Mutation

Mutation
• A stable and heritable change in a DNA sequence

• Create a phenotype different from that normally

expressed by the wildtype allele of that gene.
– Base substitutions,
– Duplications,
– Deletions
– Chromosomal aberrations (invarsions,
translocations)
Classification of Mutations in Genetics
Dominant and Recessive
Wildtype = +, Mutation =m1(recessive) or M2
(dominant)
1. +/+ gives wildtype phenotype
2. +/m1 gives wildtype phenotype
3. M2/+ gives Mutant phenotype
Semidominant
• a dominant mutation displays a more severe (or
extreme) phenotype as a homozygote than
heterozygote.
– M3/M3> M3/+>+/+
 Muller's Classification of Mutants
• H. J. Muller, 1932
• Based on comparing the phenotypic effects of
a mutation in homo-, hetero-, and hemizygotes.
1. Nullomorphs,
2. Hypomorphs,
3. Hypermorphs,
4. Antimorphs, and
5. Neomorphs
 Nullomorphs
• Mutants with no remaining gene function→no functional
gene product, equivalent of a full deletion of the gene in
terms of its influence on the final phenotype
• Null allele
• Defects in gene expression
– Transcriptional nulls
– Translational nulls
– Inactivating nulls

• Most nullomorphic mutants are recessive, i.e. enzymes

• Mutations in genes that code for structural or highly
concentration dependant gene->dominant
 Hypomorphs
• Hypomorphs are mutants that produce some
degree of residual activity, but not enough to
provide wildtype activity in mlm homozygotes.

• Weak allele

• Most hypomorphic mutants are recessive

• Give enough product to allow survival, but not

enough to produce a normal phenotype.
 Hypermorphs
• Hypermorphs produce either a hyperactive protein
product or a harmful excess of the normal protein
product
• mlm > m/+ > m/Df.
• Hypermorph can be valuable tool for dissecting a
genetic process
• Hypermorph can be valuable tool to identify
specific functions of a group of functionally
redundant genes
 Antimorphs
• An antimorphic mutation results in a protein
product that antagonizes, or poisons, the wildtype
protein
• Anti·morphic mutations are dominant
• Increasing the dose of the wildtype allele can often
ameliorate the phenotype of an antimorphic
mutant.
• One should be able to revert ( or more precisely
~pseudo-revert") an antimorphic mutant to a
nullomorphic mutation of the same gene
• A/A > A/Df> A/+ >>> +/Df > +/+
 Neomorphs
• A mutation that causes a gene to be active in an
abnormal time or place.
• Burkitt's lymphoma
• A dominant mutation (Antp73b) in Drosophila that
causes the antennae to be replaced by legs
 Modern mutant terminology
• Mutants that reduce the level of gene product are
often classified only as loss-of-function mutants
– Null mutants

– Partial loss-of-function mutants (amount or

functon)

– Temperature-sensitive (or other conditional)

loss-of-function mutants
 Modern mutant terminology
• Classification of Dominant Mutants
– Dominant Negative (Poisonous product)

– Gain-of-Function mutant (Inappropriately

regulated)

– Heterochronic Mutant (Wrong time)

 DNA-level terminology
1. Base pair substitution mutants

2. Missense mutants

3. Nonsense mutants

4. Silent substitutions

5. Base pair inserions or deletions

6. Frameshift mutants
Base pair substitution mutants
 The Importance of Mutations

• Mutation is the source of all genetic variation

– raw material of evolution

• Adaptation to the changing environment

• Mutations serve as important tools of genetic
analysis
 Why looking for new mutants???

 To identify genes required for a specific biological

process
 To isolate more mutations in a specific gene of
interest
 To obtain mutation tools for structure-function
analysis
 To isolate mutations in a gene so far identified only
by molecular approaches
 Mutations in Multicellular organisms
Two broad categories 1) Somatic 2) Germ-line

 Somatic mutations: Time, Rate, Effect

 In single-cell organisms, however, there is no distinction between germ-
line and somatic mutations, because cell division results in new individuals
 Gene and chromosome mutation.
Types of Gene Mutations

The number of possible

transversions is twice the
number of possible
transitions, but transitions
usually arise more
frequently.
 Phenotypic effects of mutations
• Loss-of-function mutations
• Gain-of-function mutation
• Terms to know
– Wild-type phenotype
– Forward mutation, & Reverse mutation
– Neutral mutation
 Suppressor mutations
• Intragenic

• Compensatory changes in the protein

Intergenic Suppressor mutations
 Mutation rate
• The frequency with which a wild-type allele at a locus
changes into a mutant allele is referred to as the mutation
rate. The mutation rate provides information about how often
a mutation arises.

• Generally expressed as the number of mutations per

biological unit, which may be mutations per cell division, per
gamete, or per round of replication.

