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Gene Mutations

• A gene mutation occurs when the sequence of the DNA within a

gene is altered in a permanent way
• A gene mutation can involve a base substitution or a removal or addition
of one or more base pairs in a gene sequence
• A point mutation is a change in a single base pair within the DNA
• For example, the DNA sequence shown here has been altered by a base
substitution, in which one base is substituted for another.

Gene Mutations
• Transition change of a pyrimidine to another pyrimidine, such as C to
T, or a purine to another purine, such as A to G
• More common
• Transversion, purines and pyrimidine are interchanged
• Besides base substitutions, a short sequence of DNA may be deleted
from or added to the chromosomal DNA:
• Deletion of 4 bp


Gene Mutations
• Addition of 4 bp


Gene Mutations Can Alter Coding Sequence
Within a Gene

• Silent mutations are those that do not alter amino acid sequence of
the polypeptide even though nucleotide sequence has changed
• Genetic code is degenerate, silent mutations can occur in certain
bases within a codon, such as the third base, so the specific amino
acid is not changed
• In contrast, missense mutations are base substitutions in which an
amino acid change does occur.
• sickle cell anemia
• a mutation in the β-globin gene, alters sixth amino acid is changed
from a glutamic acid to valine
• Single amino acid substitution alters the structure and function of the
hemoglobin protein
• As a result red blood cells assume a sickle shape under conditions of
low oxygen
• Nonsense mutations involves change from a normal codon to a stop codon
• It terminates translation of polypeptide earlier than expected
• Producing a truncated polypeptide
• A nonsense mutation occurs in a bacterial operon, it may also inhibit the
expression of downstream genes, termed polarity
• Frameshift mutations involve addition or deletion of a number of
nucleotides that is not divisible by three
• Codons are read in multiples of three, this shifts the reading frame
• Translation of mRNA results in a completely different amino acid sequence
downstream from mutation
• New mutations are more likely to produce polypeptides that have
reduced rather than enhanced function
• Nonsense mutations produce polypeptides that are substantially
• Shorter polypeptides are unlikely to function properly
• Frameshift mutations dramatically alter the amino acid sequence of
polypeptides and are thereby likely to disrupt function
• Missense mutations are less likely to alter function because they
involve a change of a single amino acid within polypeptides that
typically contain hundreds of amino acids.
• Neutral mutation If a missense mutation has no detectable
effect on protein function
• A missense mutation that substitutes an amino acid with a
similar chemistry as the original amino acid is likely to be neutral or
nearly neutral
• For example, a missense mutation that substitutes a glutamic acid for
an aspartic acid is likely to be neutral
• As both amino acids are negatively charged and have similar side
chain structures
• Silent mutations are also considered a type of neutral mutation
Point mutations can alter mRNA splicing
• Single-base-pair changes which inactivate proteins are due to splice
site mutations
• It will dramatically change RNA transcript by leading to large
insertions or deletions
• Neutral mutation does not alter protein function
• Does not affect survival or reproductive success.
• Deleterious mutation, decreases chances of survival and
• Extreme example of a deleterious mutation is a lethal mutation
• Results in death to a cell or organism
• Beneficial mutation enhances the survival or reproductive success
• An allele may be either deleterious or beneficial depending on
genotype and/or environmental conditions (sickle cell allele)
• In homozygous state, sickle cell allele lessens chances of survival
• In heterozygous (sickle cell allele and wild-type allele ) has an
increased chance of survival due to malarial resistance
Molecular Basis of Spontaneous Mutations
• Gene mutations can arise spontaneously or they can be induced
• Spontaneous mutations are naturally occurring mutations
• Induced mutations arise through the action of mutagens
• They increase rate at which mutations occur
• Salvadore Luria and Max Delbrück were interested in the
ability of bacteria to become resistant to infection by a bacteriophage
called T1.
• When a population of E. coli cells is exposed to T1, a small percentage of
bacteria are found to be resistant to T1 infection and pass this trait to their
• Luria and Delbrück were interested in whether such resistance, called tonr
(T one resistance), is due to the occurrence of random mutations
or whether it is a physiological adaptation that occurs at a low rate within
the bacterial population
• Do spontaneous mutations occur in response to a selecting agent or
are variants present at a low frequency in most populations?
• Ideal experimental system to address this important question was
analysis of mutations in bacteria
• Luria and Delbrück fluctuation test
• if E. coli bacteria are spread on a plate of nutrient medium in the
presence of phage T1, phages soon infect and kill bacteria
• Rarely, but regularly, colonies were seen that were resistant to phage
• These colonies were stable and so appeared to be genuine mutants
• Whether these mutants were produced
A- Spontaneously
B-Phage induced a physiological change that caused resistance
• If mutations occurred spontaneously, then mutations may occur at
different times in different cultures (Lauria)
• So numbers of resistant colonies per culture should show high
variation (fluctuation)
Fluctuation test
• 20 small cultures were inoculated, each with a few cells, and
incubated till 108 cells per millilitre
• At same time, a much larger culture also was inoculated and
incubated until 108 cells per millilitre
• 20 individual cultures and 20 samples of same size from large culture
were plated in presence of phage
• 20 individual cultures showed high variation in number of
resistant colonies: 11 plates had 0 resistant colonies, and remainder
had 1, 1,3, 5, 5, 6, 35, 64, and 107 per plate
• 20 samples from large culture showed much less variation from plate to
plate, all in the range of 14 to 26
• If phages were inducing mutations, there was no reason why fluctuation
should be higher on the individual cultures because all were exposed to
phage similarly
• Best explanation was that mutation was occurring randomly in time
• Early mutations gave the higher numbers of resistant cells because the
mutant cells had time to produce many resistant descendants
• Later mutations produced fewer resistant cells
• It led to the reigning “paradigm” of mutation
• “ Whether in viruses, bacteria, or eukaryotes, mutations can occur in
any cell at any time and their occurrence is random”
• It was suggested that resistant cells are selected by the environmental
agent (phage) rather than produced by it
• Luria and Delbrück were awarded the Nobel Prize in Physiology or
Medicine in 1969
• Interestingly, this was after Luria’s first graduate student, James
Watson, won his Nobel Prize (with Francis Crick in 1964) for discovery
of the DNA double-helix structure

