical energy of certain molecules, such as
pyrimidines, in DNA.
• UV light causes the formation of crosslinked
thymine dimers. Thymine dimers interfere with
transcription and DNA replication. This can result in
a mutation when that DNA strand is replicated.
 PHYSICAL AGENTS e.g U.V Radiations

UV light causes the formation

of crosslinked thymine dimers.
Thymine dimers interfere with
transcription and DNA
replication.
This can result in a mutation when
that DNA strand is replicated.
• Plants, in particular, must have effective ways of
preventing UV damage because they are exposed to
sunlight throughout the day.
• Also, suntanning greatly increases a person’s
exposure to UV light, raising the potential for
thymine dimers and mutation. This explains the
higher incidence of skin cancer among people who
have been exposed to large amounts of sun during
their lifetime.
• Most sunscreens contain organic compounds, such
as oxybenzone, that absorb UV light or opaque
ingredients, such as zinc oxide, that reflect UV light.
 DNA Repair mechanisms
Two types-
• Direct reversal repair system- act directly on damaged
nucleotides, converting each one back to its original structure.
• Mismatch repair
• Photoreactivation
• Repair of alkylation damage
• Excision repair system- This involves excision of a segment of
the polynucleotide containing a damaged site, followed by resynthesis of
the correct nucleotide sequence by a DNA polymerase. The segment
removed may be just one nucleotide in length or much longer.
• Base excision repair
• Nucleotide excision repair
• Post replicative repair
• SOS repair
• Mismatch repair by DNA Polymerase proofreading-
inserts incorrect nucleotides at a frequency of 10-5.
 • Light repair/ Photoreactivation
Photolyase recognize
and bind to thymine
dimer, catalyzes cleavage
of covalent bond between
the dimers.
Strains with mutations in phr
gene are defective in light
repair.

Because plants are exposed

to sunlight throughout the
day, photolyase is a critical
DNA repair enzyme for
many plant species.
 Repair of alkylation damage
• A protein known as
Alkyltransferase can remove
methyl or ethyl groups from
guanine bases that have been
mutagenized by alkylating agents
such as nitrogen mustard and
EMS.
• Called Alkyltransferase because
it transfers the methyl or ethyl
group from the base to a cysteine
side chain within the
alkyltransferase protein.
Surprisingly, this permanently
inactivates alkyltransferase, which
means it can be used only once!
e.g O6 methyl guanine transferase encoded by ada gene in
[Link]
 Conclusion
• Repair of some DNA modifications simply
reverses the damage, returning DNA directly
to its original state.
• For instance, photolyase, using near UV-visible
light, splits UV-induced pyrimidine dimers.
• Another example is the suicidal Ada protein
of E. coli, which transfers a methyl group from
the modified baseO6-methylguanine to itself.
2. Excision repair system- Repair of other types of
lesions requires removal of a segment of the DNA
strand around the lesion/damage. The
double-strandedness of DNA provides the means for
repairing the resulting single-strand gaps: the
removed bases can be resynthesized by using the
intact complementary strand as a template.
cut out damaged area, then repair the gap
• Base excision repair
• Nucleotide excision repair

• Post replicative repair

• SOS repair
 DARK REPAIR OR EXCISION REPAIR
repair of modified bases that do not cause DNA
distortion. Mutations caused by depurination or
deamination are examples of damage that may be
repaired by base excision repair.

• An enzyme called DNA glycosylase

removes a modified base to produce
an abasic site.
• Endonuclease then cleave the now
baseless sugar–phosphate backbone
and leaving a gap in the DNA chain.
• The gap is filled with the correct
nucleotide by a repair DNA polymerase
and
• sealing of the nick by DNA ligase.
• Damaged single bases or nucleotides are most
commonly repaired by removing the base or the
nucleotide involved and then inserting the correct
base or nucleotide.
• In base excision repair, a repair glycosylase enzyme
removes the damaged base from the DNA by cleaving
the bond between the base and the deoxyribose
sugar.
• Endonuclease then cleave the now baseless
sugar–phosphate backbone and leaving a gap in the
DNA chain.
• The gap is filled with the correct nucleotide by a repair
DNA polymerase and DNA ligase, with the opposite
DNA strand used as the template.
Base excision repair
2. Nucleotide excision Search for any distortion

repair- able to deal with more

extreme forms of damage such as
intrastrand crosslinks, multiple T-T
dimers
and many bases that have become
chemically modified.

Uvr ABC
Endonucleases-
UvrAB- recognition of site
Uvr BC dimer-excision 12 nt
Uvr D- helicase that unwinds long cut

the region between the cuts

and release the short DNA
segment
• A protein complex consisting of two UvrA
molecules and one UvrB molecule tracks along the
DNA in search of damaged DNA.
• Such DNA has a distorted double helix, which is
sensed by the UvrA/UvrB complex.

• When a damaged segment is identified, the two

UvrA proteins are released, and UvrC binds to the
site.