• Mutation frequency is defined as the incidence of a specific

type of mutation within a group of individual organisms.
 Mutation Rates
• Three factors affects mutation rates
– The frequency with which primary changes take
place in DNA
– The probability
– Our ability to calculate mutation rates
• Viral and Bacterial:1 to 100 mutations per 10 billion
cells (1X10-8 to 1X10-10)
• Eukaryotic genes 1 to 10 mutations per million
gametes (1X10-5 to 1X10-6)
– The mutation rate for achondroplasia (a type of hereditary dwarfism) is about 4
mutations per 100,000 gametes, usually expressed more simply as 4X10 5
– For achondroplasia, the mutation frequency in the United States is about 2X10 4,
which means that about 1 of every 20,000 persons in the U.S. population carries this
mutation
The higher values in eukaryotes may be due to the fact that the rates are calculated
per gamete, and several cell divisions are required to produce a gamete, whereas
mutation rates in prokaryotic cells and viruses are calculated per cell division.
 Causes of Mutations
• Spontaneous Replication Errors
– Tautomeric shifts
– Wobble formation
– Strand slippage
– Unequal crossing over

• Spontaneous Chemical Changes

– Depurination
– Deamination
Tautomeric shifts Mispairing
Wobble formation→ Incorporated error
The original incorporated error leads to a
replication error, which creates a
permanent mutation, because all the base
pairings are correct and there is no
mechanism for repair systems to detect the
error.
Strand slippage Unequal
crossing over
 Expanding trinucleotide repeats

Mutations in which copies of a trinucleotide may increase greatly in number are called
expanding trinucleotide repeats. Strand slippage in DNA replication and crossing
over between misaligned repeats are two possible sources of expansion.
 Depurination

Depurination is a common
cause of spontaneous
mutation; a mammalian Deamination
cell in culture loses
approximately 10,000
purines every day.
 Chemically Induced Mutations
• Mutagen Chemicals can produce mutations by a
number of mechanisms.
– Chemical
• Base analogs are inserted into DNA and
• Base analogs frequently pair with the wrong base.
• Hydroxylamine
• Alkylating agents, deaminating
• Alkylating agents chemicals, hydroxylamine, and oxidative
• Deamination radicals change the structure of DNA
bases, thereby altering their pairing
• Oxidative reactions properties.
• Intercalating agents • Intercalating agents wedge between the
• Radiation bases and cause single-base insertions
and deletions in replication.
Base analogs
Ethyl group
to T and G

Deaminates
C to U
& A to HX

Only Cytosine
EXTRA SLIDE
EXTRA SLIDE
Oxidative reactions
How natural processes can change the information stored in DNA
Radiation
How natural processes can change the information stored in DNA
 What Causes Mutation
 Spontaneous mutation

 If mutations occurred spontaneously, then the mutations

might be expected to occur at different times in different
cultures; so the resulting numbers of resistant colonies per
culture should show high variation (or “fluctuation”)
 The early mutations gave the higher numbers of resistant
cells because they had time to produce many resistant
descendants. The later mutations produced fewer resistant
cells
 Luria and Delbrück fluctuation test

Mutations Are Chance Occurrences Modifying the Genome at Random

 Detection of Spontaneous Mutations
• Luria and Delbrück fluctuation test and replica
platting
Detection of Spontaneous Mutations
 Ames Test TESTING FOR
MUTAGENICITY

• 1974, Bruce Ames

• Both cancer and mutations result from
damage to DNA

• Salmonella typhimurium
– Defects in the lipopolysaccharide
coat
– DNA repair system has been
inactivated
• One of the 4 strains used in the Ames
test detect base-pair substitutions; the
other three detect different types of
frameshift mutations
• Only bacteria that have undergone a
reverse mutation of the histidine gene
(his-:his+) are able to synthesize
histidine and grow on the medium.
 DNA Repair
 Cause of DNA Damage?????
Radiation, chemical mutagens, heat, enzymatic errors, and
spontaneous decay
 Rate of DNA damage Vs rate of DNA Repair
fewer than one in a thousand DNA lesions becomes a
mutation; all the others are corrected
 Lower rate of Mutation
is indicative of the efficiency of these repair systems
 The repair Pathway
chemically repairs the damage to the DNA base
is redundant (Many types of DNA damage can be corrected by
more than one pathway of repair)
deletes the damaged DNA and uses an existing
complementary sequence as a template to restore the
normal sequence
 DNA Repair
 Direct reversal of damaged DNA

 Homology-dependent repair systems

Excision-repair Pathways
Base excision Repair
Nucleotide excision repair

Mismatch-repair System (Post-replication)

 Repair of double-strand breaks

NONHOMOLOGOUS END-JOINING

HOMOLOGOUS RECOMBINATION
 Direct reversal of damaged DNA

ENZYMATIC REPAIR
Alkyltransferases

The methyltransferase from E. Coli

enzyme transfers the methyl group
from O-6-methylguanine to a
cysteine residue on the protein.
PHOTOREPAIR The transfer inactivates the enzyme,
so this repair system can be
saturated if the level of alkylation is
 Base excision repair

 Numerous DNA glycosylases exist.