Mutation is a random process. Any allele in

any cell may mutate at any time
Mechanisms of spontaneous mutations
1-DNA replication process:
• Spontaneous mutations arise from a variety of sources
• 1- DNA replication process: remarkably accurate process, mistakes
are made in copying of millions, billions, of bp
• Spontaneous mutations also arise in part because DNA is a very labile
molecule….Cellular environment itself can damage it
• Errors in DNA replication
• Error in DNA replication due to illegitimate nucleotide pair ( A–C)
formed in DNA synthesis, leading to base substitution (transition/
• INDEL results in a frameshift mutation
• Errors are corrected by proofreading of DNA pol III
• otherwise all mismatches lead to transition mutations
• Repair systems correct many of mismatched bases that escape
correction by polymerase
• Creation of a transversion by a replication error require, mispairing of a
purine with a purine or a pyrimidine with a pyrimidine.
• Dimensions of DNA double helix do not favours such mismatches
• X-ray diffraction studies shows G–A pairs, purine–purine pairs,
2- Spontaneous lesions (SL)
• Naturally occurring damage to DNA create mutations
• SL result from depurination and deamination
• Depurination, loss of a purine base
• Interruption of glycosidic bond between base and deoxyribose
• Subsequent loss of a guanine or an adenine residue from DNA
• DNA backbone(S+P) remains intact
2-Spontaneous lesions (SL)
• apurinic sites cannot specify a base complementary to
original purine
• Mammalian cells loses about 10,000 purines 20-hour cell-
cycle 37°C
• If lesions were persist, they result in significant genetic
• A base can be inserted across from an apurinic site and result
in a mutation
• Efficient repair systems remove apurinic sites
• Deamination of cytosine yields uracil
• Unrepaired uracil will pair with adenine in replication
• Resulting in conversion of a G • C pair into an A • T pair
• (a G • C → A • T transition).
Molecular Basis of Induced Mutations
• Sources of mutation are present in environment
• Whether intentionally applied in laboratory or accidentally
• Production of mutations in laboratory through exposure to mutagens
is called mutagenesis, and organism is said to be mutagenized
Mechanisms of mutagenesis
• Mutagens induce mutations by at least three different mechanisms
• 1- Replace a base in DNA
• 2- Alter a base that mispairs with another base
• 3- Damage a base so that it can no longer pair with any base under
normal conditions

