CHAPTER 7

DNA Mutation, DNA Repair and

Transposable Elements

Micky Vincent
Mutations
• A mutation is a change in a DNA base-pair or a chromosome:
a. Somatic mutations affect only the individual in which they arise.
b. Germ-line mutations alter gametes, affecting the next generation.
• Mutations are quantified in two different ways:
a. Mutation rate is the probability of a particular kind of mutation as a function of
time (e.g. number per gene per generation).
b. Mutation frequency is number of times a particular mutation occurs in
proportion to the number of cells or individuals in a population (e.g. number per
100,000 organisms).
• Mutations that are common are point mutation. There are two types:
a. base-pair substitutions.
b. base-pair deletions or insertions.
Mutations - Base-pair Substitutions
• A base-pair substitution replaces 1 base-pair with another. There are two types:
a. Transitions convert a purine-pyrimidine pair to
the other purine-pyrimidine pair (e.g. AT to GC
or TA to CG).
b. Transversions convert a purine-pyrimidine pair
to a pyrimidine-purine pair (e.g. AT to TA, or AT
to CG).
• Base-pair substitutions are also defined by their effect on the protein sequence.
- Effects vary from none to severe.
a. Missense mutations have a base-pair change
resulting in a different mRNA codon, and
therefore a different amino acid in the
b. protein.
Nonsense mutations change a codon to a
stop (nonsense) codon, resulting in
premature termination of translation and a
truncated (often nonfunctional) protein.
Mutations - Base-pair Substitutions
Mutations - Base-pair Substitutions
• Base-pair substitutions may also cause phenotypic effects, depending on the
specific amino acid change :
a. Neutral mutations change a codon, but the resulting amino acid substitution
produces no detectable change in the function of the protein.
- e.g. AAA to AGA substitutes arginine for lysine.

- The amino acids have similar properties, so the protein’s function may not
be altered.
b. Silent mutations occur when the mutant codon encodes the same amino acid
as the wild-type gene.
- e.g. AAA and AAG both encode lysine.

- No change occurs in the protein produced, so this transition would be

silent.
Mutations - Base-pair Deletions or Insertions
• Deletions and insertions can change the reading frame of the mRNA downstream
of the mutation, resulting in a frameshift mutation.
a. When the reading frame is shifted, incorrect amino acids are usually
incorporated.

b. Frameshifts may bring stop codons into the reading frame, creating a
shortened protein.
c. Frameshifts may also result in read-through of stop codons, resulting in a
longer protein.
d. Transversions convert a purine-pyrimidine pair to a pyrimidine-purine pair (e.g.
AT to TA, or AT to CG).
Reverse Mutations and Suppressor Mutations
• Point mutations are divided into two classes based on their effect on phenotype:
a. Forward mutations change the genotype from wild type to mutant.
b. Reverse mutations (reversions or back mutations) change the genotype from
mutant to wild-type or partially wild-type.
- A reversion to the wild-type amino acid in the affected protein is a true
reversion.
- A reversion to some other amino acid that fully or partly restores protein
function is a partial reversion.
• Suppressor mutations occur at sites different from the original mutation.
- Usually mask or compensate for the initial mutation without actually reversing it.
Reverse Mutations and Suppressor Mutations
• Suppressor mutations have different mechanisms depending on the site at which
they occur.
a. Intragenic suppressors occur within the same gene as the original mutation,
but at a different site. Two different types occur:
- A different nucleotide is altered in the same codon as the original mutation.
- A nucleotide in a different codon is altered (e.g. an insertion frameshift is
suppressed by a nearby deletion event).
b. Intergenic suppressors occur in a different gene (the suppressor gene) from
the original mutation.
- Suppressor genes often encode tRNAs that recognize stop codons and insert
an amino acid, preventing premature termination of translation.
- Nonsense suppressors fall into three classes, one for each stop codon (UAG,
UAA and UGA).
Spontaneous and Induced Mutations
• Most mutations are spontaneous, rather than induced by a mutagen.
• All types of point mutations can occur spontaneously, during S, G1 and G2 phases
of the cell cycle, or by the movement of transposons.
• The spontaneous mutation rate in eukaryotes is between 10-4-10-6 per gene per
generation, and in bacteria and phages 10-5-10-7/gene/generation.
a. Genetic constitution of the organism affects its mutation rate.
- In Drosophila, males and females have similar mutation rates.
- Flies of different strains, however, may have different mutation rates.
• Many spontaneous errors are corrected by the cellular repair systems, and so do
not become fixed in DNA.
DNA Replication Errors
• DNA replication errors can be either point mutations, or small insertions or
deletions.
• Base-pair substitution mutations can result
from “wobble” pairing.
DNA Replication Errors
• Bases are normally in the keto form, but sometimes can undergo tautomeric shift
to form the abnormal (enol) form. An example is a GC-to-AT transition
- During DNA replication,
G could wobble pair with
T, producing a GT pair.
- In the next round of
replication, G and A are
likely to pair normally,
producing one progeny
DNA with a GC pair, and
another with an AT pair.

- GT pairs are targets for correction by proofreading during replication, and by

other repair systems. Only mismatches uncorrected before the next round of
replication lead to mutations.
DNA Replication Errors
• Additions and deletions can occur spontaneously during replication.
- DNA loops out from the template
strand, generally in a run of the same
base. in

- DNA polymerase skips the looped out

bases, creating a deletion mutation.
- If DNA polymerase adds untemplated
base(s), new DNA looping occurs,
resulting in additional mutation.
- Insertions and deletions in structural
genes generate frameshift mutations.
DNA Replication Errors
• Spontaneous chemical changes include depurination and deamination of
particular bases, creating lesions in the DNA.
a. Depurination removes the purine (A or G) from DNA by breaking the bond with
its deoxyribose in the backbone.
- Depurination is common and if not repaired before the next round of
replication, it will result in a random base at that site.
b. Deamination removes an amino group from a base (e.g., cytosine to uracil)
- Uracil is an abnormal base in DNA, and it
will usually be repaired.
- If uracil is not replaced, it will pair with an
A during replication, resulting in a CG-to-
TA transition.
Induced Mutations
• Exposure to physical mutagens plays a role in genetic research, where they are
used to increase mutation frequencies to provide mutant organisms for study.
• Radiation (e.g. X-rays and UV) induces mutations.
a. X-rays are an example of ionizing radiation, which penetrates tissue and
collides with molecules, knocking electrons out of orbits and creating ions.
- Ions can break covalent bonds, including those in the DNA sugar-phosphate
backbone.
- Ionizing radiation is the leading cause of human gross chromosomal
mutations.
- Ionizing radiation kills cells at high doses, and lower doses produce point
mutations.
- Ionizing radiation has a cumulative effect. A particular dose of radiation
results in the same number of mutations whether it is received over a short
or a long period of time.
Induced Mutations
a. Ultraviolet (UV) causes photochemical changes in the DNA.
- UV is not energetic enough to induce ionization.
- UV has lower-energy wavelengths than X rays, and so has limited penetrating
power.
- However, UV in the 254–260 nm range is strongly absorbed by purines and
pyrimidines, forming abnormal chemical bonds.
- A common effect is dimer formation between adjacent pyrimidines,
commonly thymines (designated T^T)

- Most pyrimidine dimers are

repaired, because they produce a
bulge in the DNA helix. If enough are
unrepaired, cell death may result.
Chemical Mutagens
• Chemical mutagens may be naturally occurring, or synthetic. They form different
groups based on their mechanism of action:
• Three types of chemical mutagens:
a. Base analogs.
b. Base-modifying agents.
c. Intercalating agents.
Chemical Mutagens - Base analogs
• Base analogs incorpocate a base with alternate states (tautomers) that allow it to
base pair in alternate ways
• Analogs are similar to normal nitrogen bases, and so are incorporated into DNA
readily.
• 5-bromouracil (5BU) is an example. 5BU has a bromine
residue instead of the methyl group of thymine
- Normally 5BU resembles thymine, pairs with
adenine and is incorporated into DNA during
replication.
- In its rare state, 5BU pairs only with guanine,
resulting in a TA-to-CG transition mutation.
- If 5BU is incorporated in its rare form, the switch
to its normal state results in a CG-to-TA
transition. Thus 5BU- induced mutations may be
reverted by another exposure to 5BU.
Fig. 7.11c Mutagenic effects of the base analog 5-bromouracil (5BU)
Chemical Mutagens - Base-modifying agents
• Base-modifying agents can induce mutations at any stage of the cell cycle by
modifying the chemical structure and properties of the bases.
• Three types of base-modifying agents:
a. Deaminating agents remove amino groups such as G, C and A. An example is
nitrous acid (HNO2).
- Mutations induced by HNO2 can revert with a second treatment.
b. Hydroxylating agents that include hydroxylamine (NH2OH).
- NH2OH specifically modifies C with a hydroxyl group (OH), so that it pairs
only with A instead of with G.
c. Alkylating agents are a diverse group that add alkyl groups to bases.
- Usually alkylation occurs at the 6-oxygen of G, producing O6-allcylguanine.
- An example is methylmethane sulfonate (MMS), which methylates G to
produce O6-alkyl G.
- O6-aIkylG pairs with T rather than C, causing GC-to-AT transitions.
Chemical Mutagens - Intercalating agents
• Intercalating agents are generally thin, plate-
like hydrophobic molecules that insert
themselves between adjacent bases in dsDNA.
- During replication, a template that contains
an intercalated agent will cause insertion of a
random extra base.
- The base-pair addition is complete after
another round of replication, during which
the intercalating agent is lost.
- If an intercalating agent inserts into new DNA
in place of a normal base, the next round of
replication will result in a deletion mutation.
- Point deletions and insertions result in
frameshift mutations. These mutations show
reversion with a second treatment.

END
Site-Specific in vitro Mutagenesis of DNA
• Recombinant DNA technology allows genes to be mutated at specific positions, and
then introduced back into cells.
• This allows study of genes of unknown function and sequences used to regulate
gene expression.
• A wide variety of chemicals exist in our environment, and many can have
mutagenic effects that can lead to genetic diseases and cancer. Examples include:
a. Drugs/cosmetics/food additives
b. Pesticides
c. Industrial compounds
d. Chemical warfare agents such as mustard gas
• To test for the potential mutagenicity of chemicals, an assay known as The Ames
Test is performed.
- Assays the reversion rate of mutant strains of Salmonella typhimurium back to
wild-type.
- Used routinely to screen industrial and agricultural chemicals, and shows a
good, but not perfect, correlation between mutagens and carcinogens.
The Ames Test
• Histidine (his) are tested for reversion in the presence of the chemical, by plating
on media lacking the amino acid histidine.
• Liver enzymes (the S9 extract) are mixed with the test chemical to determine
whether the liver’s detoxification pathways convert the test chemical to a
mutagenic form.
• More revertants in the region of the test
chemical than in the untreated control
indicates that it may be a mutagen, and
further tests are indicated.
Detecting Mutation
• Mutants are studied by geneticists. Mutation in haploid organisms is readily
detected, while recessive mutations in diploid organisms are harder to
• characterize.
Detecting mutation is more complex in humans, where controlled crosses cannot
be done.
• For some organisms, especially microorganisms, selection and screening
procedures exist.
• Several mutation terminologies:
a. Visible Mutations - Some mutations affect the appearance
of an organism (e.g. Drosophila eyes or wing-shape, coat
color in animals, colony size in yeast, plaque morphology of
b. phages).
Nutritional Mutations - An example is replica plating.
Cells are first grown on supplemented medium, and
then the colonies transferred to minimal medium, as
well as to a control plate of supplemented medium.
- Colonies that grow on supplemented, but not minimal,
media are selected for further study.
Fig. 7.15 Replica-plating technique to screen for mutant strains of a colony-forming
microorganism
Detecting Mutation
• Several mutation terminologies:
c. Conditional Mutations - Mutations in some genes
(e.g., DNA and RNA polymerases) are usually lethal, so
these genes are studied by isolating conditional
-
mutations. Heat sensitivity is a common
conditional mutation, in which a normal protein is
produced at permissive temperature, and a
nonfunctional protein results at the nonpermissive
temperature. Screening is generally by replica plating
d. and incubation
Resistance at different-temperatures.
Mutations Microorganisms like E. coli
and yeast are easily screened for resistance to viruses,
chemicals or drugs, because resistant cells will grow
when wild-type cells will not.
Repair of DNA Damage
• Both prokaryotes and eukaryotes have enzyme-based DNA repair systems that
prevent mutations and even death from DNA damage.
• Repair systems are grouped by their repair mechanisms. Some directly correct,
while others excise the damaged area and then repair the gap.
• Several types of DNA damage repair mechanisms:
a. Direct Reversal of DNA Damage.
b. Base Excision Repair
c. Nucleotide excision Repair
Repair of DNA Damage - Direct Reversal of DNA Damage
• DNA polymerase proofreading corrects most of the incorrect nucleotide insertions
that occur during DNA synthesis, which stalls until the wrong nucleotide is replaced
with a correct one.
a. The role of 3’-to-5’ exonuclease activity is illustrated by mutator mutations in
E. coli, which confer a much higher mutation rate on the cells that carry them.
b. The mutD gene, encoding the e subunit of DNA polymerase III, is an example.
Cells mutant in mutD are defective in proofreading.
• UV-induced pyrimidine dimers are repaired using photoreactivation (light repair).
a. Near UV light (320–370 nm) activates photolyase (product of the phr gene) to
split the dimer.
b. Photolyases are found in prokaryotes and simple eukaryotes, but not in humans.
• Damage by alkylation (usually methyl or ethyl groups) can be removed by specific
DNA repair enzymes.
- For example, O6-methylguanine methyltransferase recognizes O6-methylguanine
in DNA, and removes the methyl group, restoring the base to its original form.
• Mutations of repair enzyme genes increase the organism’s rate of spontaneous
mutations.
Repair of DNA Damage - Base Excision Repair
• Base excision repair uses a repair glycosylase enzyme to recognize and remove
damaged bases.
a. Bond between base and deoxyribose is cleaved by the glycosylase.
b. Other enzymes cleave sugar-phosphate from backbone, leaving a gap in the
DNA. DNA polymerase and DNA ligase use the opposite strand as template to
c. Repair
fill the gap.
Repair of DNA Damage - Excision of Nucleotides
• A repair system, which does not require light, was discovered in 1964.
• It is called dark repair, the excision repair system, or the nucleotide excision repair
(NER) system.
- In E. coli, NER corrects pyrimidine dimers and other
damage-induced distortions of the DNA helix.
- The proteins required are UvrA, UvrB, UvrC and UvrD.
- A complex UvrA-UvrB proteins slides along the DNA.
As it encounters a helix distortion, the UvrA subunits
dissociate, and a UvrC binds the UvrB at the lesion.
- When UvrBC forms, the UvrC cuts 4–5 nucleotides
from the lesion on the 3’ side, and eight nucleotides
away on the 5’ side. Then UvrB is released and UvrD
binds the 5’ cut end.
- UvrD is a helicase that unwinds the region between the
cuts, releasing the short ssDNA, while DNA polymerase
I fills the gap and DNA ligase seals the backbone.
• In yeast and mammalian systems, about 12 genes
encode proteins involved in excision repair.
Repair of DNA Damage - Excision of Nucleotides
• Another repair system that involves the excision of nucleotides is a methyl-directed
mismatch repair which recognizes mismatched base pairs.
• In E. coli, initial stages involve products of
the mutS, mutL and mutH genes.
- MutS binds the mismatch, and determines which
is the new strand by its lack of methylation.
- MutL and MutH bind unmethylated GATC
sequences and bring the GATC close to the
mismatch by binding MutS.
- MutH then nicks the unmethylated GATC site,
the mismatch is removed by an exonuclease
and the gap is repaired by DNA polymerase III
and ligase.
• Eukaryotes also have mismatch repair, but it
is not clear how old and new DNA strands
are identified.
- Four genes are involved in humans, hMSH2 and
hMLH1, hPMS1 and hPMS2.
Repair of DNA Damage - Excision of Nucleotides
• Translesion DNA synthesis is used to allow a cell to survive when specific base-
pairing cannot occur. Survival is usually at the cost of incurring new mutations.
- Bacteria use a system called SOS. In E. coli SOS is controlled by two genes, lexA
and recA.
- Sufficient DNA damage activates the recA protein, which stimulates lexA to
autocleave, removing repression of the DNA repair genes.
- After damage is repaired, recA is inactivated, and newly synthesized lexA again
represses the DNA repair genes.
- The SOS system is a mutagenic bypass synthesis system:
i. DNA polymerase for translesion synthesis is made in SOS response. It
replicates over and past the lesion.
ii. Nucleotides are incorporated at the lesion that may not match wild type,
leading to mutations.
iii.This is better than the alternative, death due to incompletely replicated DNA.
Human Genetic Diseases - Resulting from DNA Replication
and Repair Mutations
• Many human genetic disorders result from gene mutations, and are included in the
OMIM website (http://www3.ncbi.nlm.nih.gov/OMIM).
• An example of human genetic diseases
resulting from defects in DNA replication or
repair is Xeroderma pigmentosum.
- The disorder occurs in homozygotes for a
mutation in a repair gene.
- Affected individuals are photosensitive, and
portions of skin exposed to light show
intense pigmentation and warty growths
that may become malignant.
- The defect is in excision repair, and the
inability to repair radiation damage to DNA
often results in malignancies.