• Each removes a specific type of modified base by
cleaving the bond that links that base to the 1-
carbon atom of deoxyribose.
 Uracil glycosylase recognizes and removes uracil
produced by the deamination of cytosine
 AP (apurinic or apyrimidinic) endonuclease
 Hypoxanthine, 3-methyladenine, 7-methylguanine
Homology-Dependent
Repair Systems
Base excision repair
 Base excision repair
 Bacteria use DNA polymerase I to replace excised nucleotides
 Eukaryotes generally use DNA polymerase β, which has no
proofreading ability and tends to make mistakes
 DNA polymerase β makes one mistake per 4000 nucleotides
inserted.
 About 20,000 to 40,000 base modifications per day are
repaired by base excision
 DNA polymerase β may introduce as many as 10 mutations
per day into the human genome
 How are these errors corrected?
 Homology-Dependent Repair Systems
Nucleotide excision repair

Distortions in the DNA backbone

• Pyrimidine dimers or large hydrocarbons
attached to the DNA, bulky DNA lesions which distorts
the DNA
• Found in cells of all organisms from bacteria to
humans and is one of the most important of all
repair mechanisms
• In yeast, approximately twelve genes of RAD3
groups are involved
• In human beings, twenty-five proteins are involved;
they remove twenty-seven to twenty-nine
nucleotides, as compared to twelve to thirteen in E.
 Homology-Dependent Repair Systems
(20 proteins)

Nucleotide excision repair

 DNA repair systems in eukaryotes are highly
conserved from yeast to humans

 Yeast repairsome recognize damaged DNA

 How can the repairosome “know” which

strand of a gene is transcribed and which is
not?

 Repair in non dividing cells.

 Transcription factor TFIIH is involved in
repair of UV damage; it has helicase
 activity and is found in both processes
Transcription-coupled repair in eukaryotes
 POSTREPLICATION REPAIR
 Replication errors failed to be
corrected by the 3-to-5
proofreading function of the
replicative polymerase
 Mismatch-repair system, can
detect such mismatches,
1. Recognize mismatched base pairs
2. Determine which base in the
mismatch is the incorrect one
3. Excise the incorrect base and carry
out repair synthesis
 DNA methylation in Bacteria
 Adenine Methylase,
methylates position 6 which
does not interefere with base
pairing activity.
 Delay in the methylation
 Mismatch-repair
System
Mismatch repair in E. Coli

Excision repair triggered by

mismatches is referred to as mismatch
repair, which encompasses about 99%
of all DNA repairs
The mismatch repair system, follows
behind the replicating fork
The genes are called mut
The mutU gene is also known as
uvrD.
 Mismatch-repair
System
Mismatch repair in human

• An important target of the

human mismatch repair system
is short repeat sequences that
can be expanded or deleted
during replication by the
slipped mispairing mechanism
 Repair of double-strand breaks
 Double-strand breaks can arise Nonhomologous End-joining
spontaneously (for example, in response to
reactive oxygen species), or they can be
induced by ionizing radiation.
 Two distinct mechanisms are used to repair
these potentially lethal lesions:
Nonhomologous end joining and
Homologous recombination
 Interestingly, the ability of double-strand
breaks to initiate chromosomal instability is
an integral feature of some normal cellular
processes that require DNA rearrangements.
Antibody gene
Homologous Recombination
 Repair of double-strand breaks

Homologous Recombination
 The mechanism of homologous recombination
utilizes the sister chromatid to repair double-strand
breaks.
 A double-strand break induces an enzyme to
chew back 5` ends, leaving 3` overhangs that are
coated with proteins, including RAD51, a RecA
homolog
 The coating of these regions with proteins that
include the RecA homolog, RAD51
 A segment of the sister chromatid (blue) is used
as a template to repair the break
 The RAD51–DNA filament then takes part in a
remarkable search of the undamaged sister
chromatid for the complementary sequence that
will be used as a template for DNA synthesis
Nature Reviews: Genetics 2, 2001, 196–206
 Faulty DNA Repair
 Xeroderma pigmentosum
 autosomal recessive trait
 an inability to repair thymine dimerization
induced by UV light
 a high incidence of skin cancer
 seven genes are required for nucleotide-
excision repair in humans
 Li-Fraumeni
 p53 (halt cell division & stimulate DNA
repair)
 Initiate apoptosis
 Predispositon to many different types of
Cancer
Faulty DNA Repair