• Mutagenizing genes and observation of phenotypic consequences is

primary experimental strategies used by geneticists
1- Incorporation of base analogs
• Some chemical compounds are sufficiently similar to normal nitrogen
bases of DNA called base analogs
• So they are incorporated into DNA in place of normal bases
• These analogs have pairing properties unlike those of normal bases
• So, they can produce mutations by causing incorrect nucleotides
insertion opposite to them in replication
• Base analog exists in a single strand, but they can cause a nucleotide-
pair substitution that is replicated in all DNA copies descended from
original strand
Base analog (2-aminopurine ….2-AP)
• Widely used in research is 2-AP
• Analog of adenine
• Pairs with Thymine but can also mispair with Cytosine when
• When 2-AP is incorporated into DNA by pairing with thymine
• It can generate A • T → G • C transitions by mispairing with cytosine
in subsequent replications
• Or, if 2-AP is incorporated by mispairing with cytosine, then G • C → A
• T transitions will result
• Genetic studies have shown that 2-AP causes transitions exclusively
2- Specific mispairing
• Some mutagens are not incorporated into DNA but instead alter a
base in such a way that it will form a specific mispair
• Certain alkylating agents, such as ethylmethanesulfonate (EMS) and
widely used nitrosoguanidine (NG)
• Such agents add alkyl groups (an ethyl group in EMS and a methyl
group in NG) to many positions on all four bases
• addition of oxygen at position 6 of guanine, create an O-6-
• This addition leads to direct mispairing with thymine
• would result in G • C → A • T transitions at next round of replication
DNA Repair
• Because most mutations are deleterious, DNA repair systems are vital for
survival of all organisms
• Spontaneous from sources inside the cell (replication, reactive oxygen) and
environmentally induced mutations from sources outside cell
(environmental: UV light, ionizing radiation, mutagens)
• If DNA repair systems did not exist, mutations would be so prevalent that
few species would survive
• Necessity of DNA repair systems becomes evident when they are missing
• Bacteria contain several different DNA repair system
• In the absence of any single system, the bacteria have a much higher
rate of mutation
• Due high rate of mutations bacterial strains are called mutator strains
• Living cells contain several DNA repair systems
• They can fix different types of DNA alterations
• Each repair system is composed of one or more proteins that play
specific roles in the repair mechanism
• In most cases, DNA repair is a multistep process
• First, one or more proteins in the DNA repair system detect an
irregularity in DNA structure
• Next, the abnormality is removed by the action of DNA repair
• Finally, normal DNA is synthesized via DNA replication enzymes
Common Types of DNA Repair Systems
Direct repair. An enzyme recognizes an incorrect
alteration in DNA structure and directly
converts it back to a correct structure
recognized and removed fromDNA
Base excision repair and nucleotide excision An abnormal base or nucleotide is first
repair recognized and removed from the DNA. A
segment of DNA is excised, and then the
complementary DNA strand is used as a
template to synthesize normal DNA.
Mismatch repair Similar to excision repair except the DNA
defect is a base pair mismatch, not an
abnormal nucleotide. The mismatch is
recognized, and a segment of DNA is
removed. Parental strand is used to
synthesize a normal daughter strand of
Homologous recombination Occurs at double-strand breaks or when DNA damage
repair causes a gap in synthesis during DNA replication. The
strands of a normal chromatid are used to repair a
damaged chromatid.

Nonhomologous end joining Occurs at double-strand breaks. The broken

ends are recognized by proteins that keep the
ends together; the broken ends are eventually
1-Direct repair
• Covalent modification of
nucleotides by mutagens
can be reversed by specific
cellular enzymes
• UV light causes the
formation of thymine
• Bacteria, fungi, most plants,
and some animals produce
photolyase that recognizes
thymine dimers and splits
1-Direct repair
• The repair mechanism itself requires light and is known as
• This process directly restores the structure of DNA.
• Because plants are exposed to sunlight throughout the day,
photolyase is a critical DNA repair enzyme for many plant species
• alkyltransferase can remove
methyl or ethyl groups from
guanine bases that have been
mutagenized by nitrogen
mustard and EMS
• 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!
2- Base Excision Repair
• (BER), involves the function DNA N-glycosylases.
• Recognize an abnormal base and cleave the bond between it and
the sugar in the DNA backbone, Creating an apurinic or apyrimidinic
• Living organisms produce multiple types of DNA N-glycosylases,
• Each recognizing particular types of abnormal base structures
• Depending on the DNA N-glycosylase, this repair system can
eliminate abnormal bases such as uracil, 3-methyladenine, 7-
methylguanine, and pyrimidine dimers
• BER repairs oxidative DNA damage
2-Base Excision Repair
• General steps involved in DNA repair via N-glycosylase.
• DNA contains a uracil in its sequence.
• This could have happened spontaneously or by the action of a
chemical mutagen. N-glycosylase recognizes a uracil within the DNA
and cleaves (nicks) the bond between the sugar and base.
• This releases the uracil base and leaves behind an apyrimidinic site.
• This abnormality is recognized by a second enzyme, AP
endonuclease, which makes a cut on the 5ʹ side.
2-Base Excision Repair
• Following this cut by AP endonuclease, one of three things
can happen.
• In some species such as E. coli, DNA polymerase I, which has a 5ʹ to 3ʹ
exonuclease activity, removes a DNA segment containing the
abnormal region, replaces it with normal nucleotides.
• This process is called nick translation (DNA replication, not mRNA
translation, actually occurs).
2- Base Excision Repair
• In humans, the DNA is repaired in two possible ways.
• 1-DNA polymerase β has the enzymatic ability to remove a site, which
is missing a base, Insert a nucleotide with the correct base
• 2- DNA polymerase б or ε can synthesize a short segment of DNA,
which generates a flap
• The flap is then removed by flap endonuclease
• Final step is carried out by DNA ligase
• It closes a gap in the DNA backbone
3-Nucleotide Excision Repair (NER)
• This system can repair many different types of DNA damage
• 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.
• NER is found in all eukaryotes and prokaryotes
3-Nucleotide Excision Repair (NER)
• In E. coli, the NER system requires four key proteins, designated UvrA,
UvrB, UvrC, and UvrD, besidesDNA polymerase and DNA ligase
• UvrA, B, C, and D recognize and remove a short segment of a
damaged DNA strand.
• They are named Uvr because they are involved in Ultraviolet light
repair of pyrimidine dimers
• UvrA–D proteins are also important in repairing chemically damaged
Step -1

A protein complex consisting of 2 UvrA + 1 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.
Step -2

When a damaged segment is identified, the two UvrA

proteins are released, and UvrC binds to the site.
Step - 3

8 bp 4-5 bp

UvrC protein makes cuts in the damaged strand on

both sides of the damaged site.
Damaged strand is cut eight nucleotides from the 5ʹ end of the
damage and 4-5 nucleotides away from the 3ʹ end.
Step - 4

UvrD, which is a helicase, recognizes the region and separates

the two strands of DNA. This releases a short DNA segment that
contains the damaged region, and UvrB and UvrC are also released.
Step - 5

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
4- Mismatch Repair Systems
• Structure of the DNA double helix obeys the AT/GC rule of base pairing.
• During the normal course of DNA replication, however, an incorrect
nucleotide may be added to the growing strand by mistake.
• This produces a mismatch between a nucleotide in the parental and the
newly made strand
• If this proofreading ability fails, cells contain additional DNA repair systems
that can detect base mismatches and fix them.
• An interesting DNA repair system that exists in all species is the mismatch
repair system.
4- Mismatch Repair Systems
• Hemimethylation provides a way for a DNA repair system to
distinguish between the parental DNA strand and the daughter
• Molecular mechanism of mismatch repair has been studied
extensively in E. coli.
• Three proteins, designated MutS, MutL, and MutH, detect the
mismatch in newly made strand
• These proteins are named Mut because their absence leads to a
much higher mutation rate
4- Mismatch Repair Systems
• MutS is to locate mismatches
• MutL acts as a linker that binds to MutH
• MutH makes a cut
• 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.

4- Mismatch Repair Systems
• MutU, functioning as helicase, separates the strands
• 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.
• Mutations in two human mismatch repair genes, hMSH2 and hMLH1,
play a role in the development of a type of colon cancer known as
hereditary nonpolyposis colorectal cancer
Step 1 -MutS protein finds a mismatch. The MutS/MutL complex binds to MutH, which is
already bound to a hemimethylated sequence.
Step 2 MutH makes a cut in the nonmethylated strand. MutU separates the DNA
strands at the cleavage site and an exonuclease digests the nonmethylated strand just
beyond the base mismatch.
Step 3- DNA polymerase fills in the vacant region. DNA ligase seals the ends.
5-Homologous Recombination Repair and
Nonhomologous End Joining
• Breakage of chromosomes—called a DNA double-strand
break (DSB)—is perhaps the most dangerous
• DSBs can be caused by ionizing radiation (X-rays or gamma
rays), chemical mutagens, and certain drugs used for
• Reactive oxygen species (ROS), by-products of aerobic
metabolism can cause double-strand breaks
• Naturally occurring double-strand breaks in a human cell
occur at a rate of between 10 and 100 breaks per cell per day
5-Homologous Recombination Repair and
Nonhomologous End Joining
• DSBs can result in chromosomal rearrangements such as inversions
and translocations
• DSBs can lead to terminal or interstitial deficiencies
• Such genetic changes have the potential to result in detrimental
phenotypic effects
• How are DSBs repaired?
• 1- Homologous Recombination Repair (HRR)
2- Non-Homologous End Joining (NHEJ).
5.1 - Homologous recombination repair
• Also called homology-directed repair, occurs when homologous DNA
• A sister chromatid, are used to repair a DSB in the other sister
• 1-DSB is processed by the short digestion of DNA strands at the
break site
• 2-This processing event is followed by the exchange of DNA strands
between the broken and unbroken sister chromatids.
• 3- The unbroken strands are then used as templates to synthesize
DNA in the region where the break occurred
5.1 - Homologous recombination repair
• 4- Finally, the crisscrossed strands are resolved, which means they are
broken and then rejoined in a way that produces separate
• Advantage
• Because sister chromatids are genetically identical, an advantage is
that homologous recombination repair can be an error-free
mechanism for repairing a DSB
• Disadvantage is that sister chromatids are available only during the S
and G2 phases of the cell cycle in eukaryotes or following DNA
replication in bacteria.