• The UvrC protein makes cuts in the damaged

strand on both sides of the damaged site.
• Typically, it cleaves phosphodiester bond 4
nucleotides downstream of the dna damage and 8
nucleotides upstrwam of the dna damage.
• After this process, UvrD, which is a helicase,
recognizes the region and separates the
twostrands of DNA. This releases a short DNA
segment that contains the damaged region, and
UvrB and UvrC are also released.
• Following the excision of the damaged DNA, DNA
polymerase fills in the gap, using the undamaged
strand as a template.
• Finally, DNA ligase makes the final covalent
connection between the newly made DNA and the
original DNA strand.
• This type of system can repair many different types
of DNA damage, including thymine dimers,
chemically modified bases, missing bases, and
certain types of crosslinks.
• In NER, several nucleotides in the damaged strand
are removed from the DNA, and the intact strand is
used as a template for resynthesis of a normal
complementary strand.
 Methyl directed mismatch repair
• Direct, base excision, and nucleotide excision
repair—recognize and act upon DNA damage
caused by mutagens. This means that they
search for abnormal chemical structures
such as modified nucleotides, dimers, and
intrastrand crosslinks.
• They cannot, however, correct mismatches
resulting from errors in replication, because
the mismatched nucleotide is not abnormal
in any way. It is simply an A, C, G, or T that
has been inserted at the wrong position.
• Mismatched base pairs which remain
uncorrected by proofreading (failed) are
corrected by methyl directed mismatch repair
system after replication.
• When the mismatch is due to an error in DNA
replication, the newly made daughter strand
contains the incorrect base, whereas the
parental strand is normal. As these
nucleotides look exactly like any other
nucleotide, the mismatch repair system that
corrects replication errors has to detect not
the mismatched nucleotide itself but also has
to find out the new daughter strand.
• There is one difficulty. The repair must be
made in the daughter polynucleotide, because
it is in this newly synthesized strand that the
error has occurred.
• The parent polynucleotide, on the other hand,
has the correct sequence.
• How does the repair process know which
strand is which?
• In E. coli the answer is that the parent strand
is methylated and so can be distinguished
from the newly synthesized polynucleotide,
which is not methylated.
The molecular mechanism
of mismatch repair
• Three proteins, designated MutS,
MutL, and MutH, detect the
mismatch and direct the removal of
the mismatched base from the newly
made strand.
• These proteins are named Mut
because their absence leads to a
much higher mutation rate than
occurs in normal strains of E. coli.
The role of MutS is to locate
mismatches.
• Once a mismatch is detected, MutS
forms a complex with MutL. MutL
acts as a linker that binds to MutH by
a looping mechanism.
• This stimulates MutH, which is
bound to a hemimethylated
site, to make a cut in the
newly made, nonmethylated
DNA strand.
• After the strand is cut, MutU,
which functions as helicase,
separates the strands, and an
exonuclease then digests the
non methylated DNA strand in
the direction of the mismatch
and proceeds beyond the
mismatch site. This leaves a
gap in the daughter strand
that is repaired by DNA
polymerase and DNA ligase.
 New strand, T has to be changed, not G
• The mismatch
correction enzyme
recognizes which strand Old strand
the base mismatch is
on by reading the
methylation state of a
nearby GATC
sequence.
• If the strand is
unmethylated- that
means it is new strand
as old strand is always
methylated) a segment
of that DNA strand
containing the
mismatch is excised
and new DNA is
inserted.
POST REPLICATIVE REPAIR

Rec A mediates
pairing with the
homologous
segment of
DNA from
parent DNA.
when the lesion is in a single strand gap,
there is no complementary strand to
repair against;

Rec A proteins binds to the single

strand at the gap and mediate
pairing with the homologous
segment of DNA on other
daughter strand.
Gap on the other strand is filled
by DNA polymerase I.
 SOS repair
• In extensive DNA damage, the
cell has to make choice
between dying or attempting
to replicate the damaged
region even though this
replication may be error-prone
and result in mutated
daughter molecules.
• DNA polymerase V is a
specialized enzyme that can
replicate DNA on damaged
DNA templates.
• The SOS response is primarily
looked on as the last best
chance that the bacterium has
 SOS repair
• Activation of Pol V requires RecA
protein.
• RecA is a single-stranded
DNA-binding protein that coats
the damaged strands, enabling
DNA polymerase V to carry out
its own, error-prone DNA
synthesis until the damaged
region has been passed.
• It selects nucleotides more or
less random and carry out error
prone DNA synthesis.
• The main replicating enzyme,
DNA polymerase III, then takes
over once again.
• The process involves a special class of DNA
polymerases that are synthesized only in response
to DNA damage.
• In E. coli, such DNA damage activates a complex
system called the SOS response. (The system is called
“SOS” because it is induced as a last-resort,
emergency response to mutational damage.) The
SOS response allows the cell to survive otherwise
lethal events, although often at the expense of
generating new mutations.
• In E. coli, Upon sufficient damage to DNA, the
recA-encoded protein,RecA, is activated.
• Activated RecA stimulates the LexA protein to
cleave itself, which in turn relieves the
repression of the DNA repair genes.
• As a result, the DNA repair genes are
expressed, and DNA repair proceeds.
• After the DNA damage is dealt with, RecA is
inactivated, and newly synthesized LexA
protein again represses the DNA repair genes.
 Molecular
Biology
recA-dependent bacterial homologous recombination

1. Homologous DNA pairs

2. Nicks made near Chi

(GCTGGTGG) sites by 3’
5’
a nuclease. 3’ 5’
5’ 3’
3. ssDNA carrying the 5’ 3’ 5’
ends of the nicks is
coated by RecA to form
RecA-ssDNA dilaments.
 Molecular
Biology

4. RecA-ssDNA filaments search

the opposite DNA duplex for
corresponding sequence
(invasion).

5. form a four-branched
Holliday structure

6. Branch migration
 Molecular
Biology
Recombination based DNA
repair at replication fork, also
called post-replication repair
a. Replication encounter a DNA
lesion
b. Skip the lesion & re-initiate on
the other side of the lesion
c. Fill the daughter strand gap by
replacing it with the
corresponding section from the
parental sister strand by
recombination
d. The original lesion can be
removed later by normal
excision repair.
Why doesn’t DNA
contain any Uracil?
 Deamination of cytosine gives uracil
So if uracil was normally present in
DNA the repair system will NOT be able
to recognize the normally present
uracil from the abnormally present
uracil and therefore won’t be able to
repair the damage